Physiological responses of Physiological responses of BurkholderiaBurkholderia phytofirmansphytofirmans strain strain PsJNPsJN

colonized plantlets of grapevine (colonized plantlets of grapevine (VitisVitis viniferavinifera L.) to L.) to low nonlow non--freezing temperatures freezing temperatures

Andreas I. THEOCHARIS

Laboratoire de Stress, Défenses et

Reproduction des Plantes

Unité de Recherche Vignes et Vins de Champagne -

Stress et Environnement UFR Sciences Exactes et Naturelles

A thesis submitted for the Degree of Doctor of Philosophy

culturally appropriate

socially just

economicallyviableholistic

scientific approach

ecologicallysound

An agriculture is Sustainable

Sustainable agriculture

(ATTRA, 2003;2005)

Beneficial microorganisms

Sustainable development

General introduction

The idea of a world where people protect the environment as they carry out their day-to-day activities

Literature Review

(Bakkeret al. 2003; Dobbelaereet al. 2003; Compantet al., 2005)

Help in management ofenvironnemental Stress

Abiotic stress

Biotic stress

Directly

•Induced systemic resistance (ISR)

Indirectly

Plant growth promotion

Plant growth promoting rhizobacteria (PGPR) & plants

The beneficial effects of PGPR

•ACC deaminaseactivity

• Nitrogen fixation

• Solubilization of minerals• etc

•Synthesis of anti-fungal compounds

•Synthesis of fungal cell wall-lysing enzymes•etc

Literature Review

ISR & signaling pathways

(Pieterseet al., 1998 ;Vallad & Goodman, 2004)De Meyer et al., 1999; Timmusk &

Wagner, 1999; Park & Kloepper, 2000 ; Magnin-Robert et al. 2007

ISR

SA-, JA-, and ET

PR genes

PRproteins

NPR1

A state of increased defensive capacity developed in

by

through activation of latent resistance mechanisms

ISR-inducing PGPR

Literature Review

(Ryalset al., 1996; Doke, 1996; Sticheret al., 1997; Conrathet al., 2002; 2006; Goellner & Conrath, 2008)

Systemic resistance by primed physiological responses

The phenomenon of priming

has been associated with

a quicker and/or stronger activation

of plant cellular defenses upon exposure to environmental stress

Priming

Systemic response induced by or

colonization of plant roots by ISR-inducing PGPR

Pathogens

Literature Review

Priming mechanism : Causes and Effects

Pathogens,PGPRs

SAR-inducers

Cold acclimationa) Primed physiological stage

PrimingOsmotic stress,

Wounding

Temperature stressElicitors

Pathogens

b) Challenge with stress

Stronger responses against stress

Faster responses against stress

(Conrath et al., 2002; 2006; Goellner & Conrath, 2008)

c) Potentiated physiological reaction of plants

Literature Review

Strain PsJN is able to establish rhizosphere and endophytic populations in plants

Burkholderia phytofirmans strain PsJN

(Pillay & Nowak, 1997; Bensalimet al., 1998; Sharma & Nowak, 1998; Compantet al., 2005)

A member of the genus Burkholderia belongs to the ß-Proteobacteria

• better water management in plants• increased resistance to temperature stress

• increased resistance against pathogens

Plant growth stimulation

•phenylalanine ammonia lyase and phenolics

induction of defense mechanisms&

• ACC deaminase activity

• a characteristic larger root system

• sturdier stems & greater lignin deposition

•increased levels of chlorophyll & cytokinins

Literature Review

Fruitingcutting

Low non-freezing temperature

(after 2 weeks)

Promoted growth and physiological

activity

Enhanced level of proline, total phenolic s &

starch deposition

Improved photosynthetic

capacity

(Ait Barka et al, 2006)

Literature Review

Grapevine –B. phytofirmans strain PsJNinteraction

Significant capacity of bacterized plantlets to withstand chilling

(Compantet al., 2005, 2008)

Rhizosphere & endophytic population in grapevine roots, vegetative and

reproductive organsColonization of

strain PsJN

resistance toBotrytis cinerea

Faster growth & development with robust root system

Beneficial effects of strain PsJN in

grapevine plantlets

(Ait Barka et al, 2000, 2002)

in vitro-plants

4°C

Tropical &subtropical origin

Not able to survive in low non-freezing

temperatures(Chilling stress)

( Lyons, 1973; Raison & Lyons, 1986; Wang, 1990; Tomashow, 1999 )

Literature review

Plant sensitivity in low non-freezing temperatures(0°C - 15°C )

Plants

The biology of plantto cold

Temperate origin (e.g. grapevine)

Cold acclimation

Adaptation to low non-freezing temperatures

Freezing tolerance (< 0°C)

Literature review

(Raison & Lyons, 1970; Lyons, 1973)

