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N° d’ordre : 4439
THÈSE
Présentée à
L’UNIVERSITÉ DE BORDEAUX 1ÉCOLE DOCTORALE : Sciences et Environnements
Par
Muhammad NADEEMPour obtenir le grade de
DocteurSpécialité : Ecologie évolutive, fonctionnelle et des communautés
Remobilisation des réserves en phosphore du grain et prélèvement
du phosphore exogène pendant la germination et la croissance
juvénile du maïs (Zea mays L.)
Soutenue le : 16/12/2011
Après avis de :
M. Jean-Christophe AVICE Prof. Université Caen RapporteurM. Christophe SALON DR, INRA Dijon Rapporteur
Devant la commission d’examen formée de :M. Jean-Christophe AVICE Prof. Université Caen RapporteurM. Christophe SALON DR, INRA Dijon RapporteurM. Abraham ESCOBAR GUTIERREZ CR, INRA Lusignan ExaminateurM. Olivier TURC CR, INRA Montpellier ExaminateurM. Alain MOLLIER CR, INRA Bordeaux Co-directeur de thèseM. Sylvain PELLERIN DR, INRA Bordeaux Directeur de thèse
INRA ENITAB, UMR 1220 TCEM Transfert sol-plantes et Cycles des Éléments Minéraux dans les écosystèmes cultivés, 71 avenue Edouard Bourlaux, BP 81, 33883 Villenave d'Ornon, France.
THESIS
Presented at
UNIVERSITY OF BORDEAUX 1Sciences and Environments Graduate School
By
Muhammad NADEEMFor the Degree of
DoctorSpeciality: Functional and Community Ecology
Remobilization of seed phosphorus reserves and exogenous
phosphorus uptake during germination and early growth stages of
maize (Zea mays L.)
16th December 2011
Examination Commission M. Jean-Christophe AVICE Prof. University of Caen ExaminerM. Christophe SALON DR, INRA Dijon ExaminerM. Abraham ESCOBAR GUTIERREZ CR, INRA Lusignan ExaminerM. Olivier TURC CR, INRA Montpellier ExaminerM. Alain MOLLIER CR, INRA Bordeaux Co-superviserM. Sylvain PELLERIN DR, INRA Bordeaux Superviser
INRA ENITAB, UMR 1220 TCEM Soil-plant Transfer and the nutrient and trace element cycle in cultivated ecosystems, 71 avenue Edouard Bourlaux, BP 81, 33883 Villenave d'Ornon, France.
N° d’ordre : 4439
Dedicated
to
My Dearest Parents
who are the part of my soul and whose love,
affection and confidence enabled
me to achieve this goal
ii
Acknowledgements
If all the trees were pens and oceans were ink, the praise of almighty ALLAH (subhanahu wa ta’ala), the ultimate source of knowledge to mankind, would never end. I therefore, start my acknowledgement as a word of thank to almighty ALLAH (subhanahu wa ta’ala), WHO bestowed me with the potential and ability to contribute a drop of material to the existing ocean of scientific knowledge. I also offer my humblest thanks from the deepest core of my heart to the Holy Prophet MUHAMMAD (peace be upon him), WHO is forever a torch of guidance and knowledge for humanity as a whole.
I would like to express my sincerest gratitude to my supervisor Prof. Dr. Sylvain PELLERIN for his continue support, guidance and also for his vast knowledge that helped me to complete this project in time. I also owe a debt of gratitude for my co-supervisor Dr. Alain MOLLIER for his worthy ideas, guidance, motivation and enthusiasm that helped me a lot to accomplish the objectives of my Ph.D research work. I would never have imagined such a best advisor and mentor for my Ph.D studies. I have no hesitation to say that I learned a lot from him.
I would like to extend my sincere regards to my thesis committee members Dr. Carolyne DURR (INRA Anger), Dr. Marie-Hélène MACHEREL (University of Anger) and Dr. Marie-Pascale PRUD'HOMME (University of Caen) for their kind guidance and for giving valuable advice during my PhD studies.
Thanks are due for Dr. Christian MOREL for providing valuable suggestions and help during my experiments especially for working with 32P. I am also thankful to Mr. Alain VIVES and Mr. Loïc PRUD'HOMME for their useful suggestions and guidance during the greenhouse and growth chamber experiments and for laboratory analysis. They did their best in this regard. My sincere thanks also go to the technical staff in the laboratory and especially Ms. Anne GALLET-BUDNEK, Ms. Sylvie MILIN and Ms. Sylvie BUSSIERE who assisted me a lot during my studies.
Words do not count easy to pay reverent thanks and compliments to my father Abdul Ghafoor, mother Shameem Akhtar, brothers Amin, Anwer, Saleem, Naeem and my sisters Rubina, Farzana, Rehana for their love, affection, amicable attitude, prayers for my success, consistent encouragement and cordial cooperation through my life. I am also thankful to all my Pakistani friends in Bordeaux and to all my fellows of lab, office and coffee room who always wished me for success. The sweet memories of these three years will always be with me. I am also thankful to Higher Education Commission, Pakistan for awarding me scholarship for my Ph.D studies in France. In the end I would like to express my affection and thankfulness for TCEM INRA Bordeaux for providing me an opportunity to complete my Ph. D work in this research unit.
Muhammad Nadeem
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REMOBILIZATION OF SEED PHOSPHORUS RESERVES AND EXOGENOUS
PHOSPHORUS UPTAKE DURING GERMINATION AND EARLY GROWTH STAGES
OF MAIZE (Zea mays L.)
Summary
Phosphorus (P) is an essential element for plant growth. Many studies have shown a very early seedling response to the limitation on the availability of P. During germination and early growth, the seedling P demand may be satisfied by the remobilization of seed P reserves and exogenous P uptake by developing roots. The objective of the thesis was to study the relative contribution of remobilization of seed P reserves, the exogenous P uptake by seedling roots and the interaction between these two processes. Various experiments were conducted to i) study the kinetics of the remobilization of seed P reserves, ii) identify precisely the beginning of exogenous P uptake by seedling roots, iii) quantify the relative contribution P fluxes in developing seedlings and iv) the interaction between these two P fluxes. Seeds with low and high P reserves were cultivated at different levels of exogenous P availability for the growth period of four weeks. The exogenous P was labelled with radioactive P (32P) to identify and quantify the P flux in young seedlings coming from exogenous P uptake and seed P reserves remobilization. Initially, 86% of P in the form of phytate and 13% C of seed reserves is localised in scutellum regardless of P initial seed P reserves. Four days after germination, 98% of seed phytate reserves are hydrolyzed. The kinetics of seed phytate hydrolysis was independent of seed P reserves and exogenous P availability. The hydrolyzed forms of phytate were temporarily stored in the seed before being translocated towards newly growing seedling compartments. The exogenous P uptake started soon after the radicle emergence (4th day) and depend mainly on the availability of exogenous P in the growth medium. The beginning of exogenous P uptake and its intensity was not influenced by the seed P reserves remobilization. The proportion of distribution of remobilized seed P reserves and the exogenous P uptake was similar among seedling shoot and roots. The whole seed and seedling P budget showed the significant P losses from germinating seeds by P efflux with the beginning of phytate hydrolysis in seeds. We proposed a model for the seed P remobilization and exogenous P uptake during germination and early growth. Assuming no interaction between seed P reserves remobilization and exogenous P uptake, the simulations were found to be in close agreement with experimental data. Our results showed the importance of exogenous P availability in growth medium during early growth stages regardless of seed P reserves.
Key-words: maize, germination, phosphorus, phytate, remobilization, P-uptake, isotope, ecophysiology, mineral nutrition.
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REMOBILISATION DES RESERVES EN PHOSPHORE DU GRAIN ET
PRELEVEMENT DE PHOSPHORE EXOGENE PENDANT LES PHASES DE
GERMINATION ET DE CROISSANCE JUVENILE DU MAÏS (Zea mays L.)
Résumé
Le phosphore (P) est un élément indispensable pour la croissance des plantes. De nombreux travaux montrent des réponses très précoces à une limitation de la disponibilité en P. Pendant la germination et la croissance juvénile, la demande en P des plantules peut être satisfaite par la remobilisation des réserves en P des graines et le prélèvement racinaire. Les objectifs de la thèse sont d’étudier la contribution respective de la remobilisation des réserves en P des graines et du prélèvement racinaire de P à l’alimentation en P des plantules de maïs, et les interactions entre ces deux processus. Différentes expériences ont été conduites pour i) étudier les cinétiques de la remobilisation des réserves en P des graines, ii) identifier précisément le début du prélèvement de P exogène par les racines, iii) quantifier la contribution relative de ces flux à l’alimentation en P de la plantule, iv) comprendre les interactions entre ces flux. Des graines riches et des graines pauvres en P on été cultivées à différents niveaux de disponibilités P exogènes pendant quatre semaines. Le traçage isotopique du P exogène (32P) a été utilisé pour quantifier le flux de prélèvement et calculer le flux de remobilisation du P des graines. Initialement, 86% du P sous forme phytate et 13% du C de la graine est localisé dans le scutellum indépendamment du niveau de richesse en P de la graine. 4 jours après le semis, 98% des phytates des graines sont hydrolysés. La cinétique d’hydrolyse des phytates est indépendante de la richesse en P des graines et de la disponibilité en P dans le milieu. Le P issu de l’hydrolyse des phytates est stocké temporairement dans la graine avant d’être transporté vers les organes en croissance de la plantule. Le prélèvement de P exogène commence dès l’émergence de la radicule (4ième jour) et dépend de la disponibilité en P dans le milieu. L’initiation du prélèvement et son intensité ne dépend pas du flux de remobilisation des réserves en P de la graine. Le P issu de la remobilisation et du prélèvement est distribué dans les mêmes proportions entre les parties ariennes et racinaires. Un bilan de P à l’échelle de la plantule entière et de la graine a permis de mettre en évidence un efflux de P depuis la graine vers l’extérieur pendant la phase d’hydrolyse des phytates. La modélisation des flux de P pendant la germination et la croissance précoce permet de rendre compte des observations sous l’hypothèse d’absence d’interaction entre les flux de remobilisation et de prélèvement de P bien que ces deux processus se chevauchent dans le temps. Nos résultats démontrent l’importance de la disponibilité locale en P dans le milieu pendant les stades précoces indépendamment du niveau de richesse en P des graines.
Mots-clés: maïs, germination, phosphore, phytate, remobilisation, prélèvement de P, isotope, écophysiologie, nutrition minérale.
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TABLE OF CONTENTS
Acknowledgements ....................................................................................................................... iii
Summary ......................................................................................................................................... v
Résumé ......................................................................................................................................... vii
TABLE OF CONTENTS ................................................................................................................ 1
LIST OF ABBREVIATIONS ......................................................................................................... 5
LIST OF FIGURES ........................................................................................................................ 7
LIST OF TABLES ........................................................................................................................ 13
1. INTRODUCTION ..................................................................................................................... 15
2. BIBLIOGRAPHIC REVIEW ................................................................................................... 19
2.1. Germination ........................................................................................................................ 19
2.1.1. Types of germination ................................................................................................... 19
2.1.2. Factors affecting germination ...................................................................................... 20
2.1.2.a. Water .................................................................................................................... 20
2.1.2.b. Temperature ......................................................................................................... 21
2.1.2.c. Hydrothermal models ........................................................................................... 22
2.2. Maize seed and its structure ................................................................................................ 22
2.3. Early seedling growth and seed reserves remobilization .................................................... 24
2.3.1. Germination, early growth and seed carbon remobilization ........................................ 25
2.3.2. Germination, early growth and mineral reserves remobilization ................................ 26
2.3.2.a. Nitrogen ............................................................................................................... 26
2.3.2.b. Phosphorus .......................................................................................................... 26
[i.] Phosphorus in seeds ................................................................................................ 26
[ii.] Phytate biosynthesis and accumulation in seeds .................................................... 27
[iii.] Functions of phytate ............................................................................................... 29
[iv.] Phytate in seeds ..................................................................................................... 29
[v.] Factors effecting seed phytate and P concentrations ............................................... 29
[vi.] Activity of phytase during germination and early growth .................................... 30
[vii.] Phytase activity and effect of different factors .................................................... 31
[viii.] Seed phytate loss ................................................................................................. 33
[ix.] Mobilization of seed P reserves during germination and early growth ................. 34
1
[x.] Effect of seed P on early seedling growth ............................................................... 36
2.4. Maize seedling root growth and exogenous phosphorus uptake ........................................ 37
[i.] Growing maize seedling roots .................................................................................. 37
[ii.] Exogenous P uptake by growing seedling roots .................................................... 39
2.5. Conclusions of bibliographic review .................................................................................. 41
2.6. Objectives of thesis studies ................................................................................................. 42
2.6.1. General representation of studied system .................................................................... 42
2.6.2. Research plan ............................................................................................................... 43
3. MATERIALS AND METHODS ............................................................................................... 45
3.1. Experiment 1 (greenhouse study 2010) .............................................................................. 45
3.1.1. Experimental layout ..................................................................................................... 45
3.1.2. Seed sowing ................................................................................................................ 45
3.1.3. Nutrient solution composition ..................................................................................... 46
3.1.4. Labeling of nutrient solution ....................................................................................... 46
3.1.5. Seedling growth conditions ......................................................................................... 47
3.1.6. Irrigation ..................................................................................................................... 48
3.1.7. Seedling harvest ........................................................................................................... 48
3.1.8. Statistical analysis ........................................................................................................ 50
3.2. Experiment 2 (growth chamber study 2010) ...................................................................... 51
3.2.1. Experimental layout and selection of maize seeds ...................................................... 51
3.2.2. Seed sowing ................................................................................................................ 51
3.2.3. Nutrient solution composition ..................................................................................... 51
3.2.4. Labeling of nutrient solution ....................................................................................... 52
3.2.5. Seedling growth conditions ......................................................................................... 52
3.2.6. Irrigation ..................................................................................................................... 54
3.2.7. Seedling harvest ........................................................................................................... 54
3.2.8. Statistical analysis ........................................................................................................ 55
3.3. Growth measurements and chemical analysis .................................................................... 56
3.3.1. Morphological analysis ................................................................................................ 56
3.3.1.a. Root and leaf morphology .................................................................................... 56
3.3.1.b. Fresh and lyophilized seedling biomass .............................................................. 56
3.3.2. Chemical analysis ........................................................................................................ 56
3.3.2.a. Phosphorus determination .................................................................................... 57
3.3.2.b. Exogenous P uptake and its allocation using 32P labeling .................................. 57
2
3.3.2.c. Phytate and phytate-P determination ................................................................... 57
3.3.2.d. Carbon and nitrogen analysis ............................................................................... 58
3.3.3. Endogenous seed P exportation flux ............................................................................ 58
3.3.4. P efflux from germinating seeds and growing maize seedling roots ........................... 58
3.4. Control of experimental procedures ................................................................................... 59
3.4.1. P loss from seeds by imbibition ................................................................................... 59
3.4.2. P sorption properties of perlite ..................................................................................... 60
3.4.3. Base temperature for maize germination ..................................................................... 60
3.4.4. P sorption on seedling roots ......................................................................................... 62
3.4.5. Specific activity induced background noise ................................................................ 63
4. RESULTS AND DISCUSSIONS .............................................................................................. 65
4.1. Relative contribution of seed phosphorus reserves and exogenous phosphorus uptake to
maize (Zea mays L.) nutrition during early growth stages ........................................................ 65
4.1.1. Objectives .................................................................................................................... 65
4.1.2. Results ......................................................................................................................... 65
4.1.2.a. Seedling growth ................................................................................................... 65
4.1.2.b. Seed and seedling C content ................................................................................ 66
4.1.2.c. Remobilization of seed P reserves and exogenous P uptake ............................... 69
4.1.2.d. Remobilization of seed N reserves and seedling N accumulation ....................... 70
4.1.2.e. Phytate and phytate-P ........................................................................................... 71
4.1.2.f. Relative contribution of seed P and exogenous P uptake to seedling P nutrition 72
4.1.2.g. Relationship between P and N remobilization ..................................................... 75
4.1.3. Discussion .................................................................................................................... 76
4.1.3.a. Carbon remobilization and seedling growth ........................................................ 76
4.1.3.b. Phytate hydrolysis and relative contribution of P to growing seedlings .............. 76
4.1.3.c. Relationship between P and N remobilization ..................................................... 77
4.2. Maize (Zea mays L.) endogenous seed phosphorus remobilization is not influenced by
exogenous phosphorus during germination and early growth stages ........................................ 78
4.2.1. Objectives .................................................................................................................... 78
4.2.2. Results .......................................................................................................................... 78
4.2.2.a. Early seedling growth .......................................................................................... 78
4.2.2.b. Accumulation of P in seedlings .......................................................................... 80
4.2.2.c. Seed phytate-P hydrolysis ................................................................................... 82
4.2.2.d. Endogenous seed P remobilization and export flux ............................................ 84
3
4.2.2.e. Exogenous P uptake by growing seedling roots .................................................. 85
4.2.2.f. P efflux and whole seedling P budget .................................................................. 85
4.2.3. Discussion .................................................................................................................... 89
4.2.3.a. Effect of endogenous and exogenous P on seed P remobilization and seedling
growth 89
4.2.3.b. Effect of endogenous and exogenous P on uptake of exogenous P .................... 90
4.2.3.c. Whole seedling P budget ...................................................................................... 91
4.2.3.d. What explains early P deficiency in maize often reported in the literature? ....... 92
4.3. Modelling of seed P reserves remobilization and exogenous P uptake .............................. 93
4.3.1. Seed phytate hydrolysis module .................................................................................. 96
4.3.2. Seedling growth module .............................................................................................. 98
4.3.3. Seedling P demand module .......................................................................................... 98
4.3.4. Exogenous P uptake by roots module ........................................................................ 100
4.3.5. Effective P fluxes and total P balance module .......................................................... 101
4.4. Model output ..................................................................................................................... 104
4.4.1. Seed phytate hydrolysis ............................................................................................. 104
4.4.2. Seed non phytate-P .................................................................................................... 105
4.4.3. Seedling P demand and seedling P accumulation ...................................................... 106
4.4.4. Seed P export and root P uptake ................................................................................ 108
4.4.5. Seed P efflux .............................................................................................................. 110
4.5. Discussion ........................................................................................................................ 111
5. GENERAL DISCUSSION AND PROSPECTIVES .............................................................. 113
BIBLIOGRAPHIC REFERENCES ........................................................................................... 117
APPENDIX ................................................................................................................................. 125
Appendix I: Prélèvement et conservation de grains de maïs avec des teneurs en phosphore
différenciées ............................................................................................................................. 125
Appendix II: Dosage des phytates par chromatographie ionique en milieu HCl 0.65M. Mode
opératoire MO-ANA-65 .......................................................................................................... 127
Appendix III: Nadeem M, Mollier A, Morel C, Vives A, Prud’homme L, Pellerin S (2011)
Relative contribution of seed phosphorus reserves and exogenous phosphorus uptake to maize
(Zea mays L.) nutrition during early growth stages. Plant Soil 346 (1-2):231-244.
doi:10.1007/s11104-011-0814-y .............................................................................................. 133
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LIST OF ABBREVIATIONS
TT : Thermal time (Cumulated degree days after sowing)
DAS : Days after sowing
PAR : Photosynthetically active radiation (µmol m-2 sec-1)
RH : Relative humidity (%)
DW : Dry weight (g seedling-1)
C+M : Coleoptile+mesocotyle
SE : Standard error
LS : Seeds with low endogenous seed P reserves (506 µg P seed-1)
HS : Seeds with high endogenous seed P reserves (952 µg P seed-1)
0P : No exogenous P
LP : Low exogenous P (100 µmol P L-1)
HP : High exogenous P (1000 µmol P L-1)
Endo-P : Endogenous seed P
Exo-P : Exogenous P
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LIST OF FIGURES
Figure 1.1: Global map of agronomic P imbalances for the year 2000 expressed per unit of
cropland area in each 0.5° grid cell. The P surpluses and deficits are each classified according to
quartiles globally (0-25th, 25-50th, 50-75th and 75-100th percentiles, MacDonald et al., 2011).
.......................................................................................................................................................15
Figure 1.2: Peak phosphorus curve indicating that production will eventually reach a maximum,
after which it will decrease (Cordell et al., 2009)..........................................................................16
Figure 1.3: Effect of P on leaf area index, in maize seedlings during early growth stage (Plénet et
al., 2000)........................................................................................................................................17
Figure 2.1: Epigeal and hypogeal germination in monocot and dicot seed...................................19
Figure 2.2: Water uptake in seeds during germination (Bewley and Black 1994)........................21
Figure 2.3: Internal structure of maize seed..................................................................................23
Figure 2.4: Germination, emergence and seedling establishment.................................................25
Figure 2.5: Phytic acid (myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate or Ins. P6).....................27
Figure 2.6: Biosynthetic pathways to phytic acid (myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate
or Ins. P6) in the eukaryotic cell. A, Structure of phytic acid. B, Structure of Ins. The numbering
of the carbon atoms follows the "D-convention" (Loewus and Murthy, 2000). C, Bio-chemical
pathway of phytic acid (Raboy et al. 2000):..................................................................................28
Figure 2.7: Changes in the myo-inositol hexa- (IP6), penta- (IP5), tetrat- (IP4), and tri- (IP3)
phosphate during germination of barley seeds (Centeno et al. 2001)............................................31
Figure 2.8: Phytate level (□) and phytase activity (∇) during barley germination (Greiner et al.
