Lecture 18 – Nitrogen

or
How does an atom of nitrogen get from the
atmosphere to part of the protein Rubisco?

N N
PDB: IBXN
Dr Sonja Dunbar
Why does a plant need
nitrogen?
• Most abundant mineral nutrient in plants
• 80% of leaf N is in chloroplasts
• Up to 30% leaf N just in Rubisco
• Needed for:
Nucleotides and nucleic acids

Secondary
Amino acids metabolites
e.g. caffeine
What happens if a plant doesn’t
have enough nitrogen?
• Stunted growth
• Chlorosis (yellowing)
• Reduced photosynthetic capacity
• Senescence (ageing)
• Leaf loss
• Poor grain quality nitrogen deprivation in reproductive phase
even as they remobilise nutrients to
prioritise rep structures

• See developmental responses – lateral root growth

Find out more in Lent term!
The journey

N2
The journey

N2

Step 1:
Nitrogen
fixation
The journey

N2

Step 1:
Nitrogen
fixation

NH3 Urea
Step 2:
NO3- Uptake Amino acids
The journey

N2

Step 1:
Nitrogen
fixation Step 3:
Transport

NH3 Urea
Step 2:
NO3- Uptake Amino acids
The journey

N2

Step 1:
Nitrogen
fixation Step 3:
Transport
Step 4: Assimilation

NH3 Urea
Step 2:
NO3- Uptake Amino acids
 Step 5: Storage
The journey

N2

Step 1:
Nitrogen
fixation Step 3:
Transport
Step 4: Assimilation

NH3 Urea
Step 2:
NO3- Uptake Amino acids
 Step 1 – Making it available
• Enter the nitrogen fixers – diazotrophs
only in bacteria — diazotrophs (quite diverse evolutionarily)

• Bacteria that contain nitrogenase

Find out more
• May be free living or in a symbiosis in Lent term!

Rhizobia in legumes Anabaena (cyanobacterium) in Azolla

leaves (water fern) helping rice grow
 Step 2 – Uptake
• May be given to the plant by a symbiont
OR
• May take it up directly from the soil
• Form depends on soil environment:
hence requires diversity of transporters

Acidic Higher pH and aerobic Slow nutrient recycling

= NH4+ & amino acids = NO3- (favourite form) = amino acids
Step 2 – Uptake of NO3 -

• NO3- uptake by H+-symporters

• Constitutive low affinity transporters, LATs (1mM)
• Constitutive and inducible HATS

INSIDE
PLANT

SOIL
NO3 - uptake by LATs
• NRT1 family
(At 53 genes)

diff loc and affinity

• NRT1.2 constitutive in
epidermis
• NRT1.5 for xylem loading
NRT1.1 can convert to HATs
• NRT1.1 in epidermal PM

• Phosphorylation induced
by starvation converts it
to HATs conformational changes (exact mechanism outo fo scope)
NO3 - uptake by HATs
• NRT2 family

• NRT2.1 epidermal PM
induced by starvation
(miR169 ↓)
• NRT2.4 in lateral root
epidermal PM
Step 2 – Uptake of NH4 +

• NH4+ more challenging substrate:

• Soil NH4+ blocks K+ uptake because it breaks down + gradients

• Inside cell, breaks down H + gradients in

respiration and photosynthesis also can break down in NH3 and H+, making it even
worse for the echemical gradient

• Therefore needs rapid assimilation to avoid toxicity

NH4+ uptake by AMT
transporters
• 6 genes in Arabidopsis
• 10 genes in rice

• HATs in root hairs and

epidermis

• AMT1;2 = LATs in
endodermis
 reduced micrbial activity
do not convert inorganic all the way to nitrate or ammonium

Step 2 – Uptake of amino acids

• Important in arctic and
boreal forests – can be
up to 150 µM in soils

• Low and high affinity symporters

• Symplastic transport to xylem from root hairs and

epidermis
 bc of the div of AA

Three gene families for root

uptake of amino acids
Amino acid permeases:
• Arabidopsis AAP1
(neutral and acidic)
• AAP5 (basic)
Lysine-histidine transporters:
• LHT1 (high affinity)
• LHT6 (acidic)

Cationic amino acid transporters

e.g. CAT6 patch clamp

GUS staining for LHT6

Step 2 – Uptake of urea
• Potential for using animal
waste as fertiliser

• Soil bacterial breakdown

 DUR3: H -urea
+ symporter
• PM of Arabidopsis, rice and maize roots
• Induced on N starvation
• Characterisation: OsDUR3 supports urea uptake
in Xenopus oocytes
OsDUR3
increases
Arabidopsis
growth on
urea as sole
N source

+
Step 3 – Transport
• Assimilation may be in roots or leaves
• Depends on species – assay plant sap:
Sap has >80% organic N Sap has mainly NO3- with <2% organic N
= assimilation in ROOTS = assimilation in LEAVES
e.g. pea, radish e.g. pumpkin, Arabidopsis

Majority of temperate plants Majority of tropical plants

More examples T&Z 6th ed, Figure 13.6

Step 3 – Transport
• Nitrate is transported to shoots via the xylem
• K+ acts as the counter ion

• OsNRT2.3 in xylem parenchyma

• Loads xylem
• Function confirmed in mutants:
• Mutant shoots nitrate deficient
• Roots retain nitrate
Step 4 – Assimilation
• Overview of the pathway
First steps in nitrate assimilation
Labelling patterns of plants fed 15N are identical for
NO3- or NO2-
• Plants will convert:
15NO - → 15NH4+
3

15NO - → 15NH4+
2

• Heavy isotope detected by mass spectrometry

• Indicates nitrate assimilated via nitrite into
ammonia
Recap quiz question 1
Which of the following compounds are toxic to
plants?