Liquid-Crystalline

phase

transitionSolid gel

Solute leakage & disrupted ion

balance

Injury &death of cells and tissues

Increased permeability

Increased activation of energy-bound

enzymes

Cessation of protoplasmic

streaming

Imbalance in metabolism

Accumulation of toxic metabolites

e.g. acetaldehydes, ethanol etc

Reduced ATP supply

Return to normal

metabolism

Brief exposure and return to 20 °C

Chilling injury in sensitive plants

Prolonged exposure

Cell membranes

Literature Review

Plant biologyin cold

AOS accumulation induces chilling injuries in sensitive plants

Inactivation of enzymes

Lipid peroxidation

Protein degradation

( Raison & Lyons, 1986; McKersie & Lesham, 1994; Asada & Takahashi, 1987; Peiet al., 2000)

PS I, II

Plasma membranes

Chilling

AOS

Damage to DNA

Sensitive plants

Detrimental the prolonged

presence of high levels of AOS

membrane rigidification & cell

death

Production ofaldehydes &

malonedialdehydes

degradation of the polyunsaturated fatty

acids

( Uemura & Steponkus, 1999; Thomashow, 1999; Xin & Browse, 2000; Chang et al., 2001; Browse & Xin, 2001)

Responses of insensitive plants to low non-freezing temperatures

Accumulation of[Ca2+

cyt]

Accumulation of AOS and activation of scavenge system

Change in gene expression and protein synthesis

Accumulation of sugarsand prolines

Modification in plantmembranes

Acclimation to cold

Biochemical & physiological

changes

Elicitation of stable developmental

responses to low temperatures

Literature Review

Photosynthetic acclimation

a regulatory network of

genes

Induction of cold related

genes (COR ) Cold acclimation

Induction ofantioxidant

systemsAccumulation of

antioxidantenzymes

(Tähtiharjuet al. 1997; Peiet al., 2000; Thomashow, 2001; Fowler & Thomashow, 2002 )

The process of cold acclimation & the signal transduction pathways

H2O2

Cold acclimation

Adaptation to low temperatures

Biochemical andphysiological

changes

AtCBF2

AtCBF3

AtCBF4

AtCBF1

Transcription factors

CBF

[Ca2+cyt]

Accumulationcoffree proline and total

soluble sugars

Signaling molecule

Up-regulation of COR gene products

Freezing tolerance

Literature Review

Literature Review

Plant biologyin cold

Cold Stress

Nutritional role during acclimation

Stabilizerof membrane

Regulators of some enzymatic systems Scavengers of reactive

oxygen species

Inducer of stress-related genes

accumulation of carbohydrates

cryoprotectantsFree prolineaccumulation

Reduction of plant activegrowth

Decreased demand forthe products ofphotosynthesis

Enhanced activitiesof Calvin cycle

enzymes

(Sasaki et al., 1996; Ögrenet al., 1997; Dörfflinget al., 1997 ; Greer et al., 2000)

Accumulation of cryoprotectant contents in acclimated plants

Insensitive plants ?

How does grapevine sense the root colonization by bacteria and what are

the molecular and physiological changes that occur in grapevine by this

interaction?

Which grapevine defense mechanisms can be activated by these changes

and how could they help grapevine to better tolerate “cool” climate?

The three objectives of the project

Despite the available information by previous studies,

several questions remained regarding the beneficial

interaction between

&

Study of response of grapevine plantlets after root inoculation by Bulkholderia phytofirmans strain

PsJN

Objective I

Characterization of defense mechanisms activated in fully bacterized plantlets upon exposure to low non-freezing

temperatures

Objective II

Objective III

cDNA-AFLP differential gene expression analysis of physiological state induced by Burkholderia phytofirmans strain PsJN in grapevine upon low non-

freezing temperatures

The three objectives of the project

Study of response of grapevine plantlets after root inoculation by Bulkholderia phytofirmans strain

PsJN

Objective I

goalInvestigation whether PsJN strain is able to stimulate the defense

mechanism by induction of selected defense genes after root inoculation,

similarly to ISR-type responses.

Objective I

May this stimulation promote the plant resistance against cold?