2000)..............................................................................................................................................32
Figure 2.9: The effect of germination temperature on phytase activity during barley germination
(Sung et al. 2005)...........................................................................................................................33
Figure 2.10: Changes in levels of various phosphorus fractions in the cotyledons of young pea
seedlings during early development (Guardiola and Sutcliffe 1971)............................................35
Figure 2.11: Photographs of maize seed (a) 2 days, (b) 3 days, (c) 4 days, (d) 5 days, and (e and
f) 7 days after germination. C, coleoptile; CN, coleoptile node; CO, coleorhizae; FL, first leaf;
M, mesocotyl; NR, nodal root; PR, primary root; S, scutellum; SL, second leaf; SSR, scutellum
seminal root (Singh et al, 2010).....................................................................................................38
Figure 2.12: Maize seedling system, including seed P reserves, growing seedling and exogenous
nutrient solution P..........................................................................................................................43
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Figure 3.1: Individual pot and sowing of maize seeds..................................................................46
Figure 3.2: Outer view of greenhouse experiment 2010...............................................................47
Figure 3.3: Climatic conditions during first experiment, (photosynthetically active radiation
"PAR" on left axis, temperature "T" and relative humidity "RH" on right axis)..........................48
Figure 3.4: A growing maize seedling during early growth..........................................................49
Figure 3.5: Desorption of 32P and washing of maize seedling roots............................................50
Figure 3.6: Growing maize seedlings in pots placed in plastic trays on iron stands in growth
chamber experiment in 2010..........................................................................................................53
Figure 3.7: Climatic conditions during second experiment, (photosynthetically active radiation
"PAR" on left axis, temperature "T" and relative humidity "RH" on right axis)..........................54
Figure 3.8: Loss of seed P by imbibition (% loss of initial seed P). .............................................59
Figure 3.9: Initial P concentrations added in perlite solution suspension and final P
concentrations in perlite solution suspension after 24 hours. Dashed line is the 1:1 line. Blue dots
are measured data and the solid line is the linear regression line..................................................60
Figure 3.10: Germination rate hour-1 calculated as the inverse of the time to reach 50 %
germination as a function of the temperature. The calculated base temperature for maize
germination was 9.64°C. ...............................................................................................................61
Figure 3.11: P sorption on maize seedling roots during germination and early growth. Data are
means and vertical bars indicate ±se for n = 3 replications...........................................................62
Figure 4.1: Changes in total seedling C (■), seed C remobilization (●), seedling C accumulation
(▲) and seed C loss (∆) in maize seedlings expressed in mg seedling-1 during germination and
early growth stages. Time is expressed in cumulated degree days after sowing. Data are means
and vertical bars indicate ± se for n = 3.........................................................................................67
Figure 4.2: Changes in C content (mg C seedling-1) on the left axis (● and solid lines) and in P
content (µg P seedling-1) on the right axis (○ and dashed lines) in the endosperm (a), scutellum
(b), leaves (c) and roots (d) during early growth of maize. Time is expressed in cumulated degree
days after sowing. Data are means and vertical bars indicate ± se for n = 3.................................68
Figure 4.3: Changes in total seedling P content (●), in shoot and root P content (▲) and in
exogenous P uptake from the nutrient solution calculated according to 32P-activity measured in
the seedling (∆). Data are means and vertical bars indicate ±se for n = 3 replications.................70
Figure 4.4: Changes in total quantity of seed (●), seedling (▲), endosperm () and scutellum (○)
nitrogen (mg seedling-1) contents in maize seeds and seedlings during germination and early
8
growth stages. Time is expressed in cumulated degree days after sowing. Data are means and
vertical bars indicate ± se for n = 3................................................................................................71
Figure 4.5: Variation in total P (µg P seed-1) and phytate-P (µg phytate-P seed compartment-1)
reserves in different compartments of the maize seed during early growth stages. Remobilization
of total seed P reserves (●); Scutellum phytate-P reserves (∆) and endosperm phytate-P (□). Data
are means and vertical bars indicate ±se for n = 3 replications.....................................................72
Figure 4.6: Allocation of exogenous P uptake (gray arrows on the right) calculated using 32P
labeling of the nutrient solution and distribution of remobilized seed P reserves (dark arrows on
the left) to seedling compartments after 298 cumulated degree days (23 DAS). The width of the
arrows represents the quantity of P flowing toward different seedling compartments. Values
inside the different compartments represent P accumulation in (+) or remobilization from (-)
each compartment. Values in italics represent P content in the seed and in the seedling after 298
cumulated degree days (23 DAS). P fluxes are expressed in µg P seedling-1. Data are means and
vertical bars indicate ± se for n = 3 replications............................................................................74
Figure 4.7: Rate of P, C and N remobilization in maize seed during early growth stages
calculated as the ratio of P (○), C (●) and N (∆) content in seed to their initial content. Data are
means and vertical bars indicate ±se for n = 3 replications...........................................................75
Figure 4.8: Seed biomass remobilization (g) in maize seeds during germination and early growth
stages: Low endogenous seed P in seedlings (LS) with no exogenous P (○), low exogenous P
(□), high exogenous P (∆), high endogenous seed P seedlings HS with no exogenous P (●), low
exogenous P (■) high exogenous P (▲) treatments. Data are means and vertical bars indicate ±
se for n = 3 replications.................................................................................................................79
Figure 4.9: Seedling biomass accumulation (g) in maize seedlings during germination and early
growth stages: Low endogenous seed P in seedlings (LS) with no exogenous P (○), low
exogenous P (□), high exogenous P (∆), high endogenous seed P seedlings HS with no
exogenous P (●), low exogenous P (■) high exogenous P (▲) treatments. Data are means and
vertical bars indicate ± se for n = 3 replications............................................................................79
Figure 4.10: Phosphorus accumulation (µg P) in maize seedlings during germination and early
growth stages: A. Low endogenous seed P in seedlings (LS) with no exogenous P (○), low
exogenous P (□), high exogenous P (∆) and B. high endogenous seed P seedlings HS with no
exogenous P (●), low exogenous P (■) high exogenous P (▲) treatments. Data are means and
vertical bars indicate ± se for n = 3 replications............................................................................80
9
Figure 4.11: Seedling phosphorus concentrations (mg P g–1) in maize seedlings during
germination and early growth stages. Low endogenous seed P seedlings (LS) with no exogenous
P (○), low exogenous P (□), high exogenous P (∆) and high endogenous seed P seedlings (HS)
with no exogenous P (●), low exogenous P (■) high exogenous P (▲) treatments, grown for 530
cumulated degree days after sowing. Data are means and vertical bars indicate ± se for n = 3
replications.....................................................................................................................................82
Figure 4.12: Scutellum phytate-P hydrolysis (µg P) in maize seeds during germination and early
growth stages. Inset, Phytate-P in the scutellum (ratio of scutellum phytate-P content to initial
scutellum phytate-P content) as a function of cumulated degree days after sowing. Low
endogenous seed P seeds with no exogenous P (○), low exogenous P (□), high exogenous P (∆)
and high endogenous seed P seeds with no exogenous P (●), low exogenous P (■) high
exogenous P (▲) treatments, grown for 530 cumulated degree days after sowing. Data are means
and vertical bars indicate ± se for n = 3 replications.....................................................................83
Figure 4.13: Quantity of endogenous seed P (µg P) exported from the seed to maize seedlings
during germination and early growth stages. Low endogenous seed P seedlings with no
exogenous P (○), low exogenous P (□), high exogenous P (∆) and high endogenous seed P
seedlings with no exogenous P (●), low exogenous P (■) and high exogenous P (▲) grown for
530 cumulated degree days after sowing. Data are means and vertical bars indicate ± se for n = 3
replications.....................................................................................................................................84
Figure 4.14: Exogenous phosphorus (µg P) uptake in maize seedlings during germination and
early growth stages. Low endogenous seed P seedlings with no exogenous P (○), low exogenous
P (□), high exogenous P (∆) and high endogenous seed P seedlings with no exogenous P (●), low
exogenous P (■) high exogenous P (▲). Data are means and vertical bars indicate ± se for n = 3
replications.....................................................................................................................................86
Figure 4.15: Phosphorus efflux (µg P) from germinating maize seeds and growing seedling roots
during germination and early growth stages. A) Low endogenous seed P seedlings with no
exogenous P (○), low exogenous P (□), high exogenous P (∆) and B) high endogenous seed P
seedlings with no exogenous P (●), low exogenous P (■) high exogenous P (▲) treatments. Data
are means and vertical bars indicate ± se for n = 3 replications....................................................86
Figure 4.16: (A-C: LS treatments) Allocation of exogenous P uptake (gray arrows on the right)
calculated using 32P labeling of the nutrient solution and distribution of remobilized seed P
reserves (dark arrows on the left) to seedling compartments and calculation of efflux after 530
cumulated degree days. The width of the arrows represents the quantity of P flowing toward
10
different seedling compartments. Values inside the different compartments represent P
accumulation in (+) or remobilization from (-) each compartment. P fluxes are expressed in µg P
seedling-1. Data are means and vertical bars indicate ±se for n = 3 replications..........................87
Figure 4.17: (A-C: HS treatments). Allocation of exogenous P uptake (gray arrows on the right)
calculated using 32P labeling of the nutrient solution and distribution of remobilized seed P
reserves (dark arrows on the left) to seedling compartments and calculation of efflux after 530
cumulated degree days. The width of the arrows represents the quantity of P flowing toward
different seedling compartments. Values inside the different compartments represent P
accumulation in (+) or remobilization from (-) each compartment. P fluxes are expressed in µg P
seedling-1. Data are means and vertical bars indicate ±se for n = 3 replications..........................88
Figure 4.18: Flow diagram of main processes that determine P dynamics in maize seed during
germination and early growth. Arrows indicate the P fluxes between different seed, seedling and
exogenous P uptake.......................................................................................................................94
Figure 4.19: Graphical display of the model of P dynamic in maize seed during germination and
early growth build with ModelMaker software.............................................................................96
Figure 4.20: Observed and modelled scutellum (●) and endosperm (▲) phytate-P hydrolysis in
scutellum and endosperm of maize seeds (HPHS) during germination and early growth stages
(R² = 0.974)....................................................................................................................................97
Figure 4.21: Observed leaf P concentrations (●) in HPHS seedlings and dilution P curve obtained
by non-linear fitting in HPHS seedlings during germination and early growth stages.................99
Figure 4.22: Relationship between seedling root P influx and seedling root lengths (HPLS and
HPHS seedlings) in maize seedlings. HPHS seedlings (▲) and HPLS seedlings (∆) and non
linear fitting curve (—) of HPHS and HPLS (– –)......................................................................101
Figure 4.23: Time courses of modelled phytate-P hydrolysis (µg seed-1) in LS and HS seeds
treated with three exogenous P availabilities (0P, LP and HP) during germination and early
growth stages...............................................................................................................................104
Figure 4.24: Modelled changes in non phytate-P (µg seed-1) in LS and HS maize seeds during
germination and early growth stages. .........................................................................................105
Figure 4.25: Seedling P demand and effective seedling P accumulation rate (µg day-1) in
growing maize seedlings during germination and early growth stages. A. Seedling P demand and
seedling P accumulation in LS seedlings treated with three exogenous P availabilities (0P, LP
HP), Right. HS seedlings treated with three exogenous P availabilities (0P, LP HP).................106
11
Figure 4.26: Time courses of modelled seedling P concentrations (µg g-1 DW) in LS and HS
seedlings treated with three exogenous P treatments (0P, LP, HP) during germination and early
growth stages...............................................................................................................................107
Figure 4.27: Observed and modelled seedling P concentrations (mg g-1 DW) in growing maize
seedlings on 530 cumulated degree days after sowing................................................................107
Figure 4.28: Modelled seedling P accumulation (µg seedling-1) originated from endogenous seed
P reserves and exogenous P uptake in growing maize seedlings during germination and early
growth stages. HS seedling treated with HP and LP exogenous P availabilities (A and B), LS
seedlings treated with HP and LP exogenous P availabilities (C and D). ..................................108
Figure 4.29: Time courses of seed P export rate (µg seed-1 day-1) in LS (dashed lines) and HS
(solid lines) seeds treated with three exogenous P treatments (0P, LP, HP) during germination
and early growth stages................................................................................................................109
Figure 4.30: Time courses of observed (Obs) and modelled (Mod) exogenous P uptake in LS (A)
and HS (B) seedlings treated with two exogenous P availabilities during germination and early
growth stages in maize.................................................................................................................109
Figure 4.31: Time courses of modelled seed P efflux (µg seed-1) in LS (dashed lines) and HS
(solid lines) seed treated with three exogenous P treatments (0P, LP, HP) during germination and
early growth stages......................................................................................................................110
12
LIST OF TABLES
Table 2.1: Mean concentration of total phosphorus and phytate percentage of some crop feeds
(Bagheri and Gueguen 1983; Nelson 1980; Sauveur 1983; Simons 1979)...................................30
Table 2.2: Phytate content changes (mg 100 g–1 DW)* after soaking whole seeds for 24 hours
(Lestienne et al. 2005)...................................................................................................................34
Table 4.1: Radicle length, root length and number of visible leaves during early growth of maize
seedlings. Values are means (±SE) of 3 replications.....................................................................66
Table 4.2: Values of selected variables for maize seedlings at 530 cumulated degree days after
sowing with two rates of endogenous P (LS "low seed P " and HS "high seed P") subjected to
three rates of exogenous P (Exo-P) availability (OP "control exogenous P", LP "low exogenous
P" and HP "high exogenous"). F-test significances of Endo-P (endogenous seed P), Exo-P and
Endo-P x Exo-P interaction are indicated (NS = not significant). Different letters in the same line
indicate significant differences at the 0.05 probability level.........................................................81
Table 4.3: Different variables and parameters of maize seed phytate-P hydrolysis and exogenous
P uptake during germination and early growth stages.................................................................103
13
14
1. INTRODUCTION
Phosphorus (P) is a vital nutrient that helps the plant to complete its normal life cycle. It is the
second macro element after nitrogen which frequently limits the crop growth and development.
Because of frequent agronomic P imbalances, about 29% of the global cropland area has overall
P deficit (MacDonald et al. 2011) as shown Figure 1.1.
Figure 1.1: Global map of agronomic P imbalances for the year 2000 expressed per unit of cropland area in each 0.5° grid cell. The P surpluses and deficits are each classified according to quartiles globally (0-25th, 25-50th, 50-75th and 75-100th percentiles, MacDonald et al., 2011).
Phosphorus makes up to 0.2% of plant's dry weight (Schachtman et al. 1998). It is highly
mobile in plants therefore when deficient it may be translocated from old plant tissues to actively
growing young seedling tissues. As a structural component of many coenzymes, phospholipids
and phosphoproteins, it is involved in several key plant functions including energy generation,
nucleic acid synthesis, photosynthesis, glycolysis, respiration, membrane synthesis and stability,
enzyme activation/inactivation, carbohydrate metabolism, and nitrogen fixation.
Despite of its prime importance, P is the least mobile and available nutrient in soil.
Applied P to soils in the form of organic or inorganic fertilizer sources reacts with clay, iron and
aluminium compounds and is converted readily to less available forms by the process of fixation.
Although agronomic inputs of P fertilizers exceeds the P removal by crops at world level, P
deficit covered almost 30% of global cropland area (MacDonald et al. 2011). Inorganic P
fertilizers therefore the major input in crop production. Mined rock phosphate is the primary
15
source of P fertilizers and approximately 90% of all mined rock phosphate is used for agriculture
(Cordell et al. 2009; Tiessen 2008).
Global cereal production has doubled in the past 40 years mainly from the increased
yields resulting from greater inputs of fertilizer, water, pesticides, new crop strains and other
technologies of the green revolution (Tilman et al. 2002; Tilman et al. 2001). Double production
of cereal crops resulted in the increased consumptions of phosphorus fertilizers for 3.5 times (9
Tg in 1960 to 40 Tg today) and this high consumption of non-renewable resource of P will lead
to complete depletion of P in 50 to 100 years (Abelson 1999; Cordell et al. 2009; Vance et al.
2003). Peak P production is projected to occur in 2035 to 2040 (Cordell et al. 2009) as shown in
Figure 1.2.
The efficiency of P fertilizer to increase the soil available P and to increase the crop yield
is largely dependent on soil conditions (Johnston and Richards 2003). The use of these inorganic
P fertilizers is also quite inefficient with less than 20% of applied inorganic P being absorbed by
plants during their first growing season (Plaxton and Tran 2011). The remaining inorganic P
become immobile in the soil or leaches into pollutes nearby surface waters (Plaxton and Tran
2011), causing eutrophication in aquatic ecosystem (Bennett et al. 2001; Runge-Metzger 1995).
Figure 1.2: Peak phosphorus curve indicating that production will eventually reach a maximum, after which it will decrease (Cordell et al., 2009).
16
Considering the economic and environmental unavailability and increasing demand of P,
a more efficient use of P fertilizers seems to be very important for sustainable agricultural
production. To improve the P fertilization strategies in agriculture requires a better understanding
and modelling of the P acquisition by crops.
Many studies have demonstrated that an early P nutrition is the most critical step because
it is involved in all energy requiring processes. Although crops absorb only small quantities of P
in their 2-3 weeks of early growth, this early accumulation of P is extremely important for
maximum dry mater and grain yield at maturity. In general, under low P availability plant
biomass accumulation decreases (Lynch et al. 1991; Mollier and Pellerin 1999; Ozanne et al.
1969; Plénet et al. 2000b) and root morphology is modified (Lambers et al. 2006). Plénet et al
(2000a) showed that the leaf area index in maize seedlings was effected markedly when the
seedlings were grown with different P fertilizers availabilities in soil as shown in Figure 1.3. The
P deficiency slowed the rate of leaf appearance in the corn, as well as reducing leaf size,
particularly that of lower leaves (Assuero et al. 2004; Colomb et al. 2000; Plénet et al. 2000a).
Under these conditions, an increase in root:shoot ratio has been reported, possibly due to a higher
allocation of assimilates to roots (Mollier and Pellerin 1999), which increases the exposed root
surface area for P uptake.
0
1
2
3
4
5
6
7
0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200
Thermal time (°C days)
LAI (
m² l
eaf
m-2
)
P0P1.5P3
1996 a
Figure 1.3: Effect of P on leaf area index, in maize seedlings during early growth stage (Plénet et al., 2000).
17
Phosphorus deficiency can also reduce protein and nucleic acid synthesis that leads to the
accumulation of soluble N compounds, particularly amide, in the tissue (Glass et al. 1980). The
reduction in total dry matter and grain yield due to a limited supply of P between planting and
six leaf stage was observed in field grown maize crop (Barry and Miller 1989). The allocation of
dry matter to the grain at later development stages was enhanced by early season P nutrition in
corn (Gavito and Miller 1998). Phosphorus effect on leaf morphogenesis and expansion in early
stages of maize development could play a role in the persistent effects of reduced leaf growth
and solar radiation interception on C nutrition of plant. The indirect effect of P deficiency on C
assimilation may reduce subsequent emergence and elongation of roots, which would have an
additional impact on P uptake capacity (Pellerin et al. 2000).
Phosphorus is absorbed by the seedling roots mostly in the ionic forms of either H2PO4–1
or HPO4–2 depending upon the pH of soils, but most preferable form is H2PO4
–1 at pH 7. The P
uptake poses a problem for plant roots, since the concentration of P in the soil solution rarely
exceeds 10 µM (Bieleski 1973) but plant requirements are quite high. Several mathematical
models simulating phosphorus uptake from soil by single root to whole seedling root system and
crop response have been developed (Baldwin et al. 1973; Claassen and Barber 1976; Mollier et
al. 2008; Nye and Marriott 1969). These modelling efforts have led to a better understanding of
nutrient uptake by roots in the absence of other limiting factors and integrate the processes
controlling soil P supply, the uptake by root system and the relationship between crop growth
and P uptake. These nutrient uptake modelling approaches successfully predict the exogenous P
uptake, but generally do not account for germination and early seedling growth, although it is the
most critical period for crop P nutrition.
Germination is the first step in successful life cycle of a plant and incorporates those
events that commence with the uptake of water by quiescent dry seeds and terminate with the
elongation of embryonic axis (Bewley 1997). The demand of P increases dramatically during the
period of rapid cell division such as seed germination and early seedling growth, however the
root system development and root absorption capacities are still limited during this period. If soil
P availability is low, the root uptake capacity may not be sufficient to cope the crop P
requirements; this raises the question of seed P reserves remobilization, their relative
contribution to maize seedling P requirements versus that of exogenous P uptake by the
developing maize seedling roots during early growth stages. The general objective of this study
was to quantify the relative contributions of seed P reserves remobilisation and exogenous P
uptake by roots to the seedling P nutrition, to identify the main factors controlling both processes
and to propose a way to model them.
18
2. BIBLIOGRAPHIC REVIEW
2.1. Germination
Germination is the first step in the successful life cycle of a plant. It is a complex phenomenon in
which the mature dry seeds pass from a state of quiescence, where the metabolism is virtually
halted, in a state of intense metabolic activity. By definition, germination incorporates those
events that commence with the uptake of water by quiescent dry seed and terminate with the
elongation of embryonic axis (Bewley 1997). The visible sign that the germination is complete,
is usually the penetration of structures surrounding the embryo by radicle (Bewley 1997). Early
events associated with seed germination include solute leakage, commencement of respiration,
DNA repair and synthesis of mitochondria, and protein synthesis extant mRNAs and newly
synthesized mRNAs (Bewley 1997).
2.1.1. Types of germination
There are two types of germination based on the fate of the cotyledons, on whether the
cotyledons grow above the ground surface or remain below the ground surface (Figure 2.1). The
term epigeal is referred to germination type when the cotyledons rose above the ground surface
by hypocotyle where they continue to provide nutritive support to the growing points till
cotyledons reserves are exhausted. It is characteristics of canola, field beans, alfalfa, red clover
and pine seeds. Epigeal germination is considered evolutionarily more primitive than hypogeal
germination. The hypogeal germination is a characteristic of maize, wheat, rye, oat, broad bean
and pea seeds, where the cotyledons were remained below the ground surface and support the
seedlings (Miller 2001).
Figure 2.1: Epigeal and hypogeal germination in monocot and dicot seed.
19
2.1.2. Factors affecting germination
Seed germination depends on both internal and external factors. Besides the basic requirement
for water, oxygen and an appropriate temperature, the seed may also be sensitive to other factors
such as light or darkness. Oxygen is required for aerobic respiration which can be assisted with
anaerobic respiration if needed. The internal factors which affect the seed germination include
hormonal control and seed dormancy.
2.1.2.a. Water
The role of water is of paramount importance and basic requirement for germination. It is
essential for enzyme activation, breakdown, translocation, and use of stored reserves material
(Miller 2001). The imbibition begins as soon as the seeds are placed in the soil, provided the
water content of soil that surrounds it should be sufficient. Imbibition of seeds depends upon the
difference of water potential in seeds and the surrounding environment of seeds. The seed
imbibtion triggers the hormonal changes that will lead to reactivation of enzymes. These
processes leads to the penetration of radicle through seed covering and leads to visible
germination (Bewley and Black 1994).
Water uptake in maize seeds is triphasic, with a rapid initial uptake (phase I), and then a
plateau phase (phase II), followed by a second substantial uptake (phase III), as shown in the
Figure 2.2. The first phase corresponds to a rapid imbibition, during which the tissues of the dry
seed absorb water quickly. One of the first change upon imbibition is the resumption of
respiratory activity, which can be detected within minutes (Bewley 1997). The influx of water
into cell of dry seeds during phase I results in temporary structural perturbations, particularly to
membranes, which lead to an immediate and rapid leakage of solutes and low molecular weight
metabolites into the surrounding imbibition solution (Bewley 1997). This is symptomatic of a
transition of the membrane phospholipids components from the gel phase achieved during
maturation drying to the normal, hydrated liquid-crystalline state (Crowe and Crowe 1992).
20
Figure 2.2: Water uptake in seeds during germination (Bewley and Black 1994).
The length of phase II is affected by imbibition temperature and water potential of the
medium in which the seeds are imbibed (Manz et al. 2005; Schopfer and Plachy 1984). The
phase III occurs only when germination is completed as the embryo elongates and breaks
through its covering structures (Bewley 1997; Manz et al. 2005). In addition to the restoration of
transcriptional and translational machineries, the germination is mainly marked by the
mobilization of stored seed reserves (Lawrence et al. 1990; Le Deunff 1975), which have been
accumulated throughout the ripening period. By the end of imbibition phase (phase I), the
reserves of the cotyledons or endosperm began to be hydrolyzed (Obroucheva and Antipova
1997), to be truly mobilized during the post-germinative growth (Bewley 1997) and used in the
development of the future seedling.
2.1.2.b. Temperature
Germination is a complex process involving many individual reactions and phases, each of
which is affected by temperature as it is widely accepted that temperature regulates germination
(Miller 2001). The relationship between germination rate or elongation of the seedling tissues as
a function of temperature have been widely studied in many species (Carberry and Campbell
1989; Covell et al. 1986; Gummerson 1986; Marshall and Squire 1996; Squire 1999). On the
basis of these results, the effect of temperature on seed germination can be expressed in terms of
cardinal temperature: that is minimum, optimum, and maximum temperature at which
germination will occur. The rate of germination increases as the temperature increase from
minimal to optimum range but it again goes decreasing when temperature goes higher than
optimum to maximum range (Covell et al. 1986; Gummerson 1986) and particularly in maize,
21
seed germination and vigour increased when day/night growth temperature was increased from
22/16 to 27/21 °C, but they decreased in response to high temperatures 33/27 °C (Modi and
Asanzi 2008).
2.1.2.c. Hydrothermal models
Temperature plays a critical role in the timing of many plant processes including seed
germination and thermal time is frequently considered an acceptable term used for complex
biological time period. Water uptake is clearly essential for seed germination and initial water
uptake into dry seeds is a physical process, viewed as satisfying the seed matric potential. At
reduced water potentials, progress towards germination is progressively reduced (Allen 2003).
Analogue to thermal time “hydrotime” quantitatively describes the rate of progress towards
germination as function seed water potential.
For the seed germination modelling, the effect of temperature and water potential can be
combined into a single term “hydrothermal time” (Allen 2003; Alvarado and Bradford 2002;
Bradford 1990; Finch-Savage et al. 2005; Gummerson 1986; Rowse and Finch-Savage 2003).
Hydrothermal models form the basic for many current efforts to predict the seed germination. A
key feature of these models is that each seed accumulates hydrothermal time (quantified progress
towards germination) according to the temperature and water potential of incubation in relation
to minimum temperature and water potential at which seed can progress. A key advantage of
hydrothermal models is that their equations apply to the entire seed population and lead to
simultaneous predictions of germination rate and percentage (Allen 2003). Hydrothermal
modelling efforts provide a theoretical framework for quantifying the effects of temperature and
water potential on seed and seedling processes under both controlled and natural conditions.
2.2. Maize seed and its structure
Seed occupies a critical position in the life history of higher plants (Bewley and Black 1994). A
seed is a small embryonic plant enclosed in covering called seed coat, with some stored food.
The seed is the product of ripening ovule of gymnosperm and angiosperm plants which occur
after pollination and some growth within mother plants. The formation of the seed completes the
process of reproduction of seed plants.
Basically the seed is composed on three main parts including embryo, stored food
materials and seed coat. The embryo is an immature plant from which a new plant will grow
under proper conditions. The embryo has one cotyledon or seed leaf in monocotyledonous seeds
22
(maize, rice, rye etc.) whereas has two cotyledons in almost all dicotyledonous seeds (pea, mung,
beans etc.). The radicle is the embryonic root, while the plumule is the embryonic shoot. The
embryonic stem above the point of attachment of the cotyledon is the epicotyle and the
embryonic stem below the point of attachment is termed as hypocotyle.
Maize seed is a monocotyledonous and in mature maize seeds, the embryo and
endosperm alike are enclosed in the dry covering of the seed, whereas the embryo is triply
protected (Fincher and Stone 1986; Sargant and Robertson 1905). During seed development, the
outer endosperm is differentiated into the aleurone layer, a morphologically and functionally
distinct layer that may be one to several cells thickness, depending upon the species (Fincher and
Stone 1986), as shown in Figure 2.3. Although aleurone and starchy endosperm share a common
origin, only the cells of the aleurone layer remain alive after storage reserves of endosperm are
deposited and the seed matures and dries (Fincher 1989).
The whole embryo is seen lying against one face of the endosperm. The endosperm is a
starchy structure and occupies the bulk of seed and represents the nutrient store that mobilizes
during germination to nourish the growing seedlings. Thus starchy endosperm constitutes the
targeted tissue for enzymes secreted by the aleurone and scutellum (Fincher 1989). The second
covering is formed of the scutellum, a cushion like structure which is wrapped round the
embryonic axis and still conceals the greater part of it in the first days of germination (Figure
2.3). Finally, the each growing part of embryo has its own sheath: the coleoptile encloses the
plumule and the coleorhiza the radicle (Figure 2.3). The insertion of these sheaths on the axis,
and of the axis on the scutellum is shown in Figure 2.3.
Figure 2.3: Internal structure of maize seed.
23
The scutellum is in contact with endosperm over the whole of its dorsal surface. It
supplies food to other parts of embryo from the stores laid up in its own tissues, and from those
of the endosperm. The contents of the endosperm are by degrees dissolved and absorbed by the
scutellum, which transmits them in solution to the growing parts of embryo (Sargant and
Robertson 1905).
2.3. Early seedling growth and seed reserves remobilization
The appearance of the radicle makes the end of germination and the beginning of establishment,
a period that ends when the seedlings have exhausted the food reserves stored in the seed.
Germination and establishment are two critical phases in the life of a plant. Germination vigour
and seedling emergence depend upon the amount and availability of seed endosperm reserves
and on soil conditions. Shoot and root growth of seedlings occurre at the expense of these seed
reserves. Seed dry weight decrease with an increase in root and shoots dry weight. The seed dry
weight loss during germination was greater than the increase in roots and shoot dry weights. The
difference was assumed to be lost due to respiration (Bouaziz and Hicks 1990).
