Nutrient Sensing & Signaling.pptx

7/31/2019 Nutrient Sensing & Signaling.pptx

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Nutrient Sensing & Signaling


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ESSENTIAL ELEMENTS (19)

MACRO NUTRIENTS

or

MAJOR ELEMENTSMICRO NUTRIENTS

or

SECONDARY ELEMENTSor

TRACE ELEMENTS

N, P, K, Ca, Mg S and SiFe, ZN, B, Cu, Mn, Mo, Cl, Ni and Na

7 ELEMENTS9 ELEMENTS

C, H, O

3 elements

( Epstein 1999 )


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Primary Nutrients

1) Nitrogen N

2) Phosphorous P

3) Potassium K


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Introduction

Nitrogen (N) - most important inorganic nutrient in plants

Major constituent - proteins, nucleic acids, many cofactors,

secondary metabolites

Nitrogen - available to plant roots in several different forms - NO3 ,

ammonium (NH4 ), and organic forms chiefly amino acids.

Nitrate - most abundant source of N - anionic form - readily

dissolved in soil water - very mobile in the soil profile


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The NO3 is converted to gaseous N2 only when the oxygen level is

depleted at that condition soil bacteria can use O2 for respiration.

concentrations of NO3 in the soil can rapidly change depending onrainfall and factors influencing microbial activity such as pH,

temperature, and oxygen concentrations

(Miller et al , 2007)


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Physiological measurements of nitrate (NO3 ) uptake by roots havedefined two systems of high and low affinity uptake as

1) HATS (> 1mM) - Up-regulation of the high-affinity transportsystem (HATS) for NO3 which stimulates lateral root (LR) growth.

2) LATS (> 6mM)

There two gene families as NRT1 & NRT2 responsible fortransporter NO3 by proton (H+) symport mechanism that driven byPH gradients across membrane.

(Reman et al, 2006)


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Transcriptome analysis, using Affymetrix ATH1 arrays and a real-timereverse transcription-PCR platform for .1,400 transcription factors.

This analysis for identifying the genes expressed during process

affected by long term or short term N deprivation

Two days of nitrogen deprivation led to coordinate repression of the

majority of the genes assigned to :

Photosynthesis

Chlorophyll synthesis


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Contnd

Plastid protein synthesis

Induction of many genes for secondary metabolism

Mitochondrial electron transport


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Physiological and Metabolic Responses to N Deprivation and Nitrate Readdition

Phenology of 9-d-old N-limited and N-replete Arabidopsis Seedlings

Plants grow on full N media for 7 days, oneculture shifted to N starvation media &

another on N replete condition for 2 days


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N starved plants shows

Increased lateral roots

Reduced chlorophyll content

Accumulation of anthocyanin

High level of sugar & starch

Decrease in Glysine & Glutamate


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Expression of genes involved in metabolism after addition of NO3

Transcript levels in N-deficient seedlings 30 min after NO3 addition relative to the level in N-deficient seedlings

Gray-Gene absent

White-No change in

gene expression

Blue- Gene

Expression

increased

Red- Gene

expressiondecreased


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Transcript levels in N-deficient seedlings 3 h after NO3 addition relative to the level in

N-deficient seedlings


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Transcript levels in N-sufficient seedlings relative to the level in N-deficient seedlings


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Expression of genes involved in RNA and protein synthesis

Three hours after adding NO3 to N-starved seedlings In N-replete seedlings


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Overview of changes in redox processes, hormone synthesis, and sensing afterNO3 addition

Transcript levels in N-deficient seedlings 30 minafter NO3 addition

Transcript levels in N-deficient seedlings 3 hafter NO3 addition


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The Arabidopsis genome probably encodes 2,000 transcription

factors or transcriptional regulators

There approximately 1,800 potential TFs on the ATH1 chip, 93showed marked changes in transcript abundance in response to N

nutrition.


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Phosphorous signaling


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Introduction

Phosphorus - essential macronutrient for growth and

development of living organisms.