A) Nitrate

B) Nitrite steals cofactors out of enzymes

C) Ammonium dissipates proton gradient

D) Glutamine
Recap quiz question 2
Which enzyme catalyses the first step of
assimilation?

A) Nitrate reductase

B) Nitrite reductase

C) Glutamine synthetase

D) GOGAT
Nitrate reductase (NR)
• Dimeric enzyme of 2 x 115kDa subunits
• Found in cytosol mini ETC

• Three domains with different cofactors:

• FAD (flavin adenine dinucleotide)
• Cytochrome b557 (haem-containing)
• Molybdopterin (Mo-containing)

NAD(P)H FAD Haem Mo NO3-

FAD Haem Mo
NAD(P)+ NO2-

Miniature electron transport chain

Recap quiz question 3
At what level is nitrate reductase regulated?

A) Only transcriptional (level of gene expression)

B) Only post-translational

C) Both transcriptional AND post-translational

Recap quiz question 4
Which of these factors increase expression of
nitrate reductase?

A) Nitrate can be overrulled by other signals

B) Sucrose

C) Glutamine

D) The circadian clock

 NR is highly regulated
things that indicate that there will be carbon skeelton acceptors (photosynthesis)

• Gene expression is induced by NO3- and light

• Repressed by glutamine
• Sucrose can induce in the dark
Triose P 14-3-3
NO3-, light Hexose P protein
glutamine ATP ADP
sucrose, clock
cytokinins P
NR kinase P

NR gene FAD Haem Mo FAD Haem Mo FAD Haem Mo

Active NR NR P’ase Active NR Inactive NR

P phosphorylation allows bindigng of 14-3-3

• Enzyme activity modulated by phosphorylation

• 14-3-3 proteins bind to P group = inactivating enzyme
• Triose-P and hexose-P inhibit NR kinase
Recap quiz question 5
Which of the following cofactors does nitrite
reductase contain?

A) FAD

B) Fe-S cluster

C) Siroheme

D) Coenzyme A
Nitrite reductase (NiR)
• Monomeric protein with siroheme and FeS cluster
• Found in plastids 6e- + 8H+ NO2-
Siroheme Fe.S

2H2O NH4+

• Uses electrons from Ferredoxin (PSI) or NADPH

• In non-photosynthetic cells, electrons come from
oxidative pentose phosphate pathway
Glucose 6-P NADP+ Ferredoxinred Nitrite
Fd-NADP NiR
G6P dehydrogenase
reductase
6-Pgluconate NADPH Ferredoxinox Ammonia
Plants lacking NiR show poor
growth and chlorosis
• Evidence from transgenic tobacco plants with
antisense for NiR
Wild type Transgenic
Nitrite reductase (nmol min-1g-1 protein) 231 none detected Low protein
content in NiR
Protein content (mg g-1 fr. wt) 6.5 0.57
nitrate red.
antisense
Nitrite content (nmol g-1 fr. wt) 102 487 still doingits plants = grew
job. poorly, were
Ammonium content (nmol g-1 fr. wt) 1761 200 chlorotic

• Toxic NO2- also accumulates

• To avoid build up of toxic NO2- regulated at NR
Assimilation of ammonia
• Initial products are amino acids
• Roots starved of N then presented with nitrate or
ammonia rapidly accumulate these amino acids:

R groups

preferential mol for N transport

N rich R-group allows it

to be a good donor

Ala Asp Asn Glu Gln

 Assimilation of ammonia
• 15N experiments showed the amide of glutamine is
labelled first

amide of
- glutamine
15NO
3
incorporation

amine of
glutamine
or glutamate

time
 Two enzymes act in concert
SEE 1A CELLS

• First into amide of glutamine, then amino group of

glutamate:
COOH ATP ADP + Pi CO.NH2 Fdred COOH COOH
| | | |
CH2 CH2 CH2 CH2
| + NH3 | | |
CH2 Glutamine CH2 GOGAT CH2 CH2
| synthetase | | |
(GS) COOH
CH.NH2 CH.NH2 CH.NH2 CH.NH2
|
| | | |
CH2
COOH COOH COOH COOH
GS has very high |
affinity for NH3 CH2 2 x Glutamate
Glutamate Glutamine |
(Km ~20 µM) Regeneration
C=O
| of acceptor
COOH
GOGAT = Glutamine-2oxoglutarate aminotransferase
2-oxoglutarate
(Fe.S cofactor)
 Incorporation into other
compounds
do plants not use the other pathway?
• All nitrogen enters the biosphere via the GS/GOGAT
cycle
GS/GOGAT
NO3- NH3
cycle

pyruvate Glutamate oxaloacetate

Transaminases

alanine 2-OG aspartate

how are the other AAs made?

Other amino acids Nucleotide bases

2-OG = 2-oxoglutarate
Step 5 – Storage
• Nitrogen stored in multiple different forms –
depends on duration of storage

Storage proteins:
Organic compounds • Globulins (in
NO3- in vacuole
e.g. amino acids legumes and potato)
• Prolamins (e.g.
glutenins in cereals)

Short term Long term

(weeks) (dormancy)
Short term storage
• Nitrate accumulates in the vacuole at maximum
growth
• Loaded by a nitrate-H+
antiporter since gradient is out of the cytoplasm

• Encoded by a member of
the chloride channel family
CLCa Here
does not safeguard against herbivory. vacuolar storage
can no longer hold
Once the plant has enough
nitrogen, it accumulates as nitrate:
Medium term storage
• Organic compounds
• Protein amino acids – esp Gln, Glu, Asn, Asp, Ala
• Non-protein amino acids defence compounds..
NH2 NH2
| |
C=NH C=NH
| |
CO.NH2 NH NH similