Plant lecture 15 - Using ammonium

Question 1

GS assimilatory role

Answer 1

• GS has ↑ Km for ammonium binding, (more likely to be involved in assimilation)
• Labelling experiments = label is 1st incorporated into glutamine amide group. Then GOGAT transfers the N from the amide to amino group in glutamate

Question 2

Compartmentalisation of GOGAT/GS cycle

Answer 2

In roots:

• GS is mainly cytosolic, some evidence for plastid
• GOGAT is plastidic, needs reducing power that comes from plastidic oxidative PPP

In leaves:

• GS = cyt (GS1), mit (GS2) + plastidic (GS3) forms
• GS1 = in phloem cells, makes glutamine
• GS2 = in mesophyll cells, recovers photorespiratory ammonia
• GOGAT is also plastidic

Question 3

Regulation of GS1 expression

Answer 3

• Regulation occurs at multiple levels

- GS = target for trying to improve N use efficiency, if overexpress GS, ↑ capture of ammonium + ↑ efficiency

Question 4

Carbon skeletons for N assimilation
(Citrate)
(Asparagine)

CHECKKKKKKKKKKK!!!!!!!!!!!!!!!!!!

Answer 4

• C skeletons drawn from variety of int. from TCA, glycolytic + PPP.
• At some point in each pathway, have aminotransferase that removes NH3 from Glu/Gln + transfers to C skeleton
• Citrate → a-ketoglut + glutamate
• Need to replace citrate
• Depends on PEPC to make OAA
• Although mostly cytosolic, some evidence of PEPC plastidic isoforms in rice

CHECK THIS!

• In illuminated leaves, C skeletons for N assimilation are derived from citrate
• Stored + synthesised during dark period
• Using Jnc + 15N/13C labelling data
• Looked at Glu-C2 + barely any molecules so glutamate is made from 2-a ketogenic x 13C
• Look at Jcn, Glu labelled w/ 13C + 15J
• Conclude most 2-aketo made overnight in dark before could incorporate any 13C into system
• Asparagine export from legume root nodules requires OAA as a C source for asp synthesis

Question 5

Glutamate dehydrogenase

Answer 5

• Reversible enzyme
• x in favour of reductive amination
• ↑ Km so ↓ affinity for ammonium
• Catabolic role e.g. detection of glutamate ox by isolated mitochondria in absence of aminotransferase activity
• ↑ GDH activity in senescence + in C starved cell suspension cultures. Both systems are running out of respiratory substrates, re-mobilise aa for other purposes

Question 6

Sources of ammonia

Answer 6

1. Ammonium uptake from the soil
   - NH4+ = main form of inorganic N in acidic soil
   - Low and high affinity ammonium transporters occur in the plasma membrane - AMTI transporters
2. Symbiotic N fixation
   - Nitrogenase
3. Photorespiration
   - Cxygenase activity of Rubisco
   - Prevents accumulation of toxic 2-phosphoglycerate
   - Ammonia release = glycine decarboxylase + serine hydroxymethyl transferase
   - Ammonium production = 10x assimilation. Re-assimilation = important
   - Evidence: photo respiratory mutants lacking GS/GOGAT die in air but grow normally in 1% O2. Shows NH3 re-assimilation x essential for growth of C3 + photoresp. x essential
4. Recycling of protein + aa
   - Oxidative de-am of glutamate is followed by re-assim. of NH4+ into transport when protein hydrolysis occurs e.g. during germination
5. Nitrate upatke + reduction
   - NO3- = main arouce of inorganic N in soil
   - Uptake is carrier mediated

Question 7

Nitrate uptake

Answer 7

• Some are constitutively expressed (low/high) + some are inducible by nitrate (high)
• 2 families
  1. NRT1/NPF = encodes low + dual affinity transporters
  2. NRT2 = high affinity transporters
• Nitrate uptake is driven by H+ cotransport
• Experiment: NRT1 expressed in Xenopus. Electrogenic NO3- transporter w/ an activity that ↑ as the external medium was acidified. Conclude NRT1 catalyses uptake w/ at least 2H+ in cotransport