Initially plant lives on seed reserves (heterotrophic stage) with external supply having
little effect and a last stage (autotrophic stage) occurs when the growth rate is determined by the
nutrient supply from surrounding environment (Grant et al. 2001). When the coleoptile breaks
through the soil surface, following by unfolding of the first cotyledon leaf, the seedling begins to
cease its dependence on endosperm (Finch-Savage 1995). The young seedlings obtain their
autotrophic independence for C, i.e. the photosynthetic ability to provide the growing tissues
with enough organic molecules, after a heterotrophic phase during which the only source of C is
located in the seminal reserve tissues. These two stages of seedling growth have been clearly
identified by Whalley et al., (1966) as illustrated in Figure 2.4., but a little is know about the
other nutrient like phosphorus and the dependence of seedling on seed P reserves and external P
uptake during early .
24
Figure 2.4: Germination, emergence and seedling establishment.
2.3.1. Germination, early growth and seed carbon remobilization
Deleens et al. (1984) conducted an experiment for 15 days at 25/22 °C day and night
temperature. She reported that decrease in maize seed reserves during germination and early
growth exhibits three phases: a rapid decrease up to the 10 th day after germination, a steady state
from the 11th to the 13th day and ultimately a very slow decrease. The main part of seed carbon
pool is used for the early development of the plant. The leaves of 7 day old seedlings are entirely
made up of carbon coming from seed reserves indicating a heterotrophic stage. Up to the 10 th
day, both sources (heterotrophic + autotrophic) participate in leaf growth; the increase of the
autotrophic part is faster and this period corresponds to transitional growth stage. In leaves of 10
days old seedling the flow of carbon from heterotrophic and autotrophic sources is quantitatively
equal. In leaves of older seedling carbon provided by photosynthetic activity (autotrophic) is
much more important, while the root growth through the 9th day results from seed originating
carbon. On the 10th day newly autotrophic carbon is incorporated, first slowly but after the 13 th
day this source becomes more and more dominant for leaves as well as for the roots (Deleens et
al. 1984). The duration of different stages including heterotrophic, transitional and autotrophic
depend upon the temperature which directly influence the germination and early growth stages
(Covell et al. 1986; Miller 2001; Modi and Asanzi 2008; Squire 1999).
A B C
See
dlin
g dr
y w
eigh
t
Stagesi l a 4f
A B C
See
dlin
g dr
y w
eigh
t
Stagesi l a 4f
A = Heterotrophic stage
B = Transition stage
C = Autotrophic stage
i = Imbibition
l = Emergence
a = Self nutrition
4f = 4 leaf stage
25
2.3.2. Germination, early growth and mineral reserves remobilization
2.3.2.a. Nitrogen
Nitrogen (N) is the key nutrient along with phosphorus for obtaining maximum final crop
harvest and quality. It plays a pivotal role in many critical functions such as photosynthesis as it
is a major component of amino acids, several vitamins and also it is necessary for enzymatic
reactions. Nitrogen is mobile in plants and it translocated from older plant parts to young plant
tissues.
The maize endosperm is a major storage house of seed nitrogen (Harvey and Oaks 1974).
The degradation of major storage proteins, zein and glutelin, in maize endosperm begins during
2nd day of germination. The protein most abundant in the mature endosperm is degraded most
rapidly. Zein and glutelin loss begun after 20 hours of germination, but total protein loss was not
apparent till 60 hours of germination (Harvey and Oaks 1974). A rapid decrease in endosperm
protein was recorded after 80 hours of germination until the endosperm protein reserves were
depleted (Harvey and Oaks 1974).
2.3.2.b. Phosphorus
[i.] Phosphorus in seeds
Phosphorus in seeds, is stored primarily in the form of phytic acid (Lott et al. 1995; Park et al.
2006; Zhu et al. 2001). Phosphorus and phytic acid concentration are closely correlated
(Lockhart and Hurt 1986). In literature the term phytic acid has been used interchangeably with
the term phytate or phytin. It occurs widely in plant tissues but is concentrated in seed or grain
tissues (Raboy 1990). As a primary storage form of phosphorus in plant seeds, phytic acid
represents 50% to 80% of total P in mature seeds and accounts for 1% to several percent of the
dry weight (Lott 1984; Raboy 1997). Inorganic P and cellular P (a component of cellular
membranes, DNA, RNA, etc) are other forms of P in seeds and generally referred to as
“available P” (Raboy 2001; Raboy et al. 2000).
Phytate is a strong chelating agent and nearlly all the phytate found in mature seeds is
bound to minerals such as K+, Mg++, Ca++, Zn+, Ba++, and Fe+++. Phytate forms a complex salt of
myo-inositol hexakisphosphoric acid (myo-inositol 1-, 2-, 3-, 4-, 5-, 6-hexakisphosphate or Ins.
P6) (O'Dell et al. 1972; Ullah and Gibson 1988). The stability of salt formed with phytate is in
the order of Cu++ > Zn+ > Mn++ > Fe++ > Ca++ (Lott et al. 2000; Sauveur 1989). Phytate is
26
associated with anti-nutrient character in seeds as it binds the calcium, magnesium and zinc
however it has a potential to contribute significantly to seed performance (Modi and Asanzi
2008). It has been estimated that about 65% of the total elemental P applied every year in world
agriculture ends up in the phytic acid molecules (Lott et al. 2000).
[ii.] Phytate biosynthesis and accumulation in seeds
The phytate molecule is composed of a glucose ring with six phosphates (Figure 2.5). Free myo-
inositol and glucose 6-phosphate play an important role in the formation of phytate as
demonstrated in Figure 2.6. Phytate has chemical formula C6H18O24P6 and molecular weight of
660 g mole–1. The biosynthetic pathway to phytic acid can be summarized as consisting of two
parts: inositol supply and subsequent inositol polyphosphate (Raboy et al. 2000).
Figure 2.5: Phytic acid (myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate or Ins. P6).
27
Figure 2.6: Biosynthetic pathways to phytic acid (myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate or Ins. P6) in the eukaryotic cell. A, Structure of phytic acid. B, Structure of Ins. The numbering of the carbon atoms follows the "D-convention" (Loewus and Murthy, 2000). C, Bio-chemical pathway of phytic acid (Raboy et al. 2000):(1), D-Ins(3)-P1 (or L-Ins[1]-P1) synthase; (2), D-Ins 3-phosphatase (or L-Ins 1-phosphatase); (3), D-Ins 3-kinase (or L-Ins 1-kinase); (4), Ins P- or polyP kinases; (5), Ins (1,3,4,5,6) P5 2-kinase or phytic acid-ADP phosphotransferase; (6), PtdIns synthase; (7), PtdIns and PtdIns P kinases, followed by PtdIns P-specific phospholipase C, and Ins P kinases; (8), D-Ins(1,2,3,4,5,6) P6 3-phosphatase; (9) D-Ins(1,2,4,5,6) P5 3-kinase; (10), D-Ins(1,2,3,4,5,6) P6 5-phosphatase; (11), D-Ins(1,2,3,4,6) P5 5-kinase; (12), pyrophosphate-forming Ins P6 kinases; (13), pyrophosphate-containing Ins PolyP-ADP phosphotransferases
Phytate rapidly accumulates in seeds during the ripening period (Abernethy et al. 1973;
Anderson and Wolf 1995; Gifford-Steffen and Clydesdale 1993) accompanied by other storage
substances such as starch and lipids. In seeds, phytate is usually found in organelles called
protein bodies, where it generally constitutes an inclusion, the globoid (Pernollet 1978). These
organelles are localized in the aleurone layer in cereals and in the endosperm and cotyledons in
legumes and oilseeds. In cereal grains, phytates are concentrated in the germ and aleurone layers
(O'Dell et al. 1972).
28
[iii.] Functions of phytate
The phytate is supposed to perform two primary functions in seeds and growing seedlings. It
plays a significant role in providing mineral nutrients and inositol used in the growth of seedlings
as well as in the maintenance of inorganic phosphate homeostasis in developing seeds and
seedlings (Lott 1984; Lott et al. 1995). Regulation of cellular inorganic phosphate concentration
may play an important role in starch synthesis, accumulation and in the function of other
metabolic pathways (Strother 1980).
[iv.] Phytate in seeds
In maize, nearly 90% of the phytic acid is accumulated in embryo (O'Dell et al. 1972) and about
10% in the aleurone layers, while maize endosperm contains only trace amount of phytic acid
(Cheng Wang et al. 1959; Lott et al. 1995; Simwemba et al. 1984). In wheat and rice about 90%
of the caryopsis phytate-P is stored in the aleurone layer and about 10% in the embryo (Lott et al.
1995). In normal and quality protein hybrids of maize, the major part of maize seed phytate-P
was concentrated in embryo tissues as compared to endosperm (Modi and Asanzi 2008).
[v.] Factors effecting seed phytate and P concentrations
Phytate and P concentrations in seeds of a given species may vary because of many factors,
including cultivar, soil fertility status, and climatic conditions (Horvatic and Balint 1996; Miller
et al. 1980; Raboy et al. 1991). The interaction of phosphorus nutrition and environmental
factors with respect to phytate accumulation in seeds has been reported (Raboy and Dickinson
1993; Steiner et al. 2007). Low temperature stress during seed development hastens the
formation of phytic acid (Horvatic and Balint 1996) and the supply of inorganic phosphorus
(Raboy and Dickinson 1993).
The concentrations of total phosphorus and their phytate percentage in different crops are
shown in Table 2.1. The phytate-P percentage ranges from 55% to 77% of total P in different
crop and especially maize 67% of the total phosphorus is in the form of phytate. High soil P
levels displayed a significantly higher phytate-P and inorganic P concentrations in maize seeds,
while increasing growth temperature caused greater accumulation of inorganic P, but it
decreased myo-inositol (Modi and Asanzi 2008). Phosphorus nutrition enhanced seed
performance of maize (Modi and Asanzi 2008), and all variations in seed total P can be
accounted by variation in phytic acid P (Raboy et al. 1989; Raboy and Dickinson 1984).
29
Table 2.1: Mean concentration of total phosphorus and phytate percentage of some crop feeds (Bagheri and Gueguen 1983; Nelson 1980; Sauveur 1983; Simons 1979).
Crops
Phosphorus total Phytate
(g kg–1) (% P total)Oat 3.4 55
Wheat 3.3 60-77Maize 2.7 67Barley 3.5 56-72
Rye 3.4 65Sorghum 3.0 60-70
[vi.] Activity of phytase during germination and early growth
Mobilization of phytate in germinating seeds is due to phytase activity, a special type of
phosphatase that can release phosphate from phytic acid as well as from other phosphorylated
substrates. Phytase (myo-inositol hexakisphosphohydrolases) is a phytate specific phosphatase
that hydrolyses phytate to inositol and free orthophosphate that releases phosphate from phytate.
Phytases are found naturally in plants and microorganisms particularly fungi (Wyss et al. 1999).
Maize is the first plant for which phytase gene isolation has been reported (Maugenest et
al. 1999). The principal function of the phytase in seeds or grains is to produce inorganic
phosphate from phytate during germination. Dephosphorylation of phytic acid to a series of myo-
inositol esters and inorganic phosphate is catalyzed by the enzyme phytase (Hegeman and
Grabau 2001; Loewus and Murthy 2000; Pilu et al. 2003; Urbano et al. 2000). This
decomposition of phytate in germination seeds may provide P, myo-inositol and mineral cations
for the rapidly growing seedlings (Loewus and Murthy 2000; Raboy et al. 1991). The young
seedling utilizes the myo-inositol as a substrate for the myo-inositol oxidation pathway and
ultimately for cell wall polysaccharides formation (Oberleas and Harland 1981).
Phytases act in a stepwise manner to carry out the hydrolysis of phytic acid (Urbano et al.
2000). They catalyze phosphate monoester hydrolysis of phytic acid, which results in the
stepwise formation of myo-inositol pentakis-, tetrakis-, tris-, bis-, and monophosphates, as well as
the liberation of inorganic phosphate. Two types of phytase have been identified which initiate
the hydrolysis of phytate at either the 3- or 6- position of the inositol ring. The plant phytases
preferentially hydrolize the phosphoester bond at 6-position of the myo-inositol residue (Greiner
et al. 2000; Greiner et al. 2001; Turk et al. 2000). Usually, but not invariably, microbial phytase
falls into the first category and plant phytase in the second category (Turk et al. 2000).
30
Centeno et al. (2001) showed that inositol hexakisphosphate was the major inositol
phosphate in rye and barley cereals, representing more than 90% of the total myo-inositol
phosphates (Figure 2.7). The concentration of myo-inositol pentaphosphate (IP5) was only 8% of
total phytate, whereas IP4 was 2 and 1% for rye and barley, respectively. IP3 was not detected in
ungerminated seeds. During the course of germination, IP6 and IP5 were rapidly degraded in rye
and barley, and IP4 was only a short living intermediate, which was increased during hydrolysis
and degraded to IP3 (Figure 2.7).
Figure 2.7: Changes in the myo-inositol hexa- (IP6), penta- (IP5), tetrat- (IP4), and tri- (IP3)
phosphate during germination of barley seeds (Centeno et al. 2001).
[vii.] Phytase activity and effect of different factors
In the non germinated seeds a low phytase activity has been detected (Greiner et al. 2001; Sung
et al. 2005). With the imbibition of water, the metabolic processes in the seeds start, and after 72
hours of germination, the phytase activity in maize, millet, sorghum, and sweet maize had
increased 3 to 6 fold as compared to the initial values. In soybean seed germination, a
pronounced increase in phytase activity accompanies a concomitant decrease in phytic acid, with
maximal phytase activity attained at approximately 10 days after germination (Ullah and Gibson
1988). The most pronounced decrease in phytic acid content was found in rice, millet, and
mungbean, where only 34%, 44%, and 50% of the initial content remained after germination for
72 hours and this is due to the phytase activity (Egli et al. 2002).
In growing maize seedlings, the phytase activity increased from day 1st to day 5th
(Laboure et al. 1993). No significant phytase activity could be detected at zero time. Phytase
activity reaches a plateau between day 5th and 7th (Laboure et al. 1993). Similar results were
31
reported by Greiner et al. (2000) in barley seedlings. He stated that maximum phytase activity in
growing barley seedlings was observed on 4th day during germination and the myo-inositol
phosphates decreased (Figure 2.8).
□ myo-Inositol phosphates Phytase activity
□ myo-Inositol phosphates Phytase activity
Figure 2.8: Phytate level (□) and phytase activity (∇) during barley germination (Greiner et al. 2000).
Temperature has a positive effect on the phytase activity. The phytase activity increases
with increase in temperature, and phytate hydrolysis quickly (Sung et al. 2005). The level of
phytase activity in barley reached its maximal value, 4 days after germination at 20-25°C and 7
days after germination at 15°C (Sung et al. 2005) as shown in Figure . The maximum phytase
activity in maize seedlings was observed at pH 4.8 in maize (Laboure et al. 1993). Phytase
activity diminished sharply on both the acid and alkaline sides of the optimum pH value and was
very low outside the range of pH 4-6. Inorganic phosphate caused some inhibition in phytase
activity even at the lowest concentration tested (0.56 mM). At concentration of 1.45 mM and
1.91 mM, phytase activity was reduced by 42 percent from the level obtained in the absence of
inorganic phosphate (Guardiola and Sutcliffe 1971).
Different cations were tested for their effect on phytase activity from 0.25 to 2 mM
(Laboure et al. 1993). Only Ca++ was found to exert a light stimulatory effect, approximately
35% over the control at 2 mM. Mg++ and Mn++ had no effect, whereas Zn++ and Fe+++ were
inhibitory for phytase activity (Laboure et al. 1993).
□ myo-Inositol phosphates
∇ Phytase activity
32
● 15°C
■ 20°C
▲ 25 °C
● 15°C
■ 20°C
▲ 25 °C
Figure 2.9: The effect of germination temperature on phytase activity during barley germination (Sung et al. 2005).
[viii.] Seed phytate loss
Different processes such as soaking, cooking, germination and fermentation cause a decrease in
phytate contents in legumes (Khalil and Mansour 1995; Reddy et al. 1978; Sandberg and
Svanberg 1991). Soaking led to a significant reduction in the phytate content of millet, maize,
rice and soybean seeds (Lestienne et al. 2005). Depending upon the botanical origin of seeds, a
significant reduction in phytate content (between 17% and 28%) was obtained by soaking whole
seeds for 24 hours at 30 °C (Lestienne et al. 2005). Two groups were distinguished on the basis
of phytate loss by soaking: millet, maize, rice and soybean which showed a significant reduction
in phytate content, and sorghum, cowpea and mungbean which showed no significant reduction
as shown in Table 2.2. The phytate was hydrolyzed either directly in seeds by phytase or in the
water after leaching into the soaking medium (Lestienne et al. 2005).
During soaking, the phytase activity of all cereals (barley, maize, millet, oat, rice, rye
sorghum, sweet maize and wheat) decreased. The decrease ranged from 10% of the initial value
for millet and rye to about 60% of the initial values for barley. Phytase activity did not change
significantly during soaking of legumes and oilseeds, with exception of a 40% reduction in lentil.
Some of this loss could be explained by leaching and, assuming that the distribution of phytase
within legume seeds is similar to the distribution of phytic acid (that is in the cotyledons), the
33
losses due to leaching can be expected to be less than in cereals where phytic acid and phytase
are primarily localized in the aleurone layer (Egli et al. 2002).
Table 2.2: Phytate content changes (mg 100 g–1 DW)* after soaking whole seeds for 24 hours (Lestienne et al. 2005).
Varieties
Phytate content
% Phytate lossUnsoaked Soaked
Millet 762 ± (66)a 550 ± (18)b 28Maize 908 ± (97)a 721 ± (16)b 21
Sorghum 925 ± (81)a 882 ± (44)a 4Rice 1084 ± (12)a 904 ± (81)b 17
Soybean 878 ± (130)a 678 ± (32)b 23Cowpea 559 ± (44)a 624 ± (42)a Apparent increase
Mung bean 236 ± (36)a 225 ± (12)a 8*Values are means±(SD). Means with a different letter in the same row are significantly different at P≤0.05, as assessed by Duncan's multiple range test.
The effect of germination is different than soaking. Germination resulted in decreased
phytase activity in most of the cereals with the exception of maize, millet, sorghum, and sweet
maize had increased 3 to 6 fold as compared to the initial values (Egli et al. 2002). The
germination process caused a significant increase of phytase activity in rye (up to 112%) and
barley (up to 212%) and a reduction in the phytate phosphorus content (up to 84%). During the
course of germination, inositol-6-phosphate and inositol 5-phosphate were rapidly degraded in
rye (88% and 79%) and barley (67% and 52%), and inositol 4-phosphate was only a short living
intermediate, which was increased during hydrolysis and degraded to inositol 3-phosphate
(Centeno et al. 2001).
In cowpea, horse gram, moth bean, mung bean, soybean and pearl millet seeds, phytate
phosphorus was between 42% and 61% of the total P of all the seeds. In general, 4%-11% of
phytate phosphorus decreased during soaking, 17%-27% during boiling and 14%-22% during
germination of seeds. Reduction in inositol 6-phosphate content was 13-19% during soaking,
27%-30% during boiling and 32%-56% during pearl millet seed germination (Dave et al. 2008).
[ix.] Mobilization of seed P reserves during germination and early growth
Changes in the levels of various phosphorus fractions and phytase activity in the cotyledons of
young pea seedlings was studied by Guardiola and Sutcliffe (1971). With the imbibition, the
phosphorus remobilization from seed reserves starts. The phosphorus lost in garden pea
cotyledons could be recovered quantitatively in the shoot and root. At first the phosphorus
remobilization from cotyledons is slow but increased gradually to a maximum rate after about 10
34
days from the time of seed soaking (Guardiola and Sutcliffe 1971) as shown in Figure 2.10. The
level of phytic acid phosphorus did not change significantly during the first 2 days of
germination but it decreased rapidly between days 4 and 10. By day 15, practically all of phytic
acid had disappeared (Guardiola and Sutcliffe 1971). Thereafter the rate of transport declined
and there was very little loss of phosphorus by the cotyledons after 22 days by which time the
amount remaining had been reduced to about 17 percent of the original content (Guardiola and
Sutcliffe 1971).
Figure 2.10: Changes in levels of various phosphorus fractions in the cotyledons of young pea seedlings during early development (Guardiola and Sutcliffe 1971).
First in the seeds, the inorganic phosphorus is a minor fraction of total seed P (Figure
2.10). There was only a small quantity of inorganic phosphate in the resting seed and after 2 days
it began to increase by the activity of phytase, which hydrolyse the phytic acid releasing their
inorganic phosphorus (Guardiola and Sutcliffe 1971). The level of inorganic phosphorus reaches
a maximum level about 10 days after germination. From then on it decreased, but at a slower rate
than did the total acid soluble phosphorus. After 25 days of germination inorganic phosphate
accounted for almost all the phosphorus in the acid soluble fraction and for three quarters of all
35
phosphorus in the cotyledons. On the other hand, nucleic acid and protein phosphorus levels
decreased from the start of germination. The rate of decrease of nucleic acid phosphorus was
most rapid at first and decreased progressively with time, whereas protein phosphorus declined
at an almost linear rate for about 15 days after germination and hardly changed thereafter
(Guardiola and Sutcliffe 1971).
[x.] Effect of seed P on early seedling growth
The seed P reserves have a significant effect on early seedling growth. In subterranean clover,
the seedling emergence was 35% greater from soil for the high P seed (0.75%; w/w), than the
lower P seed (0.48%; w/w) and this effect was independent of external P supply (Thomson and
Bolger 1993). Leaf emergence was faster and shoot dry weight was also higher in seeds with
high P reserves, but only when external P supply was deficient for the plant growth (Thomson
and Bolger 1993). Seedlings also emerge more quickly from high P seed, as after six days of
sowing, 35% of high P seed had emerged compared with 24% of low P seed. Phosphorus
concentrations in the shoot of two week old seedlings were 32-51% higher for higher P seed,
although by the four weeks plants grown from high and low P seed had similar concentrations of
P in their shoots (Thomson and Bolger 1993). Similar results were also reported by Ros et al.
(1997) in rice seedlings and De Marco (1990) in wheat.
36
2.4. Maize seedling root growth and exogenous phosphorus uptake
[i.] Growing maize seedling roots
The root system determines the capacity of a plant to access soil water and nutrients. In cereals
such as maize, the root system is formed from the seminal roots that appear at germination and
nodal, crown or adventitious roots that arise later from nodes of the shoot (Esau 1967). The root
system development of maize has been relatively well studied and roots have been variously
described as primary, seminal, nodal or axile and have often been associated with descriptors of
their point of origin, such as coleorhizae, scutellar node, coleoptillar node, and mesocotyle (Cahn
et al. 1989; Ho et al. 2004; Hochholdinger et al. 2004; Hund et al. 2007; Mollier and Pellerin
1999; Singh et al. 2010). In maize, a primary root, and a coleoptile had emerged by 2 days after
germination (Figure 2.11). The primary root emerged from the lower end of the seed through the
coleorhizae, whereas the coleoptile emerged at the upper end of the seed. Scutellar seminal roots
had also emerged from the scutellum by 2 days after germination and four scutellar seminal roots
were evident by day 3 (Singh et al. 2010). The maize produced 3-7 seminal (primary and
scutellum) roots and coleoptile nodal roots emerged at the 2nd leaf stage (Singh et al. 2010). The
studies throughout their life cycle indicate that the root system grows into the soil at about 2-3
cm day–1 (Dardanelli et al. 1997; Manschadi et al. 2008). Seminal roots play an important part in
initial water and nutrient uptake and establishment of seedlings, whereas nodal roots dominate
during the later stages of growth. It is important to note that seed P reserve mobilization and P
uptake by young root system are likely to overlap in time.
37
Figure 2.11: Photographs of maize seed (a) 2 days, (b) 3 days, (c) 4 days, (d) 5 days, and (e and f) 7 days after germination. C, coleoptile; CN, coleoptile node; CO, coleorhizae; FL, first leaf; M, mesocotyl; NR, nodal root; PR, primary root; S, scutellum; SL, second leaf; SSR, scutellum seminal root (Singh et al, 2010).
38
[ii.] Exogenous P uptake by growing seedling roots
Efficient nutrient uptake from soil by roots is a critical issue for plants given that in many
environments nutrients have poor availability especially phosphorus and may be deficient for
optimal growth. Although soils generally contain a large amount of total P, only a small
proportion is immaterially available for plant uptake. Plants obtain P as orthophosphate anions
(predominantly as H2PO4–1 or HPO4
–2 depending upon the pH of soils) from soil solution. In most
soils the concentration of orthophosphate in solution is low typically between 1 to 5 µM
(Bieleski 1973) and must therefore be replenished from other pools of soil P to satisfy the plant P
requirements. Orthophosphate is rapidly depleted in the immediate vicinity of plant roots, and as
such a large concentration gradient occurs across the rhizosphere between bulk soil and the root
surface (Gahoonia et al. 1997; Tinker and Nye 2000).
For nutrients present at low concentrations in soil solution and/or with poor diffusivity,
root growth proliferation into new regions of soil and release of root exudates are of particular
importance (Barber 1995; Darrah 1993). The availability of P in rhizosphere is influenced
significantly by changes in pH and root exudates which can either directly or indirectly affect
nutrient availability and/or microbial activity (Richardson 1994). The reactions controlling the
amount of inorganic P in solution include dissolution-precipitation of P bearing minerals,
adsorption-desorption of phosphate on soil surfaces, and the hydrolysis of organic matter
(Comerford 1999; Hinsinger 2001; Matar et al. 1992). Competition for nutrient uptake across
different plant species, between different roots and with microorganisms is also significant
(Hodge 2004). The rate of root growth and plasticity of root architecture along with development
of the rhizosphere, through either root growth or extension of root hairs, are clearly important for
effective exploration of soil and interception of nutrients (Lynch 1995).
The P uptake is generally modeled by Michaelis-Menten equation linking root P uptake
to P concentration in soil solution. The P uptake mainly occurred by diffusion process which
resulted from concentration gradient difference between soil solution and root P concentrations.
The intensity of P uptake was mainly dependent on the soil solution P availability (Bhadoria et
al. 2004; Elliott et al. 1984) and the root surface exposed (Anghinoni and Barber 1980).