Changes in pattern of defense gene expression in leaves by qRT-PCR

Objective IVitis vinifera L. cv. Chardonnay clone 7573 16-h fluorescent light at 26°C

micro-cuttings1 nodal explant

propagation

2 ml of inoculum ofstrain PsJN

6wk-old plantlets

+

Strain PsJN(3 x 108 CFU\ml of

inoculum)

two loops of strain PsJN

King’s B liquidmedium

immersion

Plant bacterizationprocess

re-suspenedin PBS

122448

Hours after root inoculation

Experiments were repeated twice

0

5

10

15

20

0 12 24 36 48

Ind

uction

Hours post-inoculation

Phenylalanine ammonia-lyase (VvPAL)

0

10

20

30

40

0 12 24 36 48

Ind

uctio

n

Hours post-inoculation

Stilbene synthase (VvStSy)

0

2

4

6

8

0 12 24 36 48

Ind

uct

ion

Hours post-inoculation

Lipoxygenase (VvLOX)

Objective I

Genes encoding enzymes from phenylopropanoid& octadecanoid pathways

0

2

4

6

8

10

0 12 24 36 48

Ind

uctio

n

Hours post-inoculation

Chitinase 4c (VvChit4c)

02468

1012141618

0 12 24 36 48

Ind

uctio

n

Hours post-inoculation

Glucanase (VvGluc)

0

50

100

150

200

0 12 24 36 48

Ind

uction

Hours post-inoculation

Protease inhibitor (VvPIN)

Objective I

Genes encoding for pathogenesis-related proteins (PRs)

Objective I

ii) Induction of systemic responses by strain PsJN

i) Induction of defense mechanism after root inoculation

•Analysis of the impact of the strain PsJN on the chilling tolerance under ISR condition

•Analysis of SA and JA levels in plants to discriminate the pathway(s) involved in the establishment of ISR

Future work for characterization of defense signal as ISR:

ISR?

PRs

Conclusion &Discussion

iii) The induction of transcript accumulation involves genes encoding for PRs, similarly to other ISR-inducing PGPR, suggesting an overlapping between ISR and SAR

1st

goal

Study of the expression pattern of well-characterized

grapevine defence genes and CBF transcription factors in

grapevine plantlet leaves

Objective II (1st goal)

Objective IICharacterization of defense mechanisms activated in fully bacterized plantlets upon exposure to low non-

freezing temperatures

strain PsJN

(3 x 108

CFU\ml)

2wk-old

plantlets

+

CBF4 transcription factors

9 h24 h

Changes in pattern of VvStSy, VvPAL, VvLOX, VvGluc, VvChit4candVvChit1b expression in leaves

Time after cold treatment

6wk-old fully bacterized

plantlets

after 4 wk

Vitis vinifera L. cv. Chardonnay clone 7573

16-h light/ 8-h dark at

26°C micro-cuttings1 node

propagationStrain PsJN

Plant bacterizationprocess

24 h48 h72 h2wk

10°C/ 4°C , 16 h light/ 8 h dark

Objective II

1st

goal

Experiments were repeated 3 times

Objective II

non-bacterized 26°C

non-bacterized 4°C

bacterized 26°C

bacterized 4°C

Time after cold treatment

Time after cold treatment

1st

goal

Genes coding enzymes from phenylopropanoid pathways

Objective II

Time after cold treatment

Time after cold treatment

non-bacterized 26°C

non-bacterized 4°C

bacterized 26°C

bacterized 4°C

Time after cold treatment

1st

goal

Genes encoding for pathogenesis-related proteins (PRs)

Objective II

non-bacterized 26°C

non-bacterized 4°C

bacterized 26°C

bacterized 4°C

1st

goal

Gene encoding enzymes from octadecanoid pathway & CBF4 transcription factor

0

20

40

60

80

100

120

140

160

180

200

9 h 24 h

Ind

uct

ion

Time after cold treatment

CBF4

Objective II

1st

goal

Conclusion &Discussion

1. In grapevine plantlets, low temperatures induced the defense-related gene

transcripts & cold specific transcription factor CBF4 according to the

phenomenon of priming

2. From analysed genes, except for CBF4, chitinases and glucanasesare of

special interest since they exhibit both antifreeze and antifungal activities

4. This induction of grapevine defense mechanism may be correlated with

previous results showing that leaf cells of bacterized plantlets are less

affected by cold, and it further indicates that B. phytofirmans strain PsJN

may improve grapevine resistance to low non-freezing temperatures (Ait

Barkaet al., 2006)

3. The clear potentiated expression of LOX in bacterized plantlets after cold

stress suggests that JA signal transduction pathway could be involved in the

process of cold acclimation induced by B. phytofirmans.