It is a constituent of key molecules such as ATP, nucleic acids, orphospholipids

As phosphate, pyrophosphate, ATP, ADP, or AMP, plays a crucial

role in energy transfer, metabolic regulation, and protein activation

(Marschner 1995).


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Phosphorus is one of the most limiting nutrients.

phosphate (Pi), is unevenly distributed in soils and >80% is immobile

and not readily available to roots

Crop yield is limited by P availability in 3040% of arable lands

(Holford, 1997)


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Plants have evolved several adaptation to low and unevenly distributed

phosphate as bellow:

A. Developmental adaptations

1. Changes in root architecture

2. Increase root-to-shoot growth ratio

3. Increase in lateral roots

4. Increase in No. & length of root hairs

5. Symbiotic association with mycorrhiza fungi


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B. Biochemical changes

1. Induction of high affinity Pi transporters

2. Increasing Pi mobilization

3. Secretion of phosphatases4. RNases

5. Organic acids

6. Alternative glycolytic or respiratory pathways

(Jose et al 2003 )


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Ethylene

Root

architecture

Auxin

P1BS

Pi starvation

responsive genes

Anthocyanin

accumulation

Root shoot ratio

Pi transporters

Signaling in plants in response to phosphate Deprivation

(Daniel et al 2007)

ROS


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Hormonal signaling in Pi deprivation

The hormones ABA, ethylene, auxin and cytokinin plays imp. role in

the control of Pi starvation responses.

The ABA accumulates anthocyanin in Pi deprivation condition.

Ethylene increases root elongation, increase in root hair density and

size in Pi - starved plants and has an opposite effect on Pi- rich

plants. Over accumulation of auxin in the primary roots & in lateral roots

which leads to stimulation of primordial emergence & lateral root

formation.


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Pi-starvation reduces Cytokinin concentration in plants which

increases root-to-shoot growth ratio and lateral root proliferation.

Exogenous addition of cytokinins represses the expression of several

pi-starvation responsive genes, such as those encoding the ACP5

phosphatase and the atpt1 pi transporter in the roots.

(Jose et al 2003 )


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The limited phosphate (Pi) supply involving biochemical, metabolic,

and developmental changes.

Arabidopsis thaliana plants harboring a reporter gene specifically

responsive to Pi starvation (AtIPS1GUS)

PHR1 encodes transcription factor related to PSR1 (Phosphate

starvation response1) from MYB family is binds to promoter of pi

responsive genes at P1BS.


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The AtIPS1 gene and other members of the Mt4/TPSI1 family, is

specifically responsive to Pi starvation.

A translational fusion between AtIPS1 and the coding region of the

GUS gene used for for identifying mutants with altered Pi starvation

responses.

PHR1 1 & PHR 1 2 mutants are produced


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Nucleotide and deduced

amino acid sequence from

the PHR1 cDNA.


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phr1-1 and phr1-2 each had a mutation in phr1 gene.

The mutation in phr1-1 in a C-to-T transition, causing the introduction of a premature

stop codon

The mutation in phr1-2 at G-to-A substitution, which impair a GT splicing donor site


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Characterization of PHR1 mutant allele :-

Histochemical analysis GUS activity driven by the AtIPS1:GUS reporter

gene in response to phosphate starvation in wild type and in the phr1-1 mutant.


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Histograms of metabolic (Anthocyanin and Pi content)


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Histograms of developmental (root/shoot growth ratio and total weight)


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Plates containing the wild-type (bottom) , the phr1-1 (left) and phr1-2 (right) mutant alleles

grown on different nutrient regimes


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Detail showing root hairs of wild type and phr1-1 grown under Pi starvation conditions


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Northern analysis of PHR1 gene expression


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Northern analysis of the effect of phr1 mutationson the expression of Pi starvation-responsive

genes.


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Potassium Signaling


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Introduction

Potassium is a macronutrient required in large quantities by plantsand is the most abundant cation in plant cells.

Potassium is one of the major nutrients, essential for plant growth

and development. It Required as a cofactor for more than 40 enzymes. Principal cation

in establishing cell turgor and maintaining cell electroneutrality.