Most of the inorganic P taken up by roots is loaded into the xylem and subsequently
translocated into shoot. Within plant cells, P is a major component of nucleic acids, membrane
lipids, and phosphorylated intermediates of energy metabolism. Thus, the cellular inorganic P
homeostasis is essential for physiological and biochemical processes. Under P deficiency, plants
can develop adaptive responses not only to facilitate efficient inorganic P acquisition and
39
translocation, but also to utilize efficiently stored P by adjusting inorganic P recycling internally,
limiting P consumption, and reallocating P from older tissues to young and actively growing
tissues.
40
2.5. Conclusions of bibliographic review
Phosphorus nutrition of maize is crucial during early growth stages as it is involved in different
metabolic processes and has a main role in the cellular energy transfer. During early growth
stages, P might be supplied by two processes including seed P reserves remobilization and
exogenous P uptake by developing maize seedling roots. The phosphorus in maize seeds is
stored in the form of phytate during the repining period. Most of the phytate is stored in germ
instead of endosperm. The P content in seeds may vary depending upon numerous factors. The
hydrolysis of phytate is controlled by phytase activity. Phytase activity is very high just after the
beginning of seed imbibition. This suggests that phytate hydrolysis may occur very early during
germination. However it was shown that phytase activity was inhibited by inorganic P. The
question also arises of possible interaction between C, N and P remobilization since they are not
localized in the same seed compartments. P losses are also likely to occur during germination
and early growth stages. The root growth is exponential during early growth stages but P uptake
per unit length of root is limited by the low diffusivity of P ions in soil solution and strongly
depends on the P concentrations. The question arises at which time the exogenous P uptake
begins. Both processes; seed P reserves remobilization and exogenous P uptake are likely to
overlap in time.
Here are some questions that arise during this study.
What is the remobilization kinetics of maize endogenous seed P reserves?
What is the possible relationship between carbon, nitrogen and phosphorus
remobilization in germinating seeds?
At which time the young growing seedling roots start the uptake of exogenous P? As
soon as the seedling radicle emerges? Or after the utilization of all seed P reserves?
Do both processes (seed P reserves remobilization and exogenous P uptake) have an
effect on each other or if are they controlled independently?
What is the relative contribution of endogenous seed P reserves and exogenous P uptake
in seedling P nutrition?
41
2.6. Objectives of thesis studies
The objective of PhD studies is to study the remobilization of endogenous seed P reserves and
the effect of exogenous P uptake to maize nutrition during early growth stages. Experimentations
with different P concentration in maize seeds and nutrient solution will be carried out to measure
the flow of P in maize seedlings from endogenous seed reserves and from nutrient solution.
Isotopic techniques will be used to quantify the P flux from exogenous nutrient solution and
from phytate decomposition.
Analysis of the experimental data may lead us to develop a mechanistic and conceptual
model. This model may explain the seed P reserves remobilization and exogenous P uptake in
maize seed and seedlings during germination and early growth stages, respectively. For better
understanding of whole system of maize growth from germination to maturity, this model will be
included in the P uptake model FUSSIM_P_Maize (Mollier et al. 2008). This model
(FUSSIM_P_Maize) integrates the processes controlling the soil P supply (mobilization and
transport in the rhizosphere), the uptake by the root system (according to the P soil availability
and crop P requirement determined by environmental conditions), and relationships between the
crop growth response and the amount of P absorbed. The different parts of the model are highly
interactive and the feedbacks are explicitly accounted for. The first version of the model was
successfully evaluated on field maize crop for the vegetative period (5 visible leaves to 16 leaves
stage) in a range of soil P availability. The results of this study will add knowledge for our
understanding about seed P reserves remobilization and start of exogenous P uptake by young
seedling roots along with relative contribution of two P sources to maize seedling nutrition
during germination and early growth stages.
2.6.1. General representation of studied system
To study the remobilization of endogenous seed phosphorus reserves and effect of exogenous P
uptake to maize nutrition during the early growth stages, the system is divided into three main
compartments namely endogenous seed P reserves, developing growing seedling and exogenous
P availability clearly defined in Figure 2.12.
42
The endogenous seed P reserves start to remobilize with the imbibition of seeds and
nourish the seedling with P. The growing developing seedling is composed of root and shoots
which develop in the response of seed reserves remobilization and exogenous nutrient uptake.
The third part of system is composed on exogenous P availability along with other necessary
nutrients (Figure 2.12).
Figure 2.12: Maize seedling system, including seed P reserves, growing seedling and
exogenous nutrient solution P.
2.6.2. Research plan
The proposed research plan combines two experimental studies and a modelling approach. In the
first step an experiment will be carried out to study the seed P reserves remobilization and
exogenous P uptake. The maize seeds will be grown with labelled nutrient solution of P to follow
the seed P reserves remobilization kinetics and start of exogenous P uptake by growing seedling
roots. The relative contribution of seed P reserves and exogenous P uptake to maize seedling P
nutrition will be determined.
43
In second step, an experiment will be carried out to know the effect of endogenous and
exogenous P availabilities on seed P reserves remobilization and exogenous P uptake. In this
experiment, maize seeds with different endogenous seed P reserves will be sown with different
exogenous P availabilities in pots in growth chamber. The start of exogenous P uptake and seed
P reserves remobilization will be followed by labelling of exogenous nutrient solution P. the
interactions between seed P reserves remobilization and exogenous P uptake will be especially
studied.
In third step, a model will be proposed. The main objective of the modelling work was to test the
overall consistency of assumptions based on the experimental work results. Comparing
simulated results with observed values obtained on independent data sets was not performed
during the PhD thesis.
44
3. MATERIALS AND METHODS
3.1. Experiment 1 (greenhouse study 2010)
A pot experiment was carried out in greenhouse, specified for radioisotope utilization between
March and April 2010, at INRA Bordeaux, France. The objectives of this study were
To know the exact time of exogenous P uptake by growing seedling roots.
o As soon as seedling radicle emerges?
o After the utilization of all endogenous seed P reserves?
To evaluate the relative contribution of maize seed P reserves and exogenous P uptake to
maize seedling nutrition during early growth stages.
3.1.1. Experimental layout
The seedlings were grown in pots for 23 days of growth period (298 cumulated degree days after
sowing). Homogenous maize seeds (cv. DKC-5783), (0.27 g to 0.35 g in weight) were selected
for this study. Before sowing, the seeds were separated into groups of nine for each pot and
imbibed for 5 minutes in a plastic tray containing filter papers and distilled water. In preliminary
experiment we verified that P loss by imbibition is negligible. See paragraph 3.4.1 for details.
3.1.2. Seed sowing
Opaque polypropylene pots (10*10*14 cm) were used to sow maize seeds (cv. DKC-5783). All
pots were filled with perlite (150 g pot–1). Perlite was chosen as substrate to sow maize seeds due
to its low buffer capacity (99.9% added P remains in the nutrient solution). On March 15, 2010
(~ 10:00 AM), the imbibed seeds were sown in perlite at 2 cm sowing depth. The sowing
position of each seed in the pot was noted as shown in Figure 3.1. To know the exact timings of
P uptake and repartitioning of total seedling P in different seedling compartments, it was
necessary to measure the accurate amount of P sorbed on seedling root surface and the
background noise of radioactivity. There were total 57 pots out of which (i) 42 pots were used to
follow the kinetics of P uptake, (ii) 9 pots to measure the amount of sorbed P on seedling root
surface and (iii) 6 pots to determine the background noise of radioactivity. These pots were
placed in four plastic trays to avoid leakage of nutrient solution and radioactivity on greenhouse
floor.
45
3.1.3. Nutrient solution composition
A complete P nutrient solution (500 µmol P L–1 i.e. 15.38 mg P L–1) containing all the necessary
micro and macro nutrients, was used to grow the maize seedlings (Anghinoni and Barber 1980;
Bhadoria et al. 2004). The nutrient solution consisted of 500 µM P as NaH2PO4.2H2O, 1 mM
NO3 as Ca(NO3)2.4H2O, 0.2 mM K as KCl, 0.1 mM Mg as MgSO4.7H2O, 46 µM B as H3BO3, 9.1
µM Mn as MnCl2.4H2O, 0.8 µM Zn as ZnSO4.7H2O, 0.3 µM Cu as CuSO4.5H2O, 0.5 µM Mo as
(NH4)6Mo7O24.4H2O; 2 mg L–1 Fe was added as Sequestrene-138 Fe (water soluble granules with
6% Fe in chelated form). The pH of the nutrient solution was adjusted to 6.4 with NaOH (1 g
100 mL–1 of distilled water) solution.
Figure 3.1: Individual pot and sowing of maize seeds.
3.1.4. Labeling of nutrient solution
Phosphorus radioisotope (32P) was used to determine the start of exogenous P uptake by growing
seedling roots and to follow its repartition within different seedling compartments during
germination and early growth stages. Accordingly, the relative contribution of remobilized P
from phytate to the P content of seed compartments was calculated. A measured quantity of 32P
(Rt0 = 1738 kBq) was added to 19 liters of nutrient solution for labelling and the initial specific
activity (SAt0) was 0.183 kBq (µmol P) –1. The labeled nutrient solution (290 mL) was added to
each pot to reach 90% saturation capacity of the perlite (150 g pot–1). The nutrient solution was
added to pots in three times within 3 minutes, so that perlite can absorb nutrient solution. The
labeled nutrient solution was added to 51 pots and the same volume of identical nutrient solution
without labeling was added to the remaining six pots.
46
3.1.5. Seedling growth conditions
The plastic trays holding the pots were placed on a table in greenhouse and the whole
experimental area was covered with a plastic sheet to keep the temperature constant throughout
the growth period (Figure 3.2). Four light lamps (High Pressure Sodium Lamp-400W) suspended
over the pots provided light at a photoperiod of 18/6 hours of light and dark. Air temperature and
the temperature inside the pots were measured with 0.2 mm diameter copper-constantan
thermocouples. Air relative humidity was measured with a relative humidity probe (HMP35AC,
Vaisala, Finland). Photosynthetically active radiation (PAR) was recorded with a PAR Sensor
(SKP 215, Skye Instruments, Llandrindod Wells, UK and JYP-1000, SDEC, France).
All sensors were connected to a data logger (21X, Campbell Scientific, UK).
Measurements were taken every 15 minutes, but only hourly average values were recorded. The
Figure 3.3 shows an example of recorded values during one experimental day. The average air
temperature, relative humidity and light intensity during the experiment were 25 °C, 43% and
246 µmol m–2 sec–1, respectively.
Figure 3.2: Outer view of greenhouse experiment 2010.
47
0
50
100
150
200
250
300
350
400
0:00 4:48 9:36 14:24 19:12 0:00
Time (One day duration)
PA
R (µ
mol
m–2
s–1
)
0
10
20
30
40
50
60
70
T (°
C) a
nd R
H (%
)
PAR
Temperature (T)Relative hum idity (RH)
Figure 3.3: Climatic conditions during first experiment, (photosynthetically active radiation "PAR" on left axis, temperature "T" and relative humidity "RH" on right axis).
Thermal time (TT) in cumulated degree days after sowing was calculated on a daily basis as
follows;
∑
−
+= bTTnTxTT
2Equation 3-1
where Tx is the maximum daily air temperature (in °C), Tn is the minimum daily air temperature
(in °C) and Tb is the base temperature (10 °C), any maximum temperature exceeding 30 °C was
set at 30 °C (Bonhomme et al. 1994).
3.1.6. Irrigation
Throughout the experiment period, all pots were irrigated on the basis of water lost from the pots
by evapotranspiration. On each day, six randomly chosen pots from plastic trays were weighed
(g) and average weight loss was compensated by adding the same volume of water to all the
pots.
3.1.7. Seedling harvest
The experiment was carried out for 298 cumulated degree days after sowing and seedlings were
harvested 14 times during this whole growth period. The initial seed biomass, total P, phytate
48
and phytate-P reserves were measured in 18 seeds (3 replications of 6 seeds). After sowing, the
seedlings were harvested each day (~ 10:00 AM) during the first week and every other day
thereafter started from 16 March 2010. At each harvest, three pots were selected from different
positions in the plastic trays and six seedlings (Figure 3.4) were sampled from each pot. When
necessary, seedlings presenting growing problems were discarded.
Figure 3.4: A growing maize seedling during early growth.
The six selected seedlings were washed with a 400 mL solution of 0.5 mM CaSO4 at 4 °C
for 1 minute to remove all the perlite from the seedling roots as shown in Figure 3.5. Next, the
seedlings were placed for 4 minutes at 4 °C in a 400 mL of solution containing high P (500 µmol
P L–1) and CaSO4 (0.5 mM) for the desorption of 32P from the seedling roots (Rubio et al. 2004).
49
Desorption of 32P
Washing with 400 mL of 0.5 mM
CaSO4 + 0, 100, 500 or 1000 µmol PL– 1
at 4°C for 4 minutes (experiment 1 and 2)
Seedling compartments1. Endosperm2. Scutellum3. Coleoptile+mesocotyle4. Roots5. Leaves
32P sorbed on roots
Seedling Removal of Perlite
Washing with 400 mL of 0.5 mM CaSO4 at
4°C for 1 minute
Root Scanning (WinRhizo)
32P determination in washing solution
1 5432
Desorption of 32P
Washing with 400 mL of 0.5 mM
CaSO4 + 0, 100, 500 or 1000 µmol PL– 1
at 4°C for 4 minutes (experiment 1 and 2)
Seedling compartments1. Endosperm2. Scutellum3. Coleoptile+mesocotyle4. Roots5. Leaves
32P sorbed on roots
Seedling Removal of Perlite
Washing with 400 mL of 0.5 mM CaSO4 at
4°C for 1 minute
Root Scanning (WinRhizo)
32P determination in washing solution
32P determination in washing solution
1 5432
Figure 3.5: Desorption of 32P and washing of maize seedling roots.
3.1.8. Statistical analysis
The mean values resulted from the measurement of three biological replications of six plants.
Statistical analyses were performed with R language environment for statistical computing and
graphics, version 2.9.1 (R Development Core Team, 2009). Means were compared using
Student’s test at the 0.05 probability level.
50
3.2. Experiment 2 (growth chamber study 2010)
The second study was conducted in growth chamber during November and December 2010, at
INRA Bordeaux, France. The objectives of this study were to know the effect of endogenous and
exogenous P availability on
The endogenous seed P reserves remobilization.
The beginning of exogenous P uptake and its intensity.
The interaction of the endogenous and exogenous P availability and effect on each other
during germination and early growth stages.
3.2.1. Experimental layout and selection of maize seeds
The maize seeds (cv. DKC-5783) with low (LS) and high (HS) endogenous seed P reserves were
used in this experiment. The seeds were harvested from long term P fertilization experiment
cultivated with irrigated maize and located at experimental site of Pierroton in Southwest of
France (lat. 44° 44′ 30″; long. 0° 46′ 59″; alt. 55m). Experimental field received 11.5 kg ha–1 and
88.40 kg ha–1 P fertilization for obtaining low and high seed P reserves, since 2003 (see for
detail, experimental protocol PX032 in Appendix I). Homogenous seeds from LS and HS
treatments with same initial seed weight (0.33 g ± 0.001) were selected for this experiment. The
initial endogenous seed P was 506 µg P seed–1 and 952 µg P seed–1 for LS and HS seeds,
respectively.
3.2.2. Seed sowing
Before sowing, the LS and HS seeds were separated into groups of nine for each pot. On 9th
November 2010 (~ 10:00 AM), these seeds were sown in perlite (120 g pot–1) at 2 cm sowing
depth in opaque polypropylene pots (10*10*14 cm) with three exogenous nutrient solution P
availabilities. The three exogenous P availabilities were 0, 100 µmol P L–1 and 1000 µmol P L–1
for no P (0P), low (LP) and high exogenous P (HP), respectively. There were total 183 pots, 90
pots were cultivated with LS seeds and other 90 pots with HS seeds. The remaining 3 pots used
to determine the radioactivity induced background noise.
3.2.3. Nutrient solution composition
Three complete nutrient solutions of P containing all the necessary micro and macro nutrients
were used to grow the maize seedlings (Bhadoria et al. 2004) with three exogenous P
51
concentrations. The nutrient solutions consisted of 1 mM NO3 as Ca(NO3)2.4H2O, 0.2 mM K as
KCl, 0.1 mM Mg as MgSO4.7H2O, 46 µM B as H3BO3, 9.1 µM Mn as MnCl2.4H2O, 0.8 µM Zn
as ZnSO4.7H2O, 0.3 µM Cu as CuSO4.5H2O, 0.5 µM Mo as (NH4)6Mo7O24.4H2O, 2 mg L–1 Fe
was added as Sequestrene-138 Fe (water soluble granules with 6% Fe in chelated form). The
exogenous P concentrations were 0, 100 µmol P L–1 or 1000 µmol P L–1 as NaH2PO4.2H2O for
0P, LP and HP, respectively. The pH of the all three nutrient solutions was adjusted to 6.4 with
NaOH (1 g 100 mL–1 of distilled water) solution.
3.2.4. Labeling of nutrient solution
In order to correctly distinguish the two P fluxes coming from endogenous seed P reserve
remobilization and exogenous P uptake in newly growing maize seedlings and their effect on
each other according to their origin, the exogenous P was labeled with 32P. The exogenous P was
labeled with radioactive P (32P) to quantify the exogenous P uptake and to follow the endogenous
seed P remobilization kinetics and the partitioning of seedling P in different seedling
compartments during early growth stages. Out of a total 183 pots i) 180 pots with 32P labeling
were used to monitor the endogenous seed P reserves remobilization kinetics and exogenous P
uptake and ii) three pots without 32P labeling sown with HS and HP to determine radioactivity
induced background noise on last seedling harvest.
An initial measured quantity (Rt0), 1561 kBq and 1669 kBq of 32P was added in 16 L of
0P and LP solutions, respectively while 1667 kBq was added in 15 liters of HP solution for
labeling of exogenous P. The initial specific activity (SAt0) was 1.043 kBq (µmol P) –1 and 0.111
kBq (µmol P) –1 in LP and HP labeled nutrient solutions, respectively. The labeled nutrient
solution (210 mL pot–1 of 0P, LP and HP) was added to total 180 pots to reach the 90% saturation
capacity of perlite and the same volume (210 mL pot–1) of HP nutrient solution without labeling
was added to the remaining three pots sown with HS seeds to know the radioactivity induced
background noise. Out of total 90 LS seed pots, each 30 pots were irrigated with labeled nutrient
solution of 0P, LP and HP, respectively. Similarly out of 90 pots treated with HS seeds, each 30
pots were irrigated with labeled nutrient solution of 0P, LP and HP, respectively.
3.2.5. Seedling growth conditions
All the pots were placed in the plastic trays on fixed iron stands with growth chamber walls as
shown in Figure 3.6. The growth chamber ground surface was covered with plastic sheet to
avoid any contamination of 32P. The photoperiod was kept as 18/6 hours of light and dark
52
throughout the seedling growth period. Air temperature and the temperature inside the pots were
measured with copper-constantan thermocouples (0.2 mm diameter). Air relative humidity was
measured with a relative humidity probe (HMP35AC, Vaisala, Finland). Photosynthetically
active radiation (PAR) was recorded with a PAR Sensor (SKP 215, Skye Instruments,
Llandrindod Wells, UK and JYP-1000, SDEC, France). All sensors were connected to a data
logger (21X, Campbell Scientific, UK).
Figure 3.6: Growing maize seedlings in pots placed in plastic trays on iron stands in growth chamber experiment in 2010.
Measurements were taken every 15 minutes, but only hourly average values were recorded. The
Figure 3.7 shows an example of recorded values during one experimental day. During the
experiment the average air temperature, relative humidity and light intensity were 28 °C, 49%
and 657 µmol m–2 sec–1, respectively. Thermal time (TT) in cumulated degree days after sowing
was calculated on daily basis as follows:
( )∑=
=
−=29
1
d
dbd TTTT
Equation 3-2
where dT is the daily air temperature (in °C) as average of hourly recorded air temperature each
day. Any hourly air temperature exceeding 30 °C was set at 30 °C (Bonhomme et al. 1994). Tb
(10 °C) is the base temperature (Eagles and Hardacre 1979).
53
0
100
200
300
400
500
600
700
800
900
00:0
0
06:0
0
11:0
0
16:0
0
21:0
0
Time (One day duration)
PA
R (µ
mol
cm
-2 s
ec-1
)
0
10
20
30
40
50
60
T (°
C) a
nd R
H (%
)
PAR
Tem perature (T)
Relative humidity (RH)
Figure 3.7: Climatic conditions during second experiment, (photosynthetically active radiation "PAR" on left axis, temperature "T" and relative humidity "RH" on right axis).
3.2.6. Irrigation
The pots were irrigated with water on the basis of water lost from the pots by evapotranspiration
throughout the experiment on daily basis. Pots of last seedling harvest were weighed (g) on each
day to estimate the average weight loss due to evapotranspiration, and weight loss was recovered
by adding same volume of water to each pot.
3.2.7. Seedling harvest
The experiment was carried out for 530 cumulated degree days after sowing and 10 seedling
harvests were taken during these four weeks of early growth period. The initial seed biomass,
total P, phytate and phytate-P reserves were determined in 18 seeds (3 replications of 6 seeds) in
each LS and HS seeds. The seedlings were harvested (~ 10:00 AM) after 16, 34, 52, 71, 89, 126,
163, 199, 309 and 530 cumulated degree days after sowing. At each seedling harvest, three pots
within each treatment were selected and six homogenous seedlings were sampled from each pot.
For all treatments, the six homogenous selected seedlings were washed first with a 400 mL
solution of 0.5 mM CaSO4 at 4 °C for 1 minute to remove all the perlite from the seedling roots
(Figure 3.5). Next the seedlings were placed at 4 °C in a 400 mL of solution containing P (0,
54
100 µmol P L–1 or 1000 µmol P L–1, depending upon the exogenous P treatment) and CaSO4 (0.5
mM) for the desorption of 32P from the seedling roots for 4 minutes (Rubio et al. 2004).
3.2.8. Statistical analysis
In second experiment the treatments were defined as the factorial combination of two
endogenous seed P levels (LS, HS) and three exogenous nutrient solutions P availabilities (0P,
LP, HP). The treatments were applied in a completely randomized block design with three
replicates and ten seedling harvests dates. Data were analyzed by ANOVA using R language
environment for statistical computing and graphics, version 2.9.1 (R Development Core Team,
2009). Means were compared using Tukey’s test at the 0.05 probability level.
55
3.3. Growth measurements and chemical analysis
In both early mentioned experiments some similar morphological and chemical analysis were
performed as explained below.
3.3.1. Morphological analysis
After washing, the seedlings were placed on a glass plate and separated into five different
seedling compartments including endosperm, scutellum, roots, coleoptile+mesocotyle and leaves
with a spatula in both experiments as shown in Figure 3.5. The following compartments were
also used for data analysis: the seed (comprising endosperm and scutellum), the seedling
(comprising leaves, roots and coleoptile+mesocotyle), and total seedling (comprising leaves,
roots, coleoptile + mesocotyle, endosperm and scutellum). All the results in this study are
expressed on the basis of each seedling in each pot.
3.3.1.a. Root and leaf morphology
At each seedling harvest, the length of the radicle (cm seedling-1) and the length of the leaf (cm
seedling-1) of each seedling were measured by scale. Then the whole root system was scanned
and root length (cm seedling-1), root volume (cm3 seedling-1) and root surface area (cm² seedling-
1) were measured using WinRHIZO Pro V.2005a (Instrument Regent, Quebec, Canada).
3.3.1.b. Fresh and lyophilized seedling biomass
After scanning, the fresh biomass (g seedling-1) of each seedling compartment (endosperm,
scutellum, leaf, root, coleoptile+mesocotyle) was determined. The seedling samples were than
lyophilized for 24 hours and their lyophilized weight (g seedling-1) was measured.
3.3.2. Chemical analysis
In both experiments, each seedling compartment was ground separately in a micro-vibrant
grinder (Retsch MM400 mixer mill, Retsch GmbH, Haan, Germany). Endosperm seed
compartment ground for 45 seconds at 25 frequency and scutellum seed part ground for 35
seconds at frequency value of 13. Leaves, roots and coleoptile+mesocotyle seedling
compartments were grinded for 45 seconds for frequency value of 20. After grinding the seedling
compartments were divided into two parts, one part was used to determine phosphorus in
seedling compartments and other for phytate, C and N analysis.
56
3.3.2.a. Phosphorus determination
Phosphorus content was determined in all seedling compartments by an adaptation of malachite
green colorimetric technique (Van Veldhoven and Mannaerts 1987). One part of each ground
sample was weighed and then reduced to ashes at 550 °C for 5 hours. The resulting ashes were
dissolved in 5 mL distilled water and placed on a hotplate to evaporate until only a few drops
were left. P content was measured colorimetrically after P mineralization with HNO3 (Van
Veldhoven and Mannaerts 1987). The total seedling P content was the sum of the respective
amounts of P in each seedling compartment.
3.3.2.b. Exogenous P uptake and its allocation using 32P labeling
Assuming that no 32P/31P-fractionation occurred during exogenous P uptake by growing seedling
roots and P transport within the seedling (Fardeau 1993; Schjørring and Jensén 1984), the
amount of P in the seedling compartment taken up from the nutrient solution (Pexo) was
calculated as follows:
=⇒
=
ns
t
texo
exo
t
ns
t
PRr
PPr
PR
Equation 3-3
where Rt/Pns is specific activity SAt measured at time t in the nutrient solution, rt is 32P-activity
measured in the mineralized seedling compartment at harvest time t. Rt and Pns are the amount of
carrier-free 32P and 31P measured in the nutrient solution, respectively. The Rt and rt values were
calculated using half life and these values were counted using a scintillation cocktail (Insta-gel
Plus Packard, PerkinElmer) using a Packard TR 2100 (Canberra Industries, Meriden, CT). The
mineralized sample of each seedling sample (10 mL) was added with scintillation cocktail liquid
(10 mL) and placed in Packard TR 2100 for the standard counting time of 20 minutes.