Objective IICharacterization of defense mechanisms activated in fully bacterized plantlets upon exposure to low non-

freezing temperatures

Determination of proline accumulation and analysis of changes in the level

of lipid peroxidation markers (aldehydes, malondialdehydes) and hydrogen

peroxide

2nd

goal

Objective II (2nd goal)

a

b

c

a

b

a

c

a

b

a

c

a

b

a

c

a

b

a

c

a

Objective II

aa

bb

aa

b b

a a

b b

ab

c

d

ab

c

d

non-bacterized 26°C

non-bacterized 4°C

bacterized 26°C

bacterized 4°C

2nd

goal

Analysis of stress markers (Prolines & Hydrogen peroxide)

Objective II

non-bacterized 26°C

non-bacterized 4°C

bacterized 26°C

bacterized 4°C

ab

a

c

a

b

a

b

a

b

a

b

a

b

a

c

a

b

a

c

a

b

a

c

a

b

a

c

a

b

a a

b

a

c

b

a

c

a

b

2nd

goal

Analysis of lipid peroxidation markers (Aldehydes & MDA)

Objective II

2nd

goal

Conclusion &Discussion

3. B. phytofirmans provokes stronger H2O2 accumulation within the first 3 days of

treatment but also speeds up the decrease of H2O2 level after 1 week. Probably, H2O2

triggers the synthesis of antioxidant enzymes such as catalaseor peroxidase that

scavenge ROS and help the plant to overcome cold conditions

2. Proline is the most well characterized stress responsive molecule, and it is not

surprizing that in grapevine, accumulation of proline appeared as a response to cold

acclimation process (Ait Barkaet al., 2006). The accumulation of proline in bacterized

plantlets according to the phenomenon of priming, reveals the protective role of

bacteria

1. Apart from gene expression, plant responds to coldness by stress-related

metabolites such as proline, hydrogen peroxide or aldehydes & malondialdehydes, in

similar way to priming phenomenon

4. Finally, aldehydes and MDA are accumulated by almost similar ways to those

reported for H2O2, confirming that B. phytofirmans speeds up grapevine reaction to

cold shift and later favours the acclimation process to cold temperatures, showing that

the presence of PsJN improves the loss of permeability of membranes as response to

cold (Barkaet al. 2006)

Objective IICharacterization of defense mechanisms activated in fully bacterized plantlets upon exposure to low non-

freezing temperatures

Determination of starch deposition and soluble sugar

(total soluble sugars, sucrose, glucose, fructose) accumulation in grapevine

plantlet leaves upon exposure to low non-freezing temperatures

3rdgoal

Objective II (3rd goal)

Objective II

b

a

ba

c

a

ba

c

a

ba

c

a

cb

a

b

cb

ab c

d

ab c

d

a

b

c

d

a

b

c

d

a

b

c

d

non-bacterized 26°C

non-bacterized 4°C

bacterized 26°C

bacterized 4°C

3rdgoal

Starch deposition & total soluble sugars accumulation

Objective II

non-bacterized 26°C

non-bacterized 4°C

bacterized 26°C

bacterized 4°Ca

ba

c

a

b

a

c

a

b

a

c

a

b

a

c

a

b

a

c

a

ba

c

a

b

a

c

a

b

a

c

a

b

aa

c

b

dc

a

b

a

c

a

b

a

c

a

b

a

c

a

b

a

c

a

b

c

b

3rdgoal

Enzymatic analysis of soluble sugars

Objective I

3rdgoal

1. According to our results strain PsJN affects carbohydrate metabolism

in grapevine plantlets in normal growth conditions related with the

stimulation of net photosynthesis (Ait Barkaet al., 2006), which may

contribute to sugar accumulation

3. By higher accumulation of carbohydrates in bacterized plantlets, we

could address that PsJN is a PGPR that primes several physiological

responses of grapevine plantlets under cold stress including the accumulation

of soluble sugars and starch, speeding up the process of cold acclimation

2. Cold acclimation induces an increase of both soluble sugars and starch

in grapevine grown in the vineyard (Ait Barka & Audran, 1996) or in our

plantlets grown in growth chamber, oppositely with starch that may be

converted into soluble saccharides during cold exposure in some species

B. Phytofirmans is an ISR-type PGPR able to potentiatethe physiological response of cold acclimation and to prime the grapevine development and growth upon low non-

freezing temperatures

B. Phytofirmans is an ISR-type PGPR able to

prime

the induction of known defense genes & genes with specific

role in cold acclimation process

StSyPAL

Chit4c

Chit1b

GlucLOX

CBF4

sucrose

the accumulation of cryoprotectant

contents

prolinetotal soluble

sugars

starch

fructoseglucose

the faster degradation of lipid peroxidation and

stress markers

malondialdehyde

hydrogen peroxide

aldehydes

General conclusions

General conclusions

Future prospects

Prospect I

Investigation of primed- physiological state of V. vinifera L. induced

by Burkholderia phytofirmans strain PsJN by transcription analysis.

The identification and analysis of gene expression profile may support

our knowledge about the signalling pathways of priming phenomenon

Prospect II

Using the molecular tools, like specific mutants of A. thaliana, for

analysis of signaling pathways induced byBurkholderia

phytofirmans strain PsJNn and for better understanding of

beneficial effects in plants

Greek Scholarship Foundation

IKY AcknowledgmentsAcknowledgments