Potassium is the fourth most abundant mineral, constituting

about 2.5% of the lithosphere.

Actual soil concentrations of this mineral vary widely, ranging from

0.04 to 3%


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Contnd..

soil potassium is available in four different pools as :

(I) soil solution(ii) exchangeable K

(iii) fixed K

(iv) lattice K

As plants can only acquire K+ from solution

concentrations of K+ in soil solution are in the range of only 0.16 mM

Concentrations of K+ in the cytosol are maintained in a range, around100 mM, which is optimal for the function of cytosolic enzymes

(Ashley et al, 2005)


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Plants rapidly sense the changes in external potassium

Two transporters are upregulated by potassium deprivation are

a high-affinity potassium uptake transporter HAK5 and KEA5

which involved in remobilization of potassium from the vacuole.

The hormonal responses to potassium deprivation include ethylene,

jasmonic acid (JA), and auxin.


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The expression of genes encoding ethylene biosynthetic enzymes

and ethylene production in potassium-deprived roots increased

Auxin play a imp. role in controlling the expression of potassium

channels

Long-term potassium starvation resulted in the conspicuous up

regulation of genes linked to JA and defense

(Schachtman et al 2007)


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Affymetrix Gene chip microarrays used to identify genes responsive

to potassium (K+)deprivation in roots of mature Arabidopsis

Many genes expression changed after subjected to 6, 48, and 96 h

ofK+ starvation

Root K + concentration reduced approximately by 60%

Potassium transporter gene from the KUP/HAK/KT family, is most

consistently and strongly up-regulated in its expression level


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Expression overview for Affymetrix Genechip experiments


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Microarray Chips spotted with oligonucleotides representing

approximately 8,300 genes (AG1-Genechip).

In addition, experiments after 48 and 96 h of starvation performed using

the ATH1 Genechip

Less than 1% of the genes showed a significant change in expression

levels compared to nonstarved roots after 6 h (21 genes) and 48 h (83

genes) of potassium starvation in both of the replicate experiments

The gene most strongly and consistently affected in all experiments for

48- and 96-h K+ starvation is the potassium transporter AtHAK5 gene.


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RT-PCR experiments confirm induction of AtHAK5 gene by

K + starvation

Roots of transgenic plants expressing the GUS and GFP reporter gene


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g p p g p g

driven by the AtHAK5 promoter

GUS +AtHAK5 promoter

K+ starved plant root After addition of K +

GFP +AtHAK5 promoter


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Reactive Oxygen Species in response to N,P, K deprivation


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Introduction

The production of ROS in roots is a common feature in response to

nitrogen, phosphorus, and potassium deprivation

Root hair cells in Arabidopsis contain a sensing system for nitrogen,

phosphorus and potassium deprivation

There N & K starvated condition ROS are formed in root hairs while

in P starvated condition ROS is formed in root cortex


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Arabidopsis root H2O2 production after 6 h nutrient deprivation


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Changes in gene expression upon nutrient deprivation in Arabidopsis

Northern analysis


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Genes mainly expressed & downregulated in wild Arabidopsis

Genes Up regulated Down regulated

AtPT2 P deprivation

AP2 K deprivation

AtHak5 K deprivation

unknown gene K deprivation

AtMyb77 K deprivation

AtrbohC N, P, K deprivation

AtrbohA P deprivation

C


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Changes in gene expression upon nutrient deprivation in the Arabidopsis rhd2 mutant

Northern analysis


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Genes Up regulated Down regulated

AP2 +

At3g03670 +

At3g49960 +

WRKY9 +

Hak5 +

AtrbohC +

Unknown gene +

Myb77

Genes mainly expressed & down regulated in Arabidopsis mutant rhd2

L li ti f ROS i A bid i t d i t i t d fi i t diti


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Localization of ROS in Arabidopsis roots during nutrient deficient conditio

Red fluorescence indicate presence of ROS


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Nutrient Sensing & Signaling.pptx