3.3.2.c. Phytate and phytate-P determination
Phytate contents were measured in the scutellum and endosperm compartments of maize seeds at
each harvest in both experiments. The second part of lyophilized ground samples of endosperm
(50 mg) and scutellum (30 mg) were placed in 1.5 mL Eppendorf tubes. Phytate was extracted
from the endosperm with 1 mL of 0.65 M HCl and from the scutellum with 0.60 mL of 0.65 M
HCl (Lorenz et al. 2007; Pontoppidan et al. 2007; Reichwald and Hatzack 2008). The samples
were shaken (SRT-2 Roller Mixer) overnight at room temperature and then centrifuged at 12000
rpm for 20 minutes. The samples were filtered with a 2 mL syringe and filter (0.2 µm cellulose
57
acetate membrane). Phytate was determined by ion chromatography (IC) using a hydroxide
eluent prepared by an eluent generator with a column designed to determine common inorganic
anions (for details see Appendix II). Phytate content was converted into phytate-P by dividing
phytate content by 3.548 (Raboy and Dickinson 1984).
3.3.2.d. Carbon and nitrogen analysis
C and N was analyzed in all seedling compartments on each seedling harvest in first experiment
and only on last seedling harvest in second experiment. The C and N analysis was performed
with a C and N analyser (Flash EA-1112, Organic Elemental Analyser) using the dynamic flash
combustion technique (modified Dumas method). A ground lyophilized sample of each seedling
compartment (3 mg) was weighed in a small tin capsule and placed in the combustion reactor by
an autosampler. Samples were combusted at 900 °C to release their C and N in gaseous form. C
and N concentration were then measured using a separation column and a TCD detector.
3.3.3. Endogenous seed P exportation flux
In second experiment, the P exportation flux from endogenous seed P towards newly growing
seedlings and P efflux was calculated on the basis of difference between initial endogenous seed
P and endogenous seed P at each seedling harvest in all P treatments.
Exported endogenous seed P = (Endogenous Seed P)t0 – (Endogenous seed P)t
Equation 3-4
3.3.4. P efflux from germinating seeds and growing maize seedling
roots
In second experiment, the P efflux from germinating seeds and growing seedling roots was
calculated considering the exogenous P uptake and endogenous seed P remobilization at each
seedling harvest. The cumulated P efflux was calculated as the difference between the P lost
from seed plus accumulated exogenous P uptake assessed by 32P activity measured in seedling,
and the accumulated P in seedlings.
Cumulated P efflux = (P lost from seed + Exogenous P uptake) – Seedling accumulated P
Equation 3-5
58
3.4. Control of experimental procedures
3.4.1. P loss from seeds by imbibition
Phosphorus loss during imbibition of seeds was reported in previous studies. Dave et al., (2008)
reported that 4-11% of phytate phosphorus decreased during soaking of whole legume seeds. A
preliminary experiment was carried out to know the effect of maize seed imbibition on seed P
loss. The 20 maize seeds (cv. DKC-5783) of homogenous seed size were selected for this
experiment. 100 mL distilled water was taken in a glass beaker (200 mL capacity) and 3 mL of
this distilled water was taken as initial reading of P determination (S0). The maize seeds were
imbibed in remaining 97 mL of distilled water for the duration of 180 minutes. The seeds were
stirred, so that all the seeds should be mixed thoroughly. The first sampling from seed imbibition
solution (S1) was carried out after 5 minutes of imbibition of seeds in distilled water. In the same
pattern, the 7 samplings (S2 …. S7) were carried out after 30, 60, 90, 120, 150, and 180 minutes
of imbibition. For each sampling 3 mL solution was taken from imbibition solution and the
remaining solution was considered as final for the next sampling for final calculations of volume
of water. The phosphorus was determined in each sample of seed imbibed water and in seeds
after 180 minutes of imbibition. After 180 minutes of imbibition, the seed P loss was less than
0.4% of initial seed P reserves as explained in the Figure 3.8, therefore the imbibition of seeds
for 5 minutes was considered as good to initiate the germination during the further studies.
0.00%
0.10%
0.20%
0.30%
0.40%
0.50%
0 50 100 150 200
Seed imbibition time (minutes)
µg P
rele
ased
/ µg
P in
itial
in th
e gr
ain
(%)
Figure 3.8: Loss of seed P by imbibition (% loss of initial seed P).
59
3.4.2. P sorption properties of perlite
Perlite was used as a substrate to sow maize seedlings during the studies. A preliminary
experiment was carried out to determine the buffer capacity of perlite. Different concentrations
of P were added to substrate/solution suspension (1 g perlite 10 mL-1 solution suspension) in
small flacons. The flacons with perlite and solution suspension were left for agitation for 24
hours at room temperature.
y = 1.0053x - 0.3201R2 = 0.9995
0
20
40
60
80
100
120
0 20 40 60 80 100 120
Initial P concentration (µg P mL-1)
Fina
l P c
once
ntra
tion
(µg
P m
L-1) 1:1 line
Figure 3.9: Initial P concentrations added in perlite solution suspension and final P concentrations in perlite solution suspension after 24 hours. Dashed line is the 1:1 line. Blue dots are measured data and the solid line is the linear regression line.
The P was determined in solution suspension after 24 hours. The added P in perlite
solution suspension was found in solution after 24 hours showing that there was no sorption of P
on perlite and all the added P remained in solution after 24 hours as shown in Figure 3.9. Due to
the very low buffer capacity, the perlite was used for further studies for sowing of maize
seedlings.
3.4.3. Base temperature for maize germination
A preliminary experiment was conducted to determine the base temperature for maize seed
germination. The experiment was carried out at four different temperatures and three endogenous
seed P levels over a growth period of 7 days after imbibition. The maize seeds with three
endogenous seed phosphorus levels ( low P, intermediate P and high P) were sown at
60
temperature range of 15 °C, 20 °C, 25 °C and 30 °C. In this study, the germination was defined
as when the seed has a visible seedling radicle. The maize seeds were placed in petri dishes on
moist filter papers and covered with one layer of filter paper to keep them moist. The petri dishes
were placed in the incubators at four different temperatures (15 °C, 20 °C, 25 °C and 30 °C).
Each phosphorus level was replicated three times in each temperature.
y = 0.0018x - 0.0169R2 = 0.8783
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0 5 10 15 20 25 30 35
Temperature (°C)
Ger
min
atio
n ra
te h
our-1
50Linéaire (50)
9.64°C
Figure 3.10: Germination rate hour-1 calculated as the inverse of the time to reach 50 % germination as a function of the temperature. The calculated base temperature for maize
germination was 9.64°C.
The distilled water was added to petri dishes from time to time to keep the moisture level at
optimum. The temperature was maintained uniform in each treatment during the experiment in
incubators. One each day, germination was noted at different times. The percent germination is
noted on the basis of total number of seeds germinated on each time. Germination percentage is
about more than 88% in all temperature and phosphorus treatments. The results showed that at
15 °C the maximum seeds were germinated on 4th day, at 20 °C on 3 rd day, on 25 °C and 30 °C
on 2nd day after imbibition, respectively. The germination is favoured at more than 20 °C, and
the optimum temperature for maize may be between 25 °C and 30 °C. The coleoptile emergence
from germinating seed is also favoured by more temperature and minimum at 15 °C, being
optimum at 25 °C. Temperature also has a positive effect on fresh seedling biomass which is
maximum at 30 °C. The base temperature calculated on the basis of germination rate per hour
61
was 9.64 °C as shown in Figure 3.10. Therefore a base temperature of 10°C was used for
calculations.
3.4.4. P sorption on seedling roots
The amount of P sorbed on the root free space was estimated by measuring 32P release in the
rinsing solution at 10, 98 and 224 cumulated degree days after sowing only in first experiment.
Three pots were sampled and six homogenous seedlings selected from each pot. Seedlings were
washed with 0.5 mM CaSO4 at 4 °C for 1 minute to remove the perlite.
0
2
3
5
6
0 50 100 150 200 250 300 350
Thermal time (cumulated degree days after sowing)
P s
orbe
d (µ
g se
edlin
g-1)
Figure 3.11: P sorption on maize seedling roots during germination and early growth. Data are
means and vertical bars indicate ±SE for n = 3 replications.
The seedlings were then placed in a high P solution (500 µmol P L–1) also containing 0.5
mM CaSO4 for 4 minutes at 4 °C to desorb 32P from the root free space. Seedlings were then
removed from the washing solution and 32P was counted in the washing solution. The root length
was measured by scanning the whole root system as described earlier. The total P sorption
calculated from specific activity and 32P counting was 5 µg P seedling-1 as shown in Figure 3.11.
The average amount of P sorbed on the root free space was always less than 0.009 µg P cm–1 of
seedling root, so it was considered as negligible in the second experiment. In second experiment,
the 32P sorbed on seedling roots was not determined in washing solution because it was
negligible during first experiment.
62
3.4.5. Specific activity induced background noise
As the calculation of exogenous P uptake by 32P labelling might be influenced by background
noise caused by specific activity in the seedlings, the specific activity induced background noise
was measured in all seedling compartments at two seedling harvests in first and on last seedling
harvest in second study. Background noise of specific activity was measured at 70 and 273
cumulated degree days after sowing in first experiment and on 530 cumulated degree days in
second experiment in pots that had received nutrient solution without 32P labeling. The six
homogenous seedlings were selected from each pot and these seedlings were washed first with a
400 mL solution of 0.5 mM CaSO4 at 4 °C for 1 minute to remove all the perlite from the
seedling roots.
Next the seedlings were placed at 4 °C in a 400 mL of solution containing P (0, 100 µmol
P L–1, 500 µmol P L–1 or 1000 µmol P L–1, depending upon the exogenous P treatment in
experiment 1 and 2) and CaSO4 (0.5 mM) for the desorption of 32P from the seedling roots for 4
minutes (Rubio et al. 2004). Seedlings were separated into different seedling compartments.
The seedling compartments were reduced to ashes at 550°C and mineralized with HNO3
(Van Veldhoven and Mannaerts 1987). The mineralized solution (10 mL) was placed in Packard
TR 2100 along with scintillation cocktail liquid (10 mL) for 20 minutes. As no significant 32P
activity (<47 cpm) was detected in the different compartments of seedlings grown in the nutrient
solution without labeling, specific activity induced background noise was considered to be zero
in both experiments.
63
64
4. RESULTS AND DISCUSSIONS
4.1. Relative contribution of seed phosphorus reserves and exogenous
phosphorus uptake to maize (Zea mays L.) nutrition during early growth
stages
4.1.1. Objectives
The objective of this first study was to characterize the kinetics of the two processes that supply
phosphorus to the developing maize seedling i.e. the remobilization of seed P reserves and
exogenous P uptake, and to evaluate their relative contribution to the young maize seedling P
supply. Although P, carbon (C) and nitrogen (N) are stored in different compartments of the seed
(P in the scutellum; C and N in the endosperm), we examined whether their remobilization
follows the same kinetics. The results of this study are presented here and were published in
Plant and Soil (Nadeem et al. 2011). The complete article is attached in Appendix III.
4.1.2. Results
4.1.2.a. Seedling growth
The radicle emerged from the maize seed along with the mesocotyle and coleoptile, the latter
having first enclosed seedling leaf at 26 cumulated degree days after sowing (Table 4.1).
Seminal roots and radicle appeared and elongated at 41 cumulated degree days. The first
seedling leaf emerged from the soil and became visible at this time. At the end of the experiment,
the seedlings had four visible leaves. Total root length and lateral root length were 572 cm
seedling–1 and 366 cm seedling–1, respectively at 298 cumulated degree days.
65
Table 4.1: Radicle length, root length and number of visible leaves during early growth of maize seedlings. Values are means (±SE) of 3 replications.
Thermal
time
Age
(days)
Root length
(cm/seedling)
Number of
visible
leaves
Cumulated
degree
days
Radicle
length
Total root
length
Root
length
(Ø<0.04)
Root
length
(Ø>0.04)
10 126
2 3 ± 0.2 4 ± 0.441 3 4 ± 0.3 11 ± 3 1 ± 0.2 10 ± 1 156 4 6 ± 0.5 25 ± 2 7 ± 1 18 ± 1 170
5 8 ± 0.2 39 ± 6 11 ± 4 28 ± 3 198 7 9 ± 2 133 ± 22 76 ± 12 56 ± 10 2126
9 11 ± 0.6 206 ± 4 114 ± 3 92 ± 2 3152 11 12 ± 0.9 294 ± 7 172 ± 7 122 ± 1 3176 13 18 ± 1 362 ± 9 223 ± 5 138 ± 12 3202
15 19 ± 1 456 ± 29 273 ± 19 183 ± 12 3224 17 20 ± 0.1 499 ± 44 301 ± 26 198 ± 18 3248
19 19 ± 1 543 ± 31 336 ± 26 207 ± 5 4273 21 22 ± 1 541 ± 20 329 ± 13 212 ± 7 4298
23 20 ± 0.2 572 ± 26 366 ± 16 206 ± 10 4
4.1.2.b. Seed and seedling C content
Total C reserves remained significantly unchanged during the first 26 cumulated degree days
after sowing as shown in Figure 4.1. They started to decrease from 26 until 98 cumulated degree
days after sowing and increase thereafter. The seed C reserves showed a rapid remobilization
from 26 cumulated degree days after sowing till 176 cumulated degree days which largely
translocated towards seedling compartments. The seedling C accumulation was started soon after
radicle emergence from 26 cumulated degree days after sowing and increase thereafter. The seed
C loss was calculated by the difference of initial seed C reserves and seed C reserves at each
seedling harvest. The difference of seed C loss and seedling C accumulation was higher up to 89
66
cumulated degree days after sowing (Figure 4.1) and assumed to be lost through respiration. This
difference started to decrease after 89 cumulated degree days when the seedlings had two visible
leaves on 98 cumulated degree days after sowing. From 202 cumulated degree days after sowing,
the seedling C accumulation was higher than seed C loss and this period corresponded to the
transition from heterotrophy to autotrophy for C.
Cumulated degree days after sowing
0 50 100 150 200 250 300 350
Qua
ntity
of C
(mg
C)
0
20
40
60
80
100
120
140
Total CSeed CSeedling CSeed C loss
Figure 4.1: Changes in total seedling C (■), seed C remobilization (●), seedling C accumulation (▲) and seed C loss (∆) in maize seedlings expressed in mg seedling-1 during germination and early growth stages. Time is expressed in cumulated degree days after sowing. Data are means and vertical bars indicate ± SE for n = 3.
We considered two maize seed compartments, namely, endosperm and scutellum. Prior to
germination, 87% (w/w) of initial seed C reserves were localized in the seed endosperm and the
remainder in the seed scutellum. Endosperm C reserves remained significantly unchanged during
the first 26 cumulated degree days after sowing (Figure 4.2a). A sharp decrease in endosperm C
reserves was observed from 41 to 176 cumulated degree days and 79% of initial endosperm C
reserves were remobilized before 176 cumulated degree days. Endosperm C reserves remained
unchanged thereafter with a minimum content of around 20 mg C in the endosperm. In contrast,
scutellum C reserves were much lower than those in the endosperm and were remobilized
rapidly during the first 41 cumulated degree days after sowing and slowly thereafter (Figure 4.2
b). The scutellum was in close contact with the embryo.
67
(a) Endosperm
mg
C s
eedl
ing-1
0
20
40
60
80
100
120
µg P
see
dlin
g-1
0
200
400
600
800C contentP content
(b) Scutellum
mg
C s
eedl
ing-
1
0
20
40
60
80
100
120
µg P
see
dlin
g-1
0
200
400
600
800
(c) Leaf
mg
C s
eedl
ing-
1
0
20
40
60
80
100
120
µg P
see
dlin
g-1
0
200
400
600
800
(d) Root
Cumulated degree days after sowing
0 50 100 150 200 250 300 350
mg
C s
eedl
ing-
1
0
20
40
60
80
100
120µg
P s
eedl
ing-1
0
200
400
600
800
Figure 4.2: Changes in C content (mg C seedling-1) on the left axis (● and solid lines) and in P content (µg P seedling-1) on the right axis (○ and dashed lines) in the endosperm (a), scutellum (b), leaves (c) and roots (d) during early growth of maize. Time is expressed in cumulated degree days after sowing. Data are means and vertical bars indicate ± SE for n = 3.
68
The fast initial remobilization of scutellum C reserves was associated with the demand
for C by the growing embryo. Of the total seed C remobilized, 68% was remobilized from the
endosperm and only 8% from the scutellum up to 176 cumulated degree days after sowing.
Seedling leaf C content started to increase with the emergence of first visible leaf on the
seedling at 41 cumulated degree days (Figure 4.2 c), while seedling root C content started to
increase on 26 cumulated degree days when the radicle emerged (Figure 4.2 d). From 41 to 98
cumulated degree days, the shoot to root C ratio increased sharply to 0.89 and remained constant
thereafter indicating that the distribution of remobilized C reserves and C assimilated
photosynthetically between shoot and root compartments remained unchanged. The
coleoptile+mesocotyle (C+M) is a smaller but important seedling compartment that helps the
first leaf to emerge and reach the light. C+M C content increased from 41 cumulated degree days
and after the emergence of the first leaf from the surface, then C content remained constant
throughout the growth period. After 298 cumulated degree days after sowing, 45%, 53% and 3%
of accumulated seedling C (104 mg seedling–1) was distributed among seedling leaves, roots and
C+M compartments, respectively.
4.1.2.c. Remobilization of seed P reserves and exogenous P uptake
Before germination, a large proportion of maize seed P reserves were located in the scutellum
rather than in the endosperm, i.e. the reverse of C reserves. The initial P content of the scutellum
was about 86% (w/w) of total maize seed P reserves, and the remaining 14% was localized in the
endosperm. The endosperm P reserves were remobilized rapidly at 26 cumulated degree days
after sowing and thereafter, a slow remobilization was observed until 202 cumulated degree days
after sowing (Figure 4.2 a), after which endosperm P content remained unchanged.
Slow remobilization of scutellum P reserves was observed until 26 cumulated degree
days after sowing. Immediately after 41 cumulated degree days, scutellum P reserves were
rapidly remobilized until 202 cumulated degree days (Figure 4.2 b). Remobilization of total seed
P reserves increased from 10 cumulated degree days to reach its maximum value at 70 cumulated
degree days (data not shown), thereafter the remobilization rate decreased as the seed P reserves
were exhausted. At 202 cumulated degree days, 92% and 84% of initial scutellum and
endosperm P reserves were remobilized; 78% of total seed remobilized P originated from the
scutellum and only 12% from the endosperm. All the P remobilized in the maize seed
compartments was quantitatively recovered in different seedling compartments.
69
Maize seedling leaf P content started to increase from 41 cumulated degree days after sowing
with the emergence of the first visible leaf (Figure 4.2 c), while seedling root P content started to
increase from 26 cumulated degree days with the emergence of the radicle (Figure 4.2 d).
Cumulated degree days after sowing
0 50 100 150 200 250 300 350
µg P seedling
-1
0
200
400
600
800
1000
Total seedling PShoot and root PShoot and root exogenous P
Figure 4.3: Changes in total seedling P content (●), in shoot and root P content (▲) and in exogenous P uptake from the nutrient solution calculated according to 32P-activity measured in the seedling (∆). Data are means and vertical bars indicate ±SE for n = 3 replications.
Total maize seedling P content remained significantly unchanged up to 98 cumulated degree
days after sowing (Figure 4.3) and then started to increase significantly from 126 cumulated
degree days, suggesting that exogenous P uptake from nutrient solution had begun. The exact
time of the beginning of exogenous root P uptake from the nutrient solution was established and
P uptake quantified by 32P-activity measurements in the seedling. Up to 56 cumulated degree
days, no 32P-activity was detected in maize seedlings. 32P-activity was detected in seedlings at 70
cumulated degree days, indicating that exogenous root P uptake was underway. Exogenous root
P uptake increased continuously thereafter. After 298 cumulated degree days, 24% of seedling P
content was provided by exogenous P uptake whereas 76% by seed P reserves remobilization.
4.1.2.d. Remobilization of seed N reserves and seedling N accumulation
The total seed N reserves started to remobilize from 26 cumulated degree days after sowing as
shown in Figure 4.4. A rapid decrease in total seed N reserves was observed from 26 cumulated
70
degree days after sowing to 176 cumulated degree days after sowing and thereafter remained
almost unchanged. Before germination, 77% of these total seed N reserves in maize were
concentrated in the endosperm and the remaining 23% in the scutellum (Figure 4.4).
Cumulated degree days after sowing
0 50 100 150 200 250 300 350
Qua
ntity
of N
(mg
N)
0
1
2
3
4
Seed NEndosperm NScutellum NSeedling N
Figure 4.4: Changes in total quantity of seed (●), seedling (▲), endosperm ( ) and scutellum (○) nitrogen (mg seedling-1) contents in maize seeds and seedlings during germination and early growth stages. Time is expressed in cumulated degree days after sowing. Data are means and vertical bars indicate ± SE for n = 3.
The remobilized seed N reserves were translocated towards seedling compartments. The
seedling N accumulation started to increase from 26 cumulated degree days after sowing and
increase thereafter. The seedling N accumulation followed the similar pattern of C and up to 298
cumulated degree days after sowing, 3 mg N was accumulated in seedling.
4.1.2.e. Phytate and phytate-P
In the maize seed most P is stored in the scutellum mainly in the form of phytate. Initially, the
maize seed had 2.61 mg g–1 DW of phytate-P. As shown in Figure 4.5, 98% of seed phytate-P
was initially localized in the scutellum and remaining 2% in the endosperm. Phytate-P started to
hydrolyze from the 1st DAS via phytase activity. The phytate-P stored in maize scutellum and
endosperm was rapidly converted into organic phosphorus and mineral nutrients, which became
available for newly developing seedlings.
71
Cumulated degree days after sowing
0 50 100 150 200 250 300 350
P an
d ph
ytat
e-P
con
tent
(µg
P se
edlin
g-1)
0
200
400
600
800
Endosperm phytate-PScutellum phytate-PTotal seed P
Figure 4.5: Variation in total P (µg P seed-1) and phytate-P (µg phytate-P seed compartment-1) reserves in different compartments of the maize seed during early growth stages. Remobilization of total seed P reserves (●); Scutellum phytate-P reserves (∆) and endosperm phytate-P (□). Data are means and vertical bars indicate ±SE for n = 3 replications.
The rapid depletion of the phytate-P fractions in the maize scutellum was observed from 10 to
126 cumulated degree days after sowing and practically all the phytate-P had been depleted by
224 cumulated degree days in the maize scutellum. Whereas in the maize endosperm, all the
phytate-P was hydrolyzed during the 1st DAS. The phytate-P in the scutellum was hydrolyzed
earlier and more rapidly than seed P was remobilized and sent to the growing seedling (Figure
4.5). Non phytate-P, calculated as the difference between total seed P and the phytate-P
remaining in the seed, increased sharply between 41 and 98 cumulated degree days and
decreased thereafter, showing that phytate-P was hydrolyzed and remained there in seeds. Later
on, when the seedling organs were developing, it was translocated to them.
4.1.2.f. Relative contribution of seed P and exogenous P uptake to
seedling P nutrition
A flow chart representing the relative contribution of seed P reserve remobilization and
exogenous P uptake (calculated from 32P-activity measurements) to seedling P nutrition is shown
72
in Figure 4.6. At the end of the experiment (298 cumulated degree days after sowing), total
maize seedling P contents were 811 µg P distributed most among seedling leaves and roots
rather than seedling C+M. Exogenous P uptake from the nutrient solution was 197 µg P (arrows
on the right) while remobilized seed P was 614 µg P (arrows on the left). The width of arrows
indicates the quantity of P flowing to the different seedling compartments. The flow of
exogenous P to the seed endosperm and scutellum was zero. Exogenous P passed directly from
seedling roots to seedling leaves. Remobilized seed P reserves played a significant role in
fulfilling seedling P requirements and contributed more to seedling P nutrition than uptake of
exogenous P. More than 95% of scutellum phytate was hydrolysed while 60% of total seed P
reserves were exported towards seedlings till 98 cumulated degree days after sowing. Up to 176
cumulated degree days after sowing, 87% of total seed P reserves were exported towards the
young seedlings. A total of 77% of the P in leaves originated from remobilized seed P reserves
and only 23% of P in leaves came from exogenous P uptake. Similarly, 73% of seedling root P
came from remobilized seed P reserves and the remaining 27% from exogenous P uptake. C+M
was a small part of the seedling and was mainly composed of remobilized seed P (83%) rather
than exogenous P uptake. Respectively 54%, 44% and 2% of exogenous P uptake from the
nutrient solution was distributed to the seedling leaves, roots and C+M, while 60%, 37% and 3%
of the remobilized seed P flux was allocated to seedling leaves, roots and C+M, respectively.
The flow of remobilized seed P was calculated as the sum of seed scutellum and seed endosperm
P remobilized from the whole seed. The flow of remobilized P from the seed after 298 cumulated
degree days was 628 µg P seed–1 i.e. not significantly different from 614 µg P seed–1, while 34 µg
P remained in the seed.
73
Figure 4.6: Allocation of exogenous P uptake (gray arrows on the right) calculated using 32P labeling of the nutrient solution and distribution of remobilized seed P reserves (dark arrows on the left) to seedling compartments after 298 cumulated degree days (23 DAS). The width of the arrows represents the quantity of P flowing toward different seedling compartments. Values inside the different compartments represent P accumulation in (+) or remobilization from (-) each compartment. Values in italics represent P content in the seed and in the seedling after 298 cumulated degree days (23 DAS). P fluxes are expressed in µg P seedling -1. Data are means and vertical bars indicate ± SE for n = 3 replications.
