Plant lecture 16 - Integration of C and N metabolism Flashcards
Nitrate reductase regulation
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- NIA = sensitive to conditions plant is in
- Reversible activation occurs when leaves = exposed to light or high CO2
- Phosphorylated/dephosh. forms of NIA are both active; Ca2+ dependent phosphorylation of a Ser permits binding of inhibitory 14-3-3 protein
- In light, leads to production of reduced ferredoxin. Reduced ferredoxin also reduces thioredoxin + involved in activation of ↑ E e.g. Calvin cycle, favours reduction of nitrate → nitrite → ammonium
- Signals prevent phosphorylation of NIA + inactivation by 14-3-3
- In the dark, photosystems x active, thioredoxin is oxidised. Activates OPP + produces NADPH which reduces ferredoxin which supplies reductants to Nitrite reductase +GS
- Inhibitory signals from chloroplast lead to phosph. of nitrate reductase + inhibition by 14-3-3
- Rate of flux nitrate → nitrate is reduced, slower assimilation of nitrate
- Store carbon skeletons for use in light
Nitrate availability + role in regulation
- ↑ changes in gene expression depending on N-containing metabolites
- Availability of NO3- → signals from hormones, aa, nitrate
- Evidence that nitrate itself plays a role in regulating gene expression:
- Transcripts that ↑ rapidly in response to NO3- (sensing nitrate itself)
- NiA mutants. Compare WT plants grown on low NO3- (N deficient) w/ NIA mutants grown on high NO3- (N deficient too).
- Find both nitrate + product of nitrate influence plant responses
Analysis of 2o metabolism in tobacco using NIA mutants
- Nitrate availability regulates shift from N-containing alkaloids to C-rich phenylpropanoids during N deficiency
- N-replete WT + nitrate-grown NIA mutants have low levels of C-rich 2o metabolism → nitrate inhibits phenypropanoid synthesis
- Genes for phenylpropanoid metabolism are induced in N-deficient WT (↓ nitrate) x nitrate-grown NIA mutants (↑ nitrate) → low nitrate is signal for switch to C-rich compounds during N-deficiency
Demonstrate that nitrate has direct effect on N metabolism:
- Get induction of NRT1/2, NIA, NII< GS/GOGAT
- Also ↑ rate of NO3- uptake, activity of nitrate reductase
- Also affects synthesis of organic acids needed for NO3- assimilation. Induces PEPC, pyruvate kinase, citrate synthase genes (↑ activity of E + levels of malate + 2-oxoglut)
- Effects = in both roots and leaves (nitrate sensing = in both tissue)
- Rapid change in gene expression = primary nitrate response
NO3- signalling
- Metabolism of NO3- also generates signals
- NO3- uptake + assimilation are inhibited by Gln, Asparagine + other aa
- WT plants show marked diol changes in transcript levels + E activities but in NIA-deificeint mutants, have ↑ levels of mRNA + E activity but no variation (nitrate alone x only signal but powerful signal)
- Sugars often complement the effect of NO3- on gene expression
- Sugars induce NRT1/2, NIA, genes for GS, PEPC
NO3- sensing
Also note plant PII homologue acts as a glutamine sensor. Different from 2 component regulatory system of bacteria
- NO3- sensor likely senses extracellular NO3- pool as cytosolic pool is generally unresponsive to changes in extracellular NO3- (excess NO3- can be stored in vacuole)
Evidence:
- NO3- transporter is involved in NO3- signalling
- Split root experiment. Single root Arabidopsis has access to ↑ NO3-
- Mutant NRT1.1 shows ↓ lateral root formation in response to external NO3-
- Either nitrate sensor or facilitator for nitrate uptake into nitrate-sensing cells (less likely)
- Mutant NRT2.1 shows lateral root formation w/o external NO3-
- Consistent of NRT2.1 as a low NO3- sensor or signal transducer
NRT1.1 as Nitrate sensor
- NRT1 mutant chl1-9 = defective in NO3- transport but still triggers intracellular response to NO3-
- At ↓ NO3-, phosphate of NRT1 at Thr101 converts transporter from low to high affinity + intracellular response to NO3- = down regulated (or change in Vmax?)
- Nrt1.1 = transporter of nitrate + receptor
Long range signalling mechanism
- In addition to uptake of NO3- at root, also need long range signalling to communicate N+C status of the root
- NRT2.1 = ↑ affinity transporter is repressed by signals from shoots (signals could be ammonium + glutamine but x established)
- NRT1.1 x sense shoot N status
Evidence
- Status of shoot reported to shoot
- Loss of function hy5-526 = in screen for impaired shoot-illumination-promoted root growth
- HY5 = TF that is a shoot-to-root mobile signal. NRT2.1 expression + N uptake depends on + maintains balance of C + N (shoot to root)
ALSO
(root to shoot)
- Thought mobile C-terminally encoded peptide (CEP) reports root N status to shoot
- CEP = transported through xylem to leaves
- x made in N-rich roots on N deficient
- When reaches leaf, CEP binds to receptor + leads to synthesis of glutaredoxin (phloem-mobile + go back to roots)
- In N-rich patch, glutaredoxin further activates NRT2.1 expression + promotes uptake of N2
Local signalling to coordinate N + C signalling
- Localised supply of nitrate stimulates elongation of lateral roots in Arabidopsis
- 1mM nitrate lateral root grows due to accumulation of auxin at apical meristem.
- Glutamine x nitrate = less root growth. Flow of auxin back through Nrt1.1 back to origin of IAA
- Thought return of IAA is impaired in nitrate x glutamine (nitrate competes for transport w/ auxin)
- Evidence = chl1 mutant. x transport either nitrate or auxin. Elongate roots irrespective of nitrate of glutamine
- Nrt1.1 has 3 functions: transports nitrate, auxin + receptor that detects external nitrate