- 614 (±23)
Remobilized P
+ 228 (±10)
+ 19 (±1)
+367 (±21)
Exogenous P
+ 86 (±4)
+ 4 (±1)
Seedling P
811 (±26)
Seed P
34 (±2)
0
+ 107 (±5)
Leaves
+ 474 (±26)
C+M
+ 23 (±2)
Roots
+ 314 (±14)
Scutellum
- 545 (±17)
Endosperm
- 83 (±4)
External P
197 (±3)
23%
%
17%
%
27%
%
77%
83%
%
73%
%
74
4.1.2.g. Relationship between P and N remobilization
The initial seed N concentration was 12 mg g–1 seed DW. The remobilization of seed N and its
accumulation in the seedling during early growth stages resembled that of C (Figure 4.4).
Phosphorus was mainly localized in the maize scutellum while C and N were localized in the
endosperm. Phosphorus, C and N were rapidly remobilized during germination and early growth
and displayed similar remobilization kinetics, although the three elements were localized in
different seedling compartments as shown in Figure 4.7.
Cumulated degree days after sowing
0 50 100 150 200 250 300 350
Rat
e of
rem
obili
zatio
n of
see
d re
serv
es
0.0
0.2
0.4
0.6
0.8
1.0
1.2
CN P
Figure 4.7: Rate of P, C and N remobilization in maize seed during early growth stages calculated as the ratio of P (○), C (●) and N (∆) content in seed to their initial content. Data are means and vertical bars indicate ±SE for n = 3 replications.
75
4.1.3. Discussion
4.1.3.a. Carbon remobilization and seedling growth
Shoot and root growth of seedlings take place at the expense of seed reserves. In our experiment,
seed C reserves, which were mainly localized in the endosperm, started to decrease from 26
cumulated degree days after sowing. From the start of the experiment until 98 cumulated degree
days, the loss of seed C reserves was greater than the accumulation of C in the seedling and the
difference was assumed to be lost through respiration. At 98 cumulated degree days, the
difference between the decrease in seed C reserves and seedling C accumulation decreased
indicating that photosynthesis had started in the two visible leaves. These results are in
accordance with those of Deleens et al. (1984) who reported a significant contribution of
photosynthetic C to maize seedling growth at 98 cumulated degree days. A net positive
accumulation of seedling C was observed after 202 cumulated degree days.
The decrease in C reserves in maize seeds during early growth occurred in three stages:
first, reserves remained unchanged until 26 cumulated degree days, second, a rapid decrease
occurred from 26 to 176 cumulated degree days, and thereafter no change was observed in seed
C reserves. Deleens et al., (1984) also reported three stages in the depletion of seed C reserves.
4.1.3.b. Phytate hydrolysis and relative contribution of P to growing
seedlings
In agreement with other studies, in our experiment phytate was found to be the main form of P
stored in the maize seeds and was localized in the scutellum rather than in the endosperm or
embryo (Lorenz et al. 2007; Lott et al. 1995; Modi and Asanzi 2008; Park et al. 2006). A sharp
drop in phytate reserves was observed between the 2nd and the 7th day after sowing (98 cumulated
degree days after sowing). This is consistent with the results of Laboure et al., (1993) who
showed that in germinating maize seeds, maximum phytase activity occurred between the 5 th and
7th day after imbibition. Hydrolyzed forms of P were temporarily stored in the seed before being
translocated to the growing organs, suggesting that the hydrolysis of phytate was not a limiting
step for seedling P supply (Figure 4.5).
All the P remobilized from seed P reserves was quantitatively recovered in different
seedling compartments. As already pointed out by Hall and Hodges (1966) that phytate break
down in seed accounts primarily for the increase in inorganic P of the roots and shoots. Four
days after sowing (56 cumulated degree days) the maize seedling only contained P originating
76
from seed P reserves. Exogenous P uptake measured by 32P enrichment in seedling roots was
only observed on the 5th day after sowing (70 cumulated degree days). From 70 cumulated
degree days on, both sources (remobilized seed P reserves and exogenous P uptake) contributed
to fulfil seedling P requirements, although the seed P reserve was the main source. After 202
cumulated degree days, only exogenous P supported seedling growth as seed P reserves were
exhausted. Like the three stages of C proposed by Deleens et al. (1984), three different growth
stages of dependence on seed P reserves and exogenous P uptake can be defined. Up to the 5 th
day after sowing (70 cumulated degree days), the maize seedling depended exclusively on seed P
reserves (heterotrophic stage for P). The transition stage started when the seedling started to
depend on seed P reserves and exogenous P uptake between the 5th and the 15th day after sowing
(from 70 to 202 cumulated degree days). Thereafter, only exogenous P (autotrophic stage for P)
fulfilled seedling P requirements. More P remobilized from seed P reserves was translocated to
seedling leaves than to seedling roots. Similarly, more exogenous P was translocated to seedling
leaves than to seedling roots. This indicates that leaf P requirements are higher than root
requirements during early growth, which corresponds to the appearance of P deficiency
symptoms on seedling leaves during early growth stages.
4.1.3.c. Relationship between P and N remobilization
Nitrogen was mainly concentrated in the maize endosperm and a sharp drop in maize endosperm
N reserves was observed from 26 cumulated degree days. Similar results were reported by
Harvey and Oaks (1974), who showed that total nitrogen in the mature endosperm decreased
most rapidly from the 2nd day after germination on. Although N and P were located in different
seed compartments (i.e. in the endosperm and scutellum, respectively), they displayed similar
remobilization kinetics during early growth of maize seedlings.
77
4.2. Maize (Zea mays L.) endogenous seed phosphorus remobilization is
not influenced by exogenous phosphorus during germination and early
growth stages
4.2.1. Objectives
As the remobilization of endogenous seed P and the uptake of exogenous P overlap in time
(Nadeem et al. 2011), the question arises whether these processes have an effect on each other or
if they are independently controlled. Our objectives were to study the effect of the availability of
endogenous and exogenous P on i) the remobilization of endogenous seed phosphorus, ii) the
beginning of uptake of exogenous P and its intensity, iii) their interaction and its effect on the
development of young maize seedlings during germination and early growth. Exogenous P was
labeled with 32P to distinguish the two fluxes of P, (one remobilized from endogenous seed
reserves and the other uptake of exogenous P) in young maize seedlings and their effect on each
other according to their origin. The results were submitted for publication in Plant and Soil. The
paper was accepted for publication with revisions on 21st October 2011.
4.2.2. Results
4.2.2.a. Early seedling growth
Prior to germination, the seed dry biomass reserves were largely localized (90%, w/w) in the
endosperm rather than in the scutellum despite endogenous seed P reserves in LS and HS seeds.
Remobilization of the seed dry biomass reserves started at 34 cumulated degree days after
sowing in all P treatments (Figure 4.8). A rapid remobilization of seed biomass reserves was
noted from 34 to 309 cumulated degree days after sowing. The availability of endogenous or
exogenous P had no significant effect on the remobilization of seed dry biomass during
germination and early growth stages.
The emergence of the seedling radicle on 34 cumulated degree days after sowing was
independent of the P availability of either endogenous or exogenous source. Seedling biomass
started to accumulate 34 cumulated degree days after sowing with the emergence of seedling
radicle. The first seedling leaf become visible at 71 cumulated degree days after sowing and at
530 cumulated degree days, seedlings had five visible leaves (Table 4.2).
78
Cumulated degree days after sowing
0 100 200 300 400 500 600
See
d bi
omas
s (g
)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0PLSLPLSHPLS0PHS LPHS HPHS
Figure 4.8: Seed biomass remobilization (g) in maize seeds during germination and early growth stages: Low endogenous seed P in seedlings (LS) with no exogenous P (○), low exogenous P (□), high exogenous P (∆), high endogenous seed P seedlings HS with no exogenous P (●), low exogenous P (■) high exogenous P (▲) treatments. Data are means and vertical bars indicate ± SE for n = 3 replications.
A regular increase in accumulated seedling biomass was observed throughout the growth
period (Figure 4.9). The availability of endogenous or exogenous P had no significant effect on
the accumulation of seedling dry biomass (Table 4.2) during germination and early growth
stages.
Cumulated degree days after sowing
0 100 200 300 400 500 600
See
dlin
g bi
omas
s (g
)
0.00
0.05
0.10
0.15
0.20
0.25
0PLSLPLSHPLS0PHS LPHS HPHS
Figure 4.9: Seedling biomass accumulation (g) in maize seedlings during germination and early growth stages: Low endogenous seed P in seedlings (LS) with no exogenous P (○), low exogenous P (□), high exogenous P (∆), high endogenous seed P seedlings HS with no exogenous P (●), low exogenous P (■) high exogenous P (▲) treatments. Data are means and vertical bars indicate ± SE for n = 3 replications.
79
4.2.2.b. Accumulation of P in seedlings
The seedlings started to accumulate P with the emergence of the seedling radicle. P accumulated
rapidly in LS seedlings from 34 cumulated degree days after sowing to 199 cumulated degree
days after sowing in all exogenous P treatments and thereafter an increase was only observed in
the HP treatment (Figure 4.10 A). A regular increase in accumulated P in the seedling was
observed in HS seedlings from 34 cumulated degree days after sowing until 530 cumulated
degree days after sowing (Figure 4.10 B).
The growing seedlings accumulated significantly more P in HP than in 0P or LP
treatments with available exogenous P despite endogenous P reserves in the seed. Less P
accumulated in the seedling than the initial amount of endogenous seed P in LS and HS
seedlings in 0P and LP treatments with exogenous P, whereas more accumulated P in the
seedling in LS and HS seedlings treated with HP exogenous P, indicating net P accumulation at
whole plant level. Accumulation of P in the seedling was significantly affected by the
availability of endogenous and exogenous P at 530 cumulated degree days. HS seedlings had
significantly more accumulated P (959 µg, 637 µg and 596 µg in HP, LP and 0P exogenous P
treatments, respectively) than LS seedlings (267 µg, 324 µg and 582 µg P in 0P, LP and HP
exogenous P treatments, respectively) as shown in Table 4.2.
Cumulated degree days after sowing
0 100 200 300 400 500 600
See
dlin
g P
(µg
P)
0
200
400
600
800
1000
1200
0PLSLPLSHPLS
Cumulated degree days after sowing
0 100 200 300 400 500 600
0PHSLPHSHPHS
0
A B
Figure 4.10: Phosphorus accumulation (µg P) in maize seedlings during germination and early growth stages: A. Low endogenous seed P in seedlings (LS) with no exogenous P (○), low exogenous P (□), high exogenous P (∆) and B. high endogenous seed P seedlings HS with no exogenous P (●), low exogenous P (■) high exogenous P (▲) treatments. Data are means and vertical bars indicate ± SE for n = 3 replications.
80
LS HS Significance
0P LP HP 0P LP HP Endo-P Exo-P
Endo-P
xExo-P
Number of visible leaves 5.3a 5.5a 5.2a 4.7a 5a 5a NS NS NS
Seedling dry weight (g) 0.18a 0.21a 0.21a 0.21a 0.23a 0.22a NS NS NS
Seedling P accumulation
(µg P)267a 324a 582b 596b 637b 959c * * NS
Table 4.2: Values of selected variables for maize seedlings at 530 cumulated degree days after sowing with two rates of endogenous P (LS "low seed P " and HS "high seed P") subjected to three rates of exogenous P (Exo-P) availability (OP "control exogenous P", LP "low exogenous P" and HP "high exogenous"). F-test significances of Endo-P (endogenous seed P), Exo-P and Endo-P x Exo-P interaction are indicated (NS = not significant). Different letters in the same line indicate significant differences at the 0.05 probability level.
81
The initial P concentration was 8 mg g-1 seedling DW in LS and HS seedlings (Figure
4.11). Concentrations of P in the seedling decreased from 8 mg g-1 to 1 - 4 mg g-1 seedling DW in
LS and HS seedlings up to 309 cumulated degree days after sowing. P concentrations in the
seedling were higher in HP than in 0P and LP exogenous P treatments despite endogenous P
seed P reserves.
Cumulated degree days after sowing
0 100 200 300 400 500 600
See
dlin
g P
con
cent
ratio
n (m
g P
g-1
)
0
2
4
6
8
10
0PLSLPLSHPLS0PHS LPHS HPHS
Figure 4.11: Seedling phosphorus concentrations (mg P g–1) in maize seedlings during germination and early growth stages. Low endogenous seed P seedlings (LS) with no exogenous P (○), low exogenous P (□), high exogenous P (∆) and high endogenous seed P seedlings (HS) with no exogenous P (●), low exogenous P (■) high exogenous P (▲) treatments, grown for 530 cumulated degree days after sowing. Data are means and vertical bars indicate ± SE for n = 3 replications.
4.2.2.c. Seed phytate-P hydrolysis
Before germination, the LS and HS seeds had 1.8 mg g-1 DW and 2.7 mg g-1 DW phytate-P,
respectively. The phytate-P was mainly localized in the scutellum rather than in the endosperm
(79% w/w in LS scutellum, vs. 91% w/w in HS scutellum) and one reverse case of seed dry
biomass distribution among seed compartments (91% w/w in LS endosperm, vs. 88% w/w in HS
endosperm). Phytate-P in the scutellum and endosperm started to hydrolyze at 16 cumulated
degree days after sowing. In LS and HS seeds, a rapid hydrolysis of phytate-P reserves in
seedling scutellum was observed up to 89 cumulated degree days after sowing (Figure 4.12).
Almost 98% of phytate-P in the scutellum and endosperm was hydrolyzed at 89 cumulated
82
degree days after sowing despite the availability of endogenous or exogenous P. The speed of
hydrolysis of phytate-P in the seed was slightly faster in LS than in HS seeds but was clearly
independent of exogenous P (Figure 4.12, Inset). The hydrolysis of phytate-P in the scutellum
and endosperm was not strongly affected by the availability of endogenous P in the seed during
germination and early growth stages.
Cumulated degree days after sowing
0 100 200 300 400 500 600
Scu
tellu
m P
hyta
te-P
(µg
P)
0
200
400
600
800
0PLSLPLSHPLS0PHS LPHS HPHS
Cumulated degree days after sowing
0 100 200 300 400 500 600
Rel
ativ
e P
hyta
te-P
0.0
0.2
0.4
0.6
0.8
1.0
Figure 4.12: Scutellum phytate-P hydrolysis (µg P) in maize seeds during germination and early growth stages. Inset, Phytate-P in the scutellum (ratio of scutellum phytate-P content to initial scutellum phytate-P content) as a function of cumulated degree days after sowing. Low endogenous seed P seeds with no exogenous P (○), low exogenous P (□), high exogenous P (∆) and high endogenous seed P seeds with no exogenous P (●), low exogenous P (■) high exogenous P (▲) treatments, grown for 530 cumulated degree days after sowing. Data are means and vertical bars indicate ± SE for n = 3 replications.
83
4.2.2.d. Endogenous seed P remobilization and export flux
The scutellum is the main P storing compartment of maize seed (86%; w/w) (before the
endosperm) irrespective of reserves of endogenous seed P (LS or HS seeds). The remobilization
of scutellum P reserves started at 16 cumulated degree days after sowing whereas the endosperm
P reserves started to remobilize later at 34 cumulated degree days after sowing. Remobilization
of endogenous seed P in LS and HS and export fluxes started from 16 cumulated degree days
after sowing in all exogenous P treatments (Figure 4.13). The export flux of endogenous seed P
was similar up to 89 cumulated degree days after sowing despite the availability of endogenous
or exogenous P; subsequent differences were due to initial endogenous seed P stocks.
Phosphorus exported from the seed displayed exactly the same kinetics regardless the
availability of exogenous P (Figure 4.13). The difference between LS and HS seeds reflected the
differences of initial endogenous seed P stocks.
Cumulated degree days after sowing
0 100 200 300 400 500 600
P exported from
the seed (µg P)
0
200
400
600
800
1000
0PLSLPLSHPLS0PHS LPHS HPHS
Figure 4.13: Quantity of endogenous seed P (µg P) exported from the seed to maize seedlings during germination and early growth stages. Low endogenous seed P seedlings with no exogenous P (○), low exogenous P (□), high exogenous P (∆) and high endogenous seed P seedlings with no exogenous P (●), low exogenous P (■) and high exogenous P (▲) grown for 530 cumulated degree days after sowing. Data are means and vertical bars indicate ± SE for n = 3 replications.
84
4.2.2.e. Exogenous P uptake by growing seedling roots
There was no significant uptake of exogenous P in the zero exogenous P treatment (0P), (Figure
4.14). No 32P activity was observed in seedling roots until 52 cumulated degree days after sowing
in either endogenous or exogenous P treatments. At 71 cumulated degree days after sowing,
significant 32P activity was observed in seedling roots in treatments with available exogenous P
despite the availability of endogenous seed P, indicating that uptake of exogenous P was already
underway, and increased thereafter (Figure 4.14). At the last sampling date, uptake of exogenous
P was slightly higher in the HS than in the LS treatment, but the difference was not statistically
significant. The uptake of exogenous seedling P was significantly affected by the availability of
exogenous P, while endogenous P in the seed had no effect on the uptake of exogenous P during
germination and early growth. The seedlings took up significantly more exogenous P in HP than
in LP.
4.2.2.f. P efflux and whole seedling P budget
The whole P budget in growing maize seedlings was calculated considering the P exported from
germinating seeds, exogenous P uptake and seedling P accumulation. In all treatments, the
amount of P that accumulated in the seedling was less than the sum of P exported from the seed
plus the P that was taken up. This result shows that a proportion of P exported from the seed was
not allocated to the growing seedling but was released outside. The P efflux started to increase
from 16 cumulated degree days after sowing with the imbibition of seeds and was higher at 52
cumulated degree days after sowing (Figure 4.15). The P efflux was 127 µg, 98 µg and 109 µg P
seedling-1 in LS seedlings whereas it was 184 µg, 181 µg and 150 µg P seedling-1 in HS seedlings
up to 52 cumulated degree days after sowing in 0P, LP and HP treatments, respectively. At 530
cumulated degree days after sowing, the P efflux was 205 µg, 189 µg and 172 µg P seedling-1 in
LS seedlings and 192 µg, 304 µg and 282 µg P seedling -1 in HS seedlings in 0P, LP and HP
treatments with available exogenous P, respectively as shown in Figure 4.16 and Figure 4.17.
Initial non phytate inorganic P was calculated in seeds based on fresh biomass, and showed that
initial concentrations of non phytate inorganic P in seeds (507 µg mL -1 and 1372 µg mL-1 in LS
and HS seeds, respectively) were much higher than the inorganic P supplied in exogenous P
treatments (3 µg mL-1 and 30 µg mL-1 in LP and HP treatments, respectively). The difference in
the concentrations of internal and external inorganic P in seeds probably resulted in the efflux of
inorganic P from seeds by diffusion to maintain homeostasis of inorganic P between the seeds
and their surroundings.
85
Cumulated degree days after sowing
0 100 200 300 400 500 600
Exo
geno
us P
upt
ake
(µg
P)
0
100
200
300
400
500
0PLSLPLSHPLS0PHS LPHS HPHS
Figure 4.14: Exogenous phosphorus (µg P) uptake in maize seedlings during germination and early growth stages. Low endogenous seed P seedlings with no exogenous P (○), low exogenous P (□), high exogenous P (∆) and high endogenous seed P seedlings with no exogenous P (●), low exogenous P (■) high exogenous P (▲). Data are means and vertical bars indicate ± SE for n = 3 replications.
Cumulated degree days after sowing
0 100 200 300 400 500 600
Cum
ulat
ed P
effl
ux (µ
g P
)
0
100
200
300
400
500
600
0PLSLPLSHPLS
Cumulated degree days after sowing
0 100 200 300 400 500 600
0PHSLPHSHPHS
A B
0
Figure 4.15: Phosphorus efflux (µg P) from germinating maize seeds and growing seedling roots during germination and early growth stages. A) Low endogenous seed P seedlings with no exogenous P (○), low exogenous P (□), high exogenous P (∆) and B) high endogenous seed P seedlings with no exogenous P (●), low exogenous P (■) high exogenous P (▲) treatments. Data are means and vertical bars indicate ± SE for n = 3 replications.
86
Figure 4.16: (A-C: LS treatments) Allocation of exogenous P uptake (gray arrows on the right) calculated using 32P labeling of the nutrient solution and distribution of remobilized seed P reserves (dark arrows on the left) to seedling compartments and calculation of efflux after 530 cumulated degree days. The width of the arrows represents the quantity of P flowing toward different seedling compartments. Values inside the different compartments represent P accumulation in (+) or remobilization from (-) each compartment. P fluxes are expressed in µg P seedling-1. Data are means and vertical bars indicate ±SE for n = 3 replications.
0
+ 304 (±26)
33%
62% 67%
5%
28%
+ 21 (±2)
+ 7 (±1)
+ 1 (±0)
+ 13 (±2)
Exogenous P uptake
P efflux: 189 µg P seedling-1
LP*LS
- 493 (±9)
Remobilised P
+ 86 (±1)
+ 15 (±2)
+ 203 (±2)
Seedling P + 325 (±27)
Seed P + 14 (±3)
Leaves + 216 (±32)
C+M + 16 (±2)
Roots +93 (±2)
Scutellum - 434 (±9)
Endosperm - 58 (±0)
94%
94%
92%
Exogenous P
6%
6%
8%
5%
0
+ 312 (±25)
32%
64% 69%
5%
26%
+ 268 (±8)
+ 85 (±5)
+ 10 (±0)
+ 173 (±3)
Exogenous P uptake
P efflux: 171 µg P seedling-1
HP*LS
- 485 (±16)
Remobilised P
+ 80 (±5)
+ 16 (±1)
+ 216 (±14)
Seedling P + 582 (±29)
Seed P + 22 (±3)
Leaves + 389 (±15)
C+M + 27 (±1)
Roots +166 (±14)
Scutellum - 431 (±10)
Endosperm - 53 (±4)
56%
59%
48%
Exogenous P
44%
37%
52%
4%
+ 267 (±10)
P efflux = 205 µg P seedling-1
OP*LS
- 471 (±16)
Remobilised P
+ 79 (±6)
+ 21 (±8)
+ 167 (±10)
Seedling P + 267 (±10)
Seed P + 35 (±11)
Leaves + 167 (±10)
C+M + 21 (±8)
Roots + 79 (±6)
Scutellum - 417 (±17)
Endosperm - 54 (±1)
63%
8%
29%
A B C
87
Figure 4.17: (A-C: HS treatments). Allocation of exogenous P uptake (gray arrows on the right) calculated using 32P labeling of the nutrient solution and distribution of remobilized seed P reserves (dark arrows on the left) to seedling compartments and calculation of efflux after 530 cumulated degree days. The width of the arrows represents the quantity of P flowing toward different seedling compartments. Values inside the different compartments represent P accumulation in (+) or remobilization from (-) each compartment. P fluxes are expressed in µg P seedling-1. Data are means and vertical bars indicate ±SE for n = 3 replications.
+ 596 (±16)
P efflux: 193 µg P seedling-1
OP*HS
- 788 (±33)
Remobilised P
+ 204 (±19)
+ 36 (±87)
+ 356 (±52)
Seedling P + 596 (±16)
Seed P + 163 (±21)
Leaves + 356 (±52)
C+M + 36 (±7)
Roots +204 (±19)
Scutellum - 749 (±18)
Endosperm - 40 (±38)
60%
6%
34%
0
+ 596 (±63)
47%
51% 53%
6%
41%
+ 41 (±8)
+ 19 (±3)
+ 1 (±0)
+ 21 (±5)
Exogenous P uptake
P efflux: 304 µg P seedling-1
LP*HS
- 899 (±12)
Remobilised P
+ 246 (±22)
+ 38 (±3)
+ 313 (±44)
Seedling P + 637 (±61)
Seed P + 53 (±8)
Leaves + 333 (±44)
C+M + 39(±3)
Roots +265 (±19)
Scutellum - 802 (±11)
Endosperm - 98 (±2)
94%
97%
93%
Exogenous P
6%
3%
7%
2%
0
+ 586 (±5)
45%
52% 52%
9%
39%
+ 373 (±68)
+ 169 (±22)
+ 10 (±1)
+ 195 (±44)
Exogenous P uptake
P efflux: 281 µg P seedling-1
HP*HS
- 867 (±32)
Remobilised P
+ 230 (±13)
+ 50 (±8)
+ 305 (±31)
Seedling P + 959 (±71)
Seed P + 85 (±17)
Leaves + 500 (±76)
C+M + 60(±7)
Roots +399 (±10)
Scutellum - 782 (±29)
Endosperm - 86 (±20)
61%
83%
58%
Exogenous P
39%
17%
42%
3%
A B C
88
4.2.3. Discussion
4.2.3.a. Effect of endogenous and exogenous P on seed P remobilization
and seedling growth
Early seedling growth occurs at the expense of seed reserves and their allocation among different
tissues of the new seedling. As the scutellum is in close contact with the embryo (Fincher and
Stone 1986), scutellum P reserves were remobilized first while endosperm reserves remains
unchanged up to 34 cumulated degree days after sowing in all P treatments. The seed biomass
reserves displayed exactly the same remobilization kinetics irrespective of the availability of
endogenous or exogenous P. In the present study no significant effect of endogenous or
exogenous P was observed on remobilization of dry biomass reserves in the seed or on the
accumulation of dry biomass in the seedling.
Seedling growth was similar in all P treatments. This result is consistent with the fact that
the concentration of P in the seedling was higher than the level (1 mg g -1 DW) at which the
increase in leaf area and in leaf length would be impaired due to P shortage during early growth
stages (Assuero et al. 2004; Plénet et al. 2000a). Although exogenous P was absent in the 0P
treatment, the P supply from remobilization of endogenous seed P satisfied seedling P
requirements during at least 530 cumulated degree days after sowing. Despite similar seedling
growth, HP seedlings accumulated much more P than LP seedlings, clearly demonstrating luxury
P accumulation.
Phytate was found to be a stored form of P in maize seeds and was localized mainly in
the scutellum rather than in the endosperm or embryo as reported by other authors (Lorenz et al.
2007; Lott et al. 1995; Modi and Asanzi 2008; Nadeem et al. 2011; Park et al. 2006). The initial
concentration of endogenous P in the seed only slightly affected the initial distribution of phytate
between the scutellum and the endosperm. The rate of hydrolysis of seed phytate-P was not
affected by the availability of exogenous P and very slightly affected by the amount of
endogenous P (Figure 4.12 inset). This is consistent with previous results which have shown that
hydrolysis of phytate-P depends on the synthesis of phytase and on phytase activity (Laboure et
al. 1993; Wyss et al. 1999), which are controlled by seed soaking, imbibition (Centeno et al.
2001; Egli et al. 2002; Lestienne et al. 2005) and temperature (Sung et al. 2005). Inorganic
phosphate was shown to reduce phytase activity (Guardiola and Sutcliffe 1971) but the absence
of effect of exogenous P concentrations on phytate hydrolysis kinetic in our experiment is
consistent with the fact that no P transfer was observed from external solution to the seed, as
89
confirmed by specific activity measurements (Figure 4.16 and Figure 4.17). Conversely the
temporary accumulations of non phytate P may explain slower kinetic of phytate P hydrolysis in
HS seeds.
The export flux of endogenous P in the seed in LS and HS seeds was similar up to 89
cumulated degree days after sowing irrespective of the availability of endogenous or exogenous
P (Figure 4.13). The difference in the export flux of endogenous P in LS and HS seeds was only
due to the concentration of endogenous P in the seed after 89 cumulated degree days after
sowing. Hall and Hodges (1966) suggested that the breakdown of phytate in germinating seeds
accounts primarily for the increase in inorganic P in the roots and shoot. The hydrolyzed forms
of P were temporarily stored in seeds (Nadeem et al. 2011) and were then translocated towards
growing maize seedlings. The rapid remobilization of endogenous P in the seed during early
growth corresponds to the dramatic increase in the demand for P by the seedling during periods
of rapid cell division, such as seed germination and early growth (Hegeman and Grabau 2001).
4.2.3.b. Effect of endogenous and exogenous P on uptake of exogenous P
Significant P uptake was observed in seedling roots at 71 cumulated degree days after sowing in
treatments with available exogenous P (in LP and HP exogenous P treatments). The seedling
roots started to take up exogenous P soon after the radicle emerged, in accordance with previous
results (Nadeem et al. 2011). The intensity of exogenous P uptake was mainly dependent on the
availability of exogenous P (Bhadoria et al. 2004; Elliott et al. 1984) and on the root surface area
exposed to exogenous P (Anghinoni and Barber 1980). In the 0P treatment, no significant gross
P uptake was detectable using labeling. In this treatment, as the concentration of exogenous P
was lower than the concentration at which net P influx ceases (e.g. Cmin~0.04 – 4 µM), P efflux
from roots may have occurred (Anghinoni and Barber 1980; Bhadoria et al. 2004). Because of
higher concentrations of exogenous P (1000 µM P) in the HP treatment, exogenous P uptake was
higher than in the LP treatment (Figure 4.14). In HP and LP exogenous treatments, similar P
absorption fluxes were observed in LS and HS seedlings, although concentrations of P were
higher in HS seedlings than in LS seedlings (Figure 4.14). There was no negative effect of the
concentration of P in the seedling on the intensity of absorption of exogenous P. This result
supports the conclusion that the demand for P by the seedlings was fully satisfied during
germination and early growth in LP and HP treatments.
90
4.2.3.c. Whole seedling P budget
By comparing accumulated P in the seedling and export of P from the seed plus uptake of
exogenous P, we demonstrated that a significant amount of P was lost via efflux. Losses of P
reached 40%, 37% and 33% in LS seedlings and 20%, 32% and 30% of initial endogenous seed
P in HS seedlings in the 0P, LP and HP exogenous P treatments, respectively. Lestienne et al.
(2005) showed that 21% of initial seed phytate was lost when whole maize seeds were soaked
for 24 hours, which released inorganic P from the seeds. The amount of P efflux depended on
initial concentration of endogenous P in the seed and occurred mainly during the first 52
cumulated degree days after sowing. This result suggests that P efflux from seeds occurs during
imbibition and germination, as seedlings have small root systems. Bewley (1997) suggested that
the influx of water into cells of dry seeds during imbibition results in temporary disturbance of
the structure, particularly that of membranes, leading to immediate and rapid leakage of solutes
and low molecular weight metabolites into the surrounding imbibition solution. The structural
disturbances associated with seed imbibition combined with high release of inorganic P due to
phytate hydrolysis during germination may contribute to P release into the external medium.
As soon as the seedlings started to accumulate biomass after leaf emergence, the P efflux
decreased, suggesting allocation of inorganic P towards seedling organs. P efflux is a component
of net P uptake in whole plants (Elliott et al. 1984) and is at least partially under metabolic
control. P efflux from roots is part of the mechanism whereby plants maintain a P balance
(Bieleski and Ferguson 1983). P releases due to phytate hydrolysis and to the export of inorganic
P from the seed were greater than seedling P requirements, resulting in losses of P from seeds.
Given that ratios of internal to external concentrations of inorganic P ranged from 16 to 457, the
diffusion process may cause loss of endogenous P from seeds. The period of P losses from
germinating seeds and growing seedling roots was similar in LS and HS seedlings despite the
availability of exogenous P, as already reported by Elliott et al. (1984).
Up to 52 cumulated degree days after sowing, remobilization of endogenous seed P
satisfied seedling P requirements in all exogenous P treatments. Similar results were reported by
Barry and Miller (1989) and Nadeem et al. (2011). From 71 cumulated degree days after sowing,
both sources of P (including endogenous and exogenous P) started to concomitantly fulfill
seedling P requirements, whereas only endogenous seed P reserves supported seedling growth in
the treatment with no exogenous P (0P). The seedling P originating from endogenous or
exogenous P sources was largely translocated towards seedling leaves rather than seedling roots
91
as already explained in Nadeem et al. (2011). This high translocation rate suggests high P
requirements by seedling leaves during early growth.
Further research is needed to improve our understanding of the remobilization of P in the
seed and its allocation towards the seedling. Our results suggest that seed P remobilization is
closely related to germination processes independently of both seed P content and external P
availability. Consequently, we need to study how external factors controlling germination (i.e.
water availability and temperature) affect both seed P remobilization and seedling P
requirements. Seedling P requirements during early growth stages also need to be characterized
for better prediction of how long seed P remobilization -including seed P release into the external
medium- is able to sustain the seedling growth.
4.2.3.d. What explains early P deficiency in maize often reported in the
literature?
Early symptoms of P deficiency have often been reported in the literature (Colomb et al.
2000; Grant et al. 2005; Grant et al. 2001; Plénet et al. 2000b; Rodriguez et al. 1998) and may be
due to low soil P and environmental conditions like cool temperatures that slow down enzyme
activity associated with anabolic metabolism, including phytase activity, as reported by Modi
and Asanzi (2008). The study results suggest that the hydrolysis of seed P reserves started soon
after imbibition. The seed phytate hydrolysis and remobilization of seed P reserves was not
influenced significantly by exogenous or endogenous P availabilities during germination and
four weeks of early growth. However a significant loss in P was observed via efflux, mainly
from germinating maize seeds. Moreover, the loss of P was independent of exogenous P
availability. On the whole, these results suggest that although hydrolysis of seed phytate-P is not
a limiting step to providing P to the growing seedling, the rapid hydrolysis of phytate favors P
losses probably by diffusion during germination. This may explain the often reported P
deficiencies in maize seedlings during germination and early stages, especially in low P soils
where root uptake is limited by exogenous P availability. An early increase in root length enables
the seedlings to explore a greater soil volume and to access more exogenous P in the soil (Goss
et al. 1993). Under low soil P availability, the early root growth enabled by hydrolysis of seed P
reserves or increased root growth associated with high seed P content may be insufficient to
overcome the limitation of soil P supply to the roots, especially when there was a significant P
loss by P efflux from germinating seeds.
92
4.3. Modelling of seed P reserves remobilization and exogenous P
uptake
Mathematical modelling has been used as a powerful research tool in many fields of scientific
activities. A model is usually a simplification of the real system designed to be more convenient
to work with. The essential characteristics of the real system should appear in the model. It
should therefore resemble the system and reproduce its dynamic behaviour (Thornley and
Johnson 1990). Modelling has been applied to a wide range of agronomic and plant
physiological processes (Brunel et al. 2009; Escobar-Gutierrez et al. 1998; Mollier et al. 2008;
Thornley and Johnson 1990). During the last four decades, several mathematical models
simulating the P uptake by roots from soil have been developed but to our knowledge, the
modelling approach has not been used in studies of remobilization of seed P reserves and
exogenous P uptake during germination and early growth stages in maize seedlings.
Our objective was to develop a model that represents the growing maize seedling which
acts as a whole system where P can flow between different compartments of the system. During
germination and early growth, the seedling acts as sink whereas seed P reserves and exogenous P
as source for seedling P demand. The whole modelled system is composed on 3 main
compartments namely i) seed P reserves (mainly in the form of phytate-P and non phytate-P)
which hydrolyse during germination, ii) a growing seedling, composed on root and shoot, iii) the
availability of exogenous P in nutrient solution and 2 main forms of P: phytate P and non
phytate-P, which is readily available for seedling use (Figure 4.18). The modelling of seed P
reserves remobilization and exogenous P uptake implies considering and integrating the
processes of seed phytate hydrolysis, P export from seed to growing seedlings as non phytate-P
and the uptake of exogenous P by developing seedling roots during germination and early
growth stages. The main features of this model are: i) the seedling system and ii) the working of
each compartment within this system. The P fluxes between different compartments of system
were expressed in µg P seedling-1 day-1. The objective of this modelling approach is to build a
mechanistic and dynamic model that would realistically describe the hydrolysis of seed phytate
reserves in germinating seeds and the uptake of exogenous P by seedling roots during
germination and early growth stages.
93
Seedling
EndospermPhytate-P
ScutellumPhytate-P
SeedNon phytate-P
P loss
Phytate hydrolysis
Seed
RootDWP
Seedling P
ShootDWP
P remobilization
P uptake
Seedling
EndospermPhytate-P
ScutellumPhytate-P
SeedNon phytate-P
EndospermPhytate-P
ScutellumPhytate-P
SeedNon phytate-P
P loss
Phytate hydrolysisPhytate
hydrolysis
Seed
RootDWP
Seedling P
ShootDWP
P remobilization
P uptakeP uptake
Figure 4.18: Flow diagram of main processes that determine P dynamics in maize seed during germination and early growth. Arrows indicate
the P fluxes between different seed, seedling and exogenous P uptake.
94
The model consists of five modules closely connected. The first one deals with the
hydrolysis of phytate reserves in seed compartments. The second and third modules describe the
seedling growth and seedling P demand considering the root-shoot growth. The fourth module
deals with the availability of exogenous P in nutrient solution and uptake by growing seedling
roots. The fifth module deals with effective P fluxes and total seedling P balance. The five
modules are integrated to simulate seed phytate-P hydrolysis, exogenous P uptake and seedling P
accumulation according to initial seed P reserves and exogenous P availabilities during
germination and 4 weeks of early growth.
The phytate hydrolysis and exogenous P uptake model was developed for the simulation
of seed phytate hydrolysis and exogenous P uptake using the software ModelMaker (Cherwell
Scientific Publishing Limited, Oxford). The software provides programming tools denominated
with the terms: “compartments,” “variables,” “flows,” “influences,” and “list of parameters”.
“Compartments” can be used to simulate changes in quantity of phytate or P and different P flow
“F” in seed and seedlings. The values in the “list of parameters” are available for the
parameterization, which means that they can be automatically varied in a desired range. For full
description of the software and its documentation, see Walker and Crout (1997). The Figure 4.19
shows a description of phytate-P hydrolysis and exogenous P uptake model in germinating maize
seeds.
95
Figure 4.19: Graphical display of the model of P dynamic in maize seed during germination and
early growth build with ModelMaker software.
4.3.1. Seed phytate hydrolysis module
We have shown that in maize seed phytate-P reserves are mainly localized in scutellum (86%)
and remaining in endosperm regardless of initial seed P reserves. The phytate hydrolysis model
was designed to test the hypothesis that there was no effect of exogenous P availabilities on
phytate hydrolysis in scutellum and endosperm. The seed imbibition caused a slow hydrolysis of
phytate followed by a rapid hydrolysis and the phytate-P reserves reached a minimal value close
to zero. A logistic model including an initial “acceleration” of hydrolysis rate was used to
simulate seed P reserves according to initial seed P reserves and a constant hydrolysis rate.
We have considered separately endosperm and scutellum with same equations. The time
course of scutellum and endosperm phytate hydrolysis during germination and early growth was
fitted to a decreasing logistic curve whose differential equations can be written:
96
( )
−××−=
EndokPhytP
PhytPEndoadt
PhytPd EndoEndo
Endo
_1_
Equation 4-6
( )
−××−=
ScutkPhytP
PhytPScutadt
PhytPd ScutScut
Scut
_1_
Equation 4-7
Where Equation 4-6 represents the rate of phytate hydrolysis in endosperm while Equation 4-7
indicates the scutellum phytate hydrolysis rate, respectively. The PhytPEndo and PhytPScut indicate
the instantaneous values of phytate-P in endosperm and scutellum in maize seeds, respectively.
The initial values of phytate stock are different in endosperm and scutellum among LS
and HS seeds. The parameters k_Endo and k_Scut represents the values of initial quantity of
phytate in endosperm and scutellum, respectively in both LS and HS seeds. The parameters
a_Endo and a_Scut are the rate of phytate hydrolysis in endosperm and scutellum, respectively.
The measured data on HPHS seedling treatment were selected to estimate the parameters
a_Endo and a_Scut, respectively (Figure 4.20). The values of parameter (K) for LS and HS seeds
are given in Table 4.3.
0
100
200
300
400
500
600
700
800
0 100 200 300 400 500 600Cumulated degree days after sowing
Phy
tate
-P (µ
g se
ed-1
)
Observed_Scutellum
Observed_Endosperm
Adjusted_Scutellum_HPHS
Adjusted_Endosperm_HPHS
Figure 4.20: Observed and modelled scutellum (●) and endosperm (▲) phytate-P hydrolysis in
scutellum and endosperm of maize seeds (HPHS) during germination and early growth stages
(R² = 0.974).
97
4.3.2. Seedling growth module
Our results showed no significant effect of endogenous or exogenous P treatments on early
seedling growth. As soon as the radicle emerged from germinating seeds, the seedlings start to
gather biomass. The shoot and root biomass showed a linear relationship with growth period in
all the P treatments during this whole growth period of 4 weeks. By considering the linear
growth curves of shoot and root, their respective growth rate can be written as Equation 4-8 and
Equation 4-9 respectively.
ShootDWadt
dDWShoot _=
Equation 4-8
RootDWadt
dDWRoot _=
Equation 4-9
The a_ShootDW and a_RootDW represents the shoot and root growth rate in g day-1. As
no significant effect of P treatments was observed on seedling growth; therefore the parameters
of HPHS seedling treatment were kept constant for all the remaining treatments for simulation
and their values are given in the Table 4.3.
4.3.3. Seedling P demand module
As seedling growth phenomenon progressed, we observed a decrease in seedling P
concentrations. This pattern of decrease in P concentration is similar to that what observed in
case of N by (Justes et al. 1994). We observed that P was not limiting in HPHS treatment,
consequently we used P dilution curve of HPHS treatment to predict the seedling P demand. The
shoot or the root P demand was calculated considering the relationship (exponential) between
shoot or root P concentration and shoot or root dry biomass accumulation as shown in Figure
4.21. The total seedling P demand is the sums of seedling shoot and roots P demands;
Seedling P demand = Shoot P demand + Root P demand
Equation 4-10
The P concentration in crops has been related to the biomass according to the general
equation:
98
bWaWP −×=
Equation 4-11
Where W is the amount dry matter (g) accumulation in shoot or roots and P is the
concentrations (µg g-1) of phosphorus in shoot or roots.
Shoot P concentrations and shoot biomass
y = 3.3134x-0.2383
R2 = 0.8714
0
2
4
6
8
10
12
14
0.00 0.02 0.04 0.06 0.08 0.10
Shoot biomass (g seedling-1)
Sho
ot P
con
(mg
g-1)
Figure 4.21: Observed leaf P concentrations (●) in HPHS seedlings and dilution P curve
obtained by non-linear fitting in HPHS seedlings during germination and early growth stages.
The dilution law expressed in Equation 4-11 corresponds to the existence of an allometric
relation between accumulated P and accumulated dry matter in shoots or roots, accounting for
the following equation:( )bWaP −×= 1
Equation 4-12
Where a indicates the amount of accumulated P in seedling shoot or roots while (1-b) shows the
allometric ratio between P accumulated and dry matter accumulation in shoot or roots. Since the
derivation of Equation 4-12 yields:
( )dt
dWWbadtdP b−−= 1
Equation 4-13
99
Where dtdP
represents the P accumulation rate in seedling shoot or roots, while dt
dWshows the
growth rate of seedling shoot or roots. The values of parameter a and b are giving in Table 4.3
and we used the parameters of HPHS for all other treatments for model simulation.
4.3.4. Exogenous P uptake by roots module
The results showed that seedlings start uptake of exogenous P soon after the radicle emergence
and we observed that seedling root growth was a linear function of the root length (cm) when
drawn against root dry weight (g). Using the equation as mentioned by Claassen and Barber
(1977), the P influx can be written as:
−
−−
=12
1
2
12luxinf RLRL
RLRL
Ln
1t2tUU
P
Equation 4-14
Where U is the amount of P uptake by seedling roots at time t, whereas RL is the seedling root
length and subscripts refer to the time 1 and 2. By using the above equation we can write:
RLbrateuptake eaRL
dtdPP ×−××==
Equation 4-15
The Equation 4-15 represents the P uptake rate, where a and b refers to the coefficient of
relationship (exponential) between P influx and root length as shown in Figure 4.22 for HPLS
and HPHS seedlings. The values of the coefficients for LP and HP seedlings in available
exogenous P treatments are given in Table 4.3.
100
P influx and seedling root length
y = 0.160365e-0.002089x
R2 = 0.802709
y = 0.1744e-0.0044x
R2 = 0.4493
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0 100 200 300 400 500 600 700
Root length (cm seedling-1)
P in
flux
(µg
P c
m-1
day
-1)
HPHSHPLSExponentiel (HPHS)Exponentiel (HPLS)
Figure 4.22: Relationship between seedling root P influx and seedling root lengths (HPLS and
HPHS seedlings) in maize seedlings. HPHS seedlings (▲) and HPLS seedlings (∆) and non
linear fitting curve (—) of HPHS and HPLS (– –).
4.3.5. Effective P fluxes and total P balance module
During germination and early growth, the seedling P demand could be satisfied by
remobilization of seed non phytate-P reserves and exogenous P uptake. We considered the two
cases where if the seedling P demand is less than the seedling exogenous P uptake and
remobilization of seed non phytate-P reserves than seed P export will be equal to:
Seed P export = Seedling P demand – exogenous P uptake
Equation 4-16
In the second case if the seedling P demand is higher than the seedling exogenous P
uptake and remobilization of non phytate-P reserves in seed, than
Seedling accumulation = P uptake + available non phytate-P from seed
Equation 4-17
The total seedling P budget was calculated in all P treatments considering the available
non phytate-P reserves in seeds, the exogenous P uptake and P loss from germinating seeds, as
already shown in previous chapter. A significant quantity of P was lost from germinating seeds
101
during early growth stages. The Equation 4-18 shows the quantity of P loss from germinating
seeds, where Emax is the maximum quantity of P loss from the seed at time t, while Km is the
time when the Emax value is ½. The values of Emax and Km for LS and HS seeds are giving in
Table 4.3.
+
×=tk
tEPlossm
max
Equation 4-18
102
Table 4.3: Different variables and parameters of maize seed phytate-P hydrolysis and exogenous P uptake during germination and early growth stages.
Symbol Variable or parameter with units Value SourceLS
seedlingsHS
seedlingsa_ShootDWT_ini_ShootDW
a_RootDWT_ini_RootDW
Shoot growth rate (g day-1)Time when shoot start to gather biomass (days)Root growth rate (g day-1)Time when roots start to gather biomass (days)
0.00341.853
0.00411.8048
FittedFittedFitted
Fitted
Ini_non_PhytEndo
PhytPEndo_ini
Ini_non_PhytScut
PhytPScut_ini
Initial non-phytate P in endosperm (µg seedling-1)Initial phytate P in endosperm (µg seedling-1)Initial non-phytate P in scutellum (µg seedling-1)Initial phytate P in scutellum (µg seedling-1)
0
153
24
413
0
167
126
693
Observed
Observed
Observed
Observed
a_Shoot_P
b_Shoot
a_Root_P
b_Root
Amount of P accumulated in shoot (µg seedling-1)Ratio between P and DW accumulation in shootAmount of P accumulated in root (µg seedling-1)Ratio between P and DW accumulation in root
3.3134
0.2383
1.65
0.3248
Fitted
FittedFitted
Fitted
a_Endo
K_Endoa_ScutK_Scut
Rate of phytate P hydrolysis in endosperm (µg day-1)Maximum phytate P in endosperm (µg seedling-1)Rate of phytate P hydrolysis in scutellum (µg day-1)Maximum phytate P in scutellum (µg seedling-1)
168
543
0.859
174
0.851
732
Fitted
Fitted
Fitted
Fitted
Emaxkm
Maximum export P from seed (µg seed-1)Time to reach Emax at ½ value
193
2.51
321
2.58
Fitted
FittedLP
TreatmentHP
Treatment
a_Uptb_Upt
Coefficients of relationship b/w P influx and root length
0.01204-0.00225
0.1557-0.0027
FittedFitted
103
4.4. Model output
4.4.1. Seed phytate hydrolysis
The time courses of predicted seed phytate-P hydrolysis in LS and HS seed during germination
and early growth are shown in Figure 4.23. As we observed no significant effect of exogenous P
availabilities (0P, LP and HP) on seed phytate hydrolysis, therefore we applied the parameters of
phytate-P hydrolysis rate (a_Endo and a_Scut) of HPHS seeds to all the remaining treatments for
simulation of observed values of phytate-P at each seedling harvest, whereas the maximum
initial values (K) were of respective seed P reserves for LS and HS seeds. The model provides a
good simulation prediction of seed phytate-P hydrolysis in scutellum and endosperm in both LS
and HS seeds, although the initial seed phytate-P reserves vary between LS and HS seeds. For
each LS and HS seeds, the fitted values of initial phytate-P stock (K_Endo and K_Scut) are giving in
Table 4.3.
0
100
200
300
400
500
600
700
800
900
1000
0 100 200 300 400 500 600
Cumulated degree days after sowing
Phy
tate
-P (µ
g se
ed-1
)
0PLS
LPLS
HPLS
OPHS
LPHS
HPHS
Figure 4.23: Time courses of modelled phytate-P hydrolysis (µg seed-1) in LS and HS seeds
treated with three exogenous P availabilities (0P, LP and HP) during germination and early
growth stages.
104
4.4.2. Seed non phytate-P
With the imbibition of maize seeds, the stored phytate-P starts to hydrolyse. The results of
experiments 1 and 2 showed that the hydrolyzed forms of phytate-P are temporarily stored in
seeds before translocation towards growing seedlings during germination and early growth
stages. The Figure 4.24 shows the modelled changes in non phytate-P in seeds treated with three
exogenous P availabilities (0P, LP, HP) during germination and early growth stages.
0
50
100
150
200
250
300
350
400
450
500
0 100 200 300 400 500 600
Cumulated degree days after sowing
Non
phy
tate
-P (µ
g P
see
d-1)
0PLS
LPLS
HPLS
0PHS
LPHS
HPHS
Figure 4.24: Modelled changes in non phytate-P (µg seed-1) in LS and HS maize seeds during
germination and early growth stages.
Before germination, there was a low quantity of non phytate-P in both LS and HS seeds.
The quantity of non phytate-P starts to increase as soon as the phytate starts to hydrolyse during
germination process. In experiment 2, we observed that phytate-P in the scutellum and
endosperm started to hydrolyze at 16 cumulated degree days after sowing. In LS and HS seeds, a
sharp increase in hydrolysis of phytate-P reserves in seedling scutellum was observed up to 89
cumulated degree days after sowing (Figure 4.12). These results are consistent with that
maximum phytate hydrolysis was observed on 89 cumulated degree days after sowing in
germinating seeds regardless of exogenous P treatments (as shown in Figure 4.12). The modelled
non phytate-P of HS seeds is almost double than the LS seeds, which is consistent with the
results that initial seed P reserves are almost double in HS seeds as compared to LS seeds. From
89 cumulated degree days after sowing, the quantity of non phytate-P seeds starts to decrease in
seeds, corresponding to the translocation towards newly growing seedlings. The predicted non
105
phytate-P in HPHS seeds showed that seed non phytate-P was over predicted as compared to the
other treatments. The non phytate-P was translocated towards newly growing seedling earlier in
LS seeds as compared to HS seeds due the initial difference in seed P reserves (Figure 4.24).
Overall the model predicts well both the initial increase in non phytate-P quantity in both LS and
HS seeds during germination and early growth stages of maize and the decrease in non phytate-P
in seed when the seedling P demand increased.
4.4.3. Seedling P demand and seedling P accumulation
The Figure 4.25 (A and B) shows the simulated seedling P demand expressed as daily amount of
P required to fulfil seedling P requirement and the effective seedling P accumulation rate in
maize seedlings during early growth stages for LS and HS treatments. The seedling P demand
was similar in all LS and HS seedlings treated with different exogenous P availabilities. The
seedling P demand was fulfilled in all treatments till 199 cumulated degree days after sowing in
LS seedlings (Figure 4.25 A). As soon as the seed P reserves were exhausted after 199
cumulated degree days after sowing, the seedling P accumulation was observed only in HP
treatment where 78% of the effective P accumulation was observed from exogenous P, while a
very small fraction (8%) in LP treatment. Being rich in endogenous seed P reserves, HS seedling
P demand was overlapped by the effective seedling P accumulation rate till 382 cumulated
degree days after sowing in all exogenous P treatments (Figure 4.25 B) and afterward its only
exogenous P from which the seedling accumulate P to fulfil their demand.
0
20
40
60
80
100
120
0 100 200 300 400 500 600
Cumulated degree days after sowing
See
dlin
g P
dem
and
and
effe
ctiv
e se
edlin
g
P a
ccum
ulat
ion
rate
(µg
day-1
)
Seedling P accum_0PLS
Seedling P demand_0PLSSeedling P accum_LPLS
Seedling P demand_LPLSSeedling P accum_HPLS
Seedling P demand_HPLS
A
0
20
40
60
80
100
120
0 100 200 300 400 500 600
Cumulated degree days after sowing
See
dlin
g P
dem
and
and
effe
ctiv
e se
edlin
g
P a
ccum
ulat
ion
rate
(µg
day-1
) Seedling P accum_0PHSSeedling P demand_0PHSSeedling P accum_LPHSSeedling P demand_LPHSSeedling P accum_HPHSSeedling P demand_HPHS
B
Figure 4.25: Seedling P demand and effective seedling P accumulation rate (µg day-1) in
growing maize seedlings during germination and early growth stages. A. Seedling P demand and
seedling P accumulation in LS seedlings treated with three exogenous P availabilities (0P, LP
HP), Right. HS seedlings treated with three exogenous P availabilities (0P, LP HP).
106
Consequently, the model predicted that seedling P concentration was firstly reduced in 0PLS and
LPLS and later for 0PHS and LPHS as compared to HP treatments (Figure 4.26). The modelled
and observed seedling P concentrations on 530 cumulated degree days after sowing in growing
maize seedlings treated with three exogenous P treatments, was shown in Figure 4.27. We
observed high seedling P concentrations in HP treatments as compared to 0P or LP in both LS
and HS seedlings. The model over predicted the seedling P concentrations in LS seedlings
whereas in HS seedlings, it seems very close to the observed values.
0
4
8
12
16
20
0 100 200 300 400 500 600
Cumulated degree days after sowing
See
dlin
g P
con
cent
ratio
n (µ
g g-1
) 0PLSLPLSHPLS0PHSLPHSHPHS
Figure 4.26: Time courses of modelled seedling P concentrations (µg g-1 DW) in LS and HS
seedlings treated with three exogenous P treatments (0P, LP, HP) during germination and early
growth stages.
0.0
0.5
1.01.5
2.0
2.5
3.0
3.54.0
4.5
5.0
0P LP HP 0P LP HP
LS LS LS HS HS HS
See
dlin
g P
con
cent
ratio
n (m
g g-1
)
Observed_seedling_Pc
Modelled_seedling_Pc
Figure 4.27: Observed and modelled seedling P concentrations (mg g-1 DW) in growing maize
seedlings on 530 cumulated degree days after sowing.
107
4.4.4. Seed P export and root P uptake
The modelled seedling P originated from endogenous seed P reserve remobilization and
exogenous P uptake during germination and early growth stages is shown in Figure 4.28 (A-D).
The seed P reserves remobilization remains a major source of seedling P in both LS and HS
seedlings regardless of exogenous P availabilities. During the early five days after sowing, the
remobilization flux has much importance as seedlings are fully dependent on seed P reserves in
all P treatments.
0
100
200
300
400
500
600
700
800
900
1000
0 100 200 300 400 500 600
Cumulated degree days after sowing
P (µ
g se
edlin
g-1)
Seedling_P_HPHS
Uptake_P_HPHS
Seed_P_HPHS
A
0
100
200
300
400
500
600
700
800
900
1000
0 100 200 300 400 500 600
Cumulated degree days after sowing
P (µ
g se
edlin
g-1)
Seedling_P_LPHS
Uptake_P_LPHS
Seed_P_LPHS
B
0
100
200
300
400
500
600
700
800
900
1000
0 100 200 300 400 500 600
Cumulated degree days after sowing
P (µ
g se
edlin
g-1)
Seedling_P_HPLS
Uptake_P_HPLS
Seed_P_HPLS
C
0
100
200
300
400
500
600
700
800
900
1000
0 100 200 300 400 500 600
Cumulated degree days after sowing
P (µ
g se
edlin
g-1)
Seedling_P_LPLS
Uptake P
Seed_P_LPLS
D
Figure 4.28: Modelled seedling P accumulation (µg seedling-1) originated from endogenous seed
P reserves and exogenous P uptake in growing maize seedlings during germination and early
growth stages. HS seedling treated with HP and LP exogenous P availabilities (A and B), LS
seedlings treated with HP and LP exogenous P availabilities (C and D).
The Figure 4.29 shows a maximum seed P export in both LS and HS seeds on 34
cumulated degree days after sowing and decrease thereafter regardless of exogenous P
treatments. The effective ratio of seedling P accumulation and seedling P demand shows that
seedling P demand was fully satisfied till 199 cumulated degree days after sowing in 0PLS and
108
LPLS seedlings whereas till 309 cumulated degree days after sowing in HPLS seedlings. After
this period it is the exogenous P which fulfils the seedling P requirements in available P
treatments.
0
20
40
60
80
100
120
0 100 200 300 400 500 600
Cumulated degree days after sowing
See
d P
exp
ort r
ate
(µg
seed
-1 d
ay-1
) 0PHS
LPHS
HPHS
0PLS
LPLS
HPLS
Figure 4.29: Time courses of seed P export rate (µg seed-1 day-1) in LS (dashed lines) and HS
(solid lines) seeds treated with three exogenous P treatments (0P, LP, HP) during germination
and early growth stages.
The Figure 4.30 (A) shows the P uptake in LS seedlings treated with LP and HP exogenous P
treatments. The model over predicts the exogenous P uptake than the observed values in LP and
HP exogenous P treatments, while the Figure 4.30 (B) shows that the model predicts accurately
the exogenous P uptake in HS seedlings.
0
100
200
300
400
500
0 100 200 300 400 500 600
Cumulated degree days after sowing
P u
ptak
e (µ
g P
see
dlin
g-1)
Mod_LPLS Obs_LPLS
Mod_HPLS Obs_HPLS
A
0
100
200
300
400
500
0 100 200 300 400 500 600
Cumulated degree days after sowing
P u
ptak
e (µ
g P
see
dlin
g-1)
Mod_LPHS Obs_LPHS
Mod_HPHS Obs_HPHS
B
Figure 4.30: Time courses of observed (Obs) and modelled (Mod) exogenous P uptake in LS
(A) and HS (B) seedlings treated with two exogenous P availabilities during germination and
early growth stages in maize.
109
4.4.5. Seed P efflux
The modelled seed P efflux in LS and HS seeds treated with three exogenous P availabilities (0P,
LP and H) was shown in Figure 4.31. The model predicts well the seed P efflux during
germination and early growth of maize (Figure 4.15). The seed P efflux start to increase from
very first day after sowing of seeds. From 89 cumulated degree days after sowing the seed P
efflux remains almost similar, as the seed P was being exported towards seedlings and the
quantity of P remaining in seeds decreased. The more seed P efflux was observed in HS seeds as
compared to LS seeds during germination.
0
50
100
150
200
250
300
350
0 100 200 300 400 500 600
Cumulated degree days after sowing
See
d P
effl
ux (µ
g se
ed-1
)
0PLS 0PHSLPLS LPHSHPLS HPHS
Figure 4.31: Time courses of modelled seed P efflux (µg seed-1) in LS (dashed lines) and HS
(solid lines) seed treated with three exogenous P treatments (0P, LP, HP) during germination and
early growth stages.
110
4.5. Discussion
The modelling is a useful approach to understand the actual processes. The phytate hydrolysis
and exogenous P uptake model gives an overall understanding of seed P reserves hydrolysis and
exogenous P uptake during germination and early growth stages in maize seedlings. The phytate
reserves in seed go under hydrolysis with the imbibition. The maximum phytate hydrolysis was
observed during the first week of maize seedling growth. This hydrolysis caused an increase in
non phytate-P in seeds. Similar results were reported by Centeno et al. (2001) in rye and barley.
He showed that the phytate (IP6) hydrolysis caused an increase of IP5, IP4 and IP3 intermediate
products during germination which ultimately produce phosphate for newly growing seedlings.
The rapid increase in non phytate-P quantity in seeds during the first week caused the P loss by
efflux from seeds. This happens due to the low seedling biomass development with less P need
in first week and the non phytate-P lost by efflux. As soon as the seedlings developed their shoot
and root compartments the P efflux starts to reduce after first week as observed and predicted in
the simulation model. The model gives a better understanding of increase in the quantity of non
phytate-P and P efflux in both LS and HS seeds.
The seedling P demand in LS and HS seedlings was fulfilled only by seed P reserves
remobilization in 0P exogenous P treatments, whereas the exogenous P supports the seedlings to
fulfil their P demand in available exogenous P treatments (LP, HP), although the model over
predict the exogenous P uptake in LS seedlings. The developed model can predict the seedling P
demand, seed P reserves remobilization and exogenous P uptake in germinating maize seedlings
during early growth stages.
The present model predicts the dynamics of seed P reserves and seedling P considering the
hypothesis that there was no interaction between phytate P hydrolysis kinetic and P uptake by
roots. However, we could not simulate the P efflux from germinating maize seeds. We used the
Michaelis Menten equation function to predict the P efflux on a mechanistic way. A further step
would be to couple the proposed model with model of seedling germination and early growth
considering the environmental variables (Temperature, humidity, radiation etc).
111
112
5. GENERAL DISCUSSION AND PROSPECTIVES
Phosphorus is an integral part of living cells and involved in all energy requiring processes like
cell division, energy transfer and photosynthesis. Therefore, P nutrition has a critical importance
in all growing crops during germination and early growth stages. Many previous studies have
shown an early P limitation on early maize crop growth and final yield. Keeping in view the
importance of early maize growth stages, we studied the remobilization of seed P reserves and
exogenous P uptake and their effect on each other during early seedling growth.
We studied two main compartments of maize seed namely endosperm and scutellum. The
maize endosperm is a starchy structure and study results have shown that endosperm contains
almost 87% of C and 77% of N of total seed C and N reserves, respectively. The scutellum is
relatively a small seed compartment; it enclosed the embryo and located on dorsal side of the
endosperm. We have shown that although scutellum is the small part of seed (containing only
13% of seed biomass), it contains almost 86% of total seed phosphorus reserves. The major part
of the maize seed P reserves either in endosperm or scutellum, is in the form of phytate. Phytate
is a complex salt of myo-inositol hexakisphosphate containing six phosphate molecules along
with minerals like calcium, magnesium and zinc etc. The results have shown that the initial seed
P status have no significant effect on the partitioning of phytate among seed scutellum and
endosperm.
During germination, as soon as the seeds are imbibed, the scutellum and endosperm
phytate reserves were started to hydrolyze by the activity of phytase. The phytate hydrolysis
provides inorganic P for the growing seedlings. The study results have shown that the hydrolysis
of phytate was almost not influenced by endogenous seed P reserves and not influenced by
exogenous P availabilities during germination and early growth stages. The remobilization of
seed P reserves displayed similar remobilization kinetics as that of C and N during germination
and early seedling growth, although they are localized in different compartments of maize seeds.
During the first two days of early maize growth, it is the scutellum P reserves which fulfil the
seedling P requirements, being in close contact with embryo. The results of both experiments
have shown that seed phytate hydrolysis was not a limiting step in seedling P requirements.
113
As soon as the seedling radicle was emerged on 2nd day after germination, the seedling P
accumulation was begun. The endogenous seed P reserves or exogenous P availabilities have no
significant effect on seedling radicle emergence. Four day after germination, the maize seedling
roots are able to uptake the exogenous P as shown in both experimental studies. The intensity of
exogenous P uptake by growing seedling roots was mainly determined by the availability of
exogenous P, while it did not depend upon seedling P status or initial endogenous seed P
reserves. Both processes, the exogenous P uptake and endogenous seed P remobilization seem to
be controlled independently during maize germination and early growth stages. A major part of
seedling P, originated from endogenous or exogenous P sources was translocated towards
seedling leaves rather than seedling roots. This suggests a very high seedling leaf P demand
during four weeks of early growth rather than seedling roots. Similarly to C, we can clearly
define the three different stages of P acquisition by newly growing maize seedlings depending
upon the endogenous seed P and exogenous P availabilities. The four day old maize seedlings
are completely made up of endogenous seed P defining it as heterotrophic stages for P
acquisition. Both sources of P including endogenous and exogenous P, provides P to the growing
seedling from 4th to 15th days after germination (transitional stage for P), and from 15 th day after
sowing its only exogenous P which fulfils the seedling P requirements (autotrophic stage for P).
Modelling seed Preserves remobilization and exogenous P uptake under the assumption that P
phytate P hydrolysis and root P uptake are independent process correctly reproduce experimental
data.
From the agronomical point of view, the rapid hydrolysis of phytate in maize seeds
during the first three four days after germination was shown to favour the inorganic P increase in
seed, whereas the four day old seedlings have not much vegetative part during this growth
period. On the whole, these results suggest that although hydrolysis of seed phytate is not a
limiting step for providing P to the growing seedlings, but this rapid hydrolysis may favours the
P losses probably by diffusion during germination. The P losses by efflux were independent of
exogenous P availabilities; therefore the idea of increasing endogenous seed P reserves for better
seedling establishment seems not to be very effective, whereas the study results have shown that
availability of exogenous P in the root zone is very important during germination and early
growth stages. These P losses from germinating maize seeds may explain the early seedling P
deficiencies during germination and early growth.
114
Further steps would be to identify external factors (temperature, water content …) which
are likely to affect seed P reserves remobilization during germination and early growth, to
include them in the proposed model and to test this model on independent data set. Because of it
unexpected importance, the mechanism underlying P efflux from the seed and the factors
controlling it also need further studies. We also suggest to test the main findings of this work and
the proposed model on other species. Eventually the proposed model might be included in a
general P uptake model (FUSSIM-P-Maize) to better understand the relationships between early
P nutrition, crop growth and yield.
115
116
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APPENDIX
Appendix I: Prélèvement et conservation de grains de maïs avec des teneurs en phosphore différenciées
UMR-TCEM
PROTOCOLE EXPERIMENTAL Réf : PX032Version : 1date : 15 octobre 2008PRÉLÈVEMENT ET CONSERVATION DE GRAINS DE MAIS AVEC DES
TENEURS EN P DIFFÉRENTIEES
Objet et domaine d’application
Récolte d'épi de maïs avec un gradient de teneur en phosphore afin de faire une étude préalable
sur les méthodes de dosage des différentes formes de P contenu dans les graines de maïs et sur la
mise au point d'un dispositif expérimental permettant le suivi de la remobilisation du P de la
graine lors de la germination (Sujet de thèse de Nadeem Muhammad 2008-2011).
Liste de diffusion et si nécessaire niveau de confidentialité
Diffusion interne INRA
Hygiène et sécurité
Précautions habituelles en expérimentation au champ (vêtements sécurité, travail en binôme si
travail en hauteur, etc …)
Principe de la méthode
Récolte d'épis sur les différentes parcelles de l'essai longue durée P au champ sur le site de
Pierroton. Les parcelles sont sélectionnées en fonction des teneurs en P des grains des épis de
façon à disposer d'une gamme de teneur en P. Les semences utilisées en 2005 avaient une teneur
en P moyenne de 3.2 mg P /g. La gamme des teneurs en P des grains produits sur le dispositif
s'étend de 1.3 à 3.2 mg P / g. Pour cette étude, nous souhaitons disposer de trois niveaux de P
dans les graines couvrant cette gamme. A partir des analyses les plus récentes disponibles (2004)
les parcelles suivantes ont été sélectionnées (voir localisation sur le plan ci-dessous):
Low P Intermediate P High PP0.5 Bloc 3 P1 Bloc 1 P4 B11.34 mg P/g 2.2 mg P/g 2.67 mg P/g
Diffusion : Contrôlée Interne TCEMRédacteur : Vérificateur Approbation : Nom : Mollier A Nom : Denoroy P. Nom : PELLERIN S Visa :Fonction : CR Fonction : ingénieur Fonction : Directeur Le : 5/01/09
125
Prélever 30 épis par parcelle de façon aléatoire au moment de la récolte (20 octobre 2008) en
dehors des rangs destinés à l'estimation du rendement des parcelles.
Conserver dans une poche en indiquant le nom de la parcelle
Identifier 5 épis par traitement et les peser. Ces épis seront utilisés pour suivre l'évolution du
séchage des épis par des pesées bihebdomadaires.
Enlever les spathes et écarter les épis montrant des attaques d'insectes ou de champignons.
Mettre les épis à sécher à l'air libre jusqu'à un poids constant. Conserver les épis à l'abri de
l'humidité et des ravageurs sans mélanger les traitements (ne pas égrainer les épis).
cases
PLAN ESSAI P PIERROTON lysi-métriques
QuantitéForage P kg/ha
En 2003 P0.5 11.05 cabaneP0.75 16.58
P1 22.10P2 44.20P4 88.40 Nord
piste
Distances 67.20 m
54.40 m6.60 m 12.80 25.60 38.40 51.20 22.50 m
75 m 38 m
P0.75 P4 P1 P0.5 P2
BLOC 4 18 m
1 m allée56 m 19 m
P1 P0.5 P4 P2 P0.75
BLOC 3 18 m
FOS
1 m allée 0 m S37 m E
G PU P2 P1 P0.75 P4 a P0.5 II s ND BLOC 2 s 18 m RA a AG gE e
R 1 m alléeA r 18 mM oP P4 P2 P0.5 P0.75 u P1E BLOC 1 e
18 m
borne 6.60 m12.80 m 12.80 m 12.80 m 12.80 m 3.20 12.80 m 22.91 m
8 rangs 16 rangs 16 rangs 16 rangs 16 rangs 4 r. 16 rangs 28 rangs
18 m surface 1 parcelle = 230.40 m²surface de l'essai = 4608 m²
bornepiste
BOIS de PINS
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Appendix II: Dosage des phytates par chromatographie ionique en milieu HCl 0.65M. Mode opératoire MO-ANA-65
TCEM
MODE OPÉRATOIRE Réf : MO-ANA-065
Version : 0
Date : 30/11/2010DOSAGE DES PHYTATES PAR CHROMATOGRAPHIE IONIQUE EN MILIEU
HCL 0.65M
Objet et domaine d’application
Le présent mode opératoire a pour but de décrire le processus de dosage des phytates (myo-
inositol 1-, 2-, 3-, 4-, 5-, 6-hexakisphosphate) par la technique de séparation des pics en
chromatographie ionique. Ce dosage est réalisé sur des compartiments de grains de mais après
une extraction par le réactif de Wade (modification de la couleur du complexe Fe- acide
sulfosalicylique en présence de phytates).
Hygiène et sécurité
Pas de mesures particulières
Principe de la méthode
La mesure des phytates est déterminée par chromatographie ionique (IC). Le principe est basé
sur un échange d’ions sur une colonne composée d’une résine chargée soit positivement (pour
séparer des anions), soit négativement (pour séparer des cations). Les ions sont entrainés par une
phase mobile (éluent) et séparés par l’action de la phase stationnaire (colonne). La détection est
réalisée par une cellule de conductivité.
Figure 1. Principe de la chromatographie ionique
Diffusion : Contrôlée Interne TCEMRédacteur Vérificateur Approbation : Nom : VIVES.A Nom : MOLLIER.A – MOREL.C Nom : PELLERIN. S Visa :Fonction: Technicien Fonction : Chercheurs Fonction : Directeur Le :17/12/2010
127
Matériels nécessaires
- Chromatographie ionique Dionex modèle ICS 2000
- Passeur d’échantillon Dionex modèle AS50
- Système d’eau ultra pure Elga
- Vial 2 ml de type 2-SV
Réactifs (chimiques et biologiques)
- Cartouche d’éluent d’hydroxyde de potassium EGC III KOH P/N 074532
- HCl : préparer 200 ml HCl à 0.65 N (2.4%) soit 10,8 ml dans 200 ml
- Acide phytique dodecasodium salt (PADDSS ; Sigma P-8810)
- Eau ultra-pure
Contraintes de la méthode
- Pas de contraintes particulières
Contenu du mode opératoire
Préparation des réactifs
Solution Standard de Phytates (maïs):
- Dissoudre 100 mg de PADDSS (Sigma P-8810) dans 10 ml d’HCL 0.65N.
- Préparer la gamme de standard Phytates d’après le tableau ci-dessous.
- Disposer ces solutions dans des vials de 2 ml de type 2-SV
Dosage en chromatographie ionique.
L'échantillon à analyser (volume de 20 µL) est injecté, par l’intermédiaire du passeur
d’échantillon AS50, en tête de la pré-colonne AG11- 4mm P/N 044078 dont le rôle est de
préserver la colonne de séparation de tout contaminant provenant des échantillons. La migration
des espèces se fait selon leur affinité (capacité de l'ion à être plus ou moins retenu) pour la résine.
PhytatesStandard HCl 0.65N Stock (10 mg / ml) Concentration.
―――μl――― μg ml
1 1000 0 02 980 20 2003 960 40 4004 920 80 8005 900 100 10006 800 200 2000
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La migration est assurée par l'éluant (EGC III KOH P/N 074532) injecté par la pompe (à un débit
variant de 0.8 ml/min).
Figure 2. Injection de l’échantillon
Création du gradient d’éluent: démarrer à moins trois minutes à 12 mM de KOH pour arriver à
90 mM au bout de quarante cinq minutes.
Ensuite, stabiliser à 12 mM pendant deux minutes avant de passer à l’échantillon suivant :
Figure 3. Analyse de l’échantillon
La séparation est effectuée sur la colonne échangeuse d’ions AS11- 4 mm P/N 044076. Pour ce
dosage, la température de la colonne doit être de 30°C. Pour augmenter la sensibilité et abaisser
le seuil de détection des éléments, un système de suppression est utilisé. Son rôle est de
supprimer la conductivité de l'éluant, afin que le pic de conductivité de l'élément à analyser soit
le plus net possible. Le suppresseur utilisé est le modèle Dionex ASRS 300 – 4mm P/N 064554
avec une intensité réglée à 179 mA.
La détection est assurée par la cellule de conductivité DS6 P/N 057985 à une température de
30°C.
Pour le dosage des phytates en milieu HCl 0.65N, le seuil de détection est de 0.116 mg/L et le
seuil de quantification 0.262 mg/L
129
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 47.0-5.0
0.0
5.0
10.0
15.0
20.0
25.0 1Phytate_08_06_10 #5 [modified by User] ST 1000 ECD_1µS
min
1 - 0.4842 - 2.0873 - 2.2444 - 2.5975 - 2.7446 - 3.4607 - 3.894
8 - 6.777
9 - 8.534 10 - 13.28711 - 13.650 12 - 20.66413 - 23.09014 - 23.69015 - 24.264
16 - Phytates - 26.710
17 - 32.19418 - 35.28019 - 37.744
Figure 4. Séparation des pics des phytates avec la colonne AS-11
130
Programme phytates chroméléon
Sampler.AcquireExclusiveAccessPressure.LowerLimit = 200 [psi]Pressure.UpperLimit = 3000 [psi]%A.Equate = « KOH »CR_TC= OnFlush Volume = 100Wait FlushStateNeedleHeight 1 [mm]CutSegmentVolume 10 [µl]SyringeSpeed = 4CycleTime = 0 [mn]WaitForTemperature= 30.0 [°C]Pump-ECD.Data_Collection_Rate 5.0 [hz]CellTemperature.Nominale= 30 [°C]ColumnTemperature.Nominale= 30 [°C]Suppressor_Type= ASRS_4mm; Pump_ECD.Carbonate = 0.0; Pump_ECD.Bicarbonate = 0.0; Pump_ECD.Hydroxyde = 90.0; Pump_ECD.Tetraborate = 0.0; Pump_ECD.Other eluent = 0.0; Pump_ECD.Recommanded Current 179Suppressor_Current 179 [mA]ECD_Total.Step = 0.20 [s]ECD_Total.Average off
Wait SampleReadyFlow 0.80 [ml/mn]
-3.000 Concentration 12.00 [mM]Curve 50.000 Pump_ECD.AutozeoConcentration 12.00 [mM]Curve 5LoadWait CycleTimeStateInjectWait InjectStateECD_1.AcqOnECD_Total.AcqOnSampler.ReleaseExclusive.AccessConcentration 12.00 [mM]Curve 545.000 Concentration 90.00 [mM]Curve 547.000 ECD_1.AcqOnECD_Total.AcqOnSampler.ReleaseExclusive.AccessConcentration 12.00 [mM]Curve 5End
131
132
Appendix III: Nadeem M, Mollier A, Morel C, Vives A, Prud’homme L, Pellerin S (2011) Relative contribution of seed phosphorus reserves and exogenous phosphorus uptake to maize (Zea mays L.) nutrition during early growth stages. Plant Soil 346 (1-2):231-244. doi:10.1007/s11104-011-0814-y
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137
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139
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141
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143
144
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REMOBILIZATION OF SEED PHOSPHORUS RESERVES AND EXOGENOUS
PHOSPHORUS UPTAKE DURING GERMINATION AND EARLY GROWTH STAGES
OF MAIZE (Zea mays L.)
Key-words: maize, germination, phosphorus, phytate, remobilization, P-uptake, isotope, ecophysiology, mineral nutrition.
REMOBILISATION DES RESERVES EN PHOSPHORE DU GRAIN ET
PRELEVEMENT EXOGENE PENDANT LE GERMINATION ET GERMINATION ET
LA CROISSANCE JUVENILE DU MAIS (Zea mays L.)
Mots-clés: maïs, germination, phosphore, phytate, remobilisation, prélèvement de P, isotope, écophysiologie, nutrition minérale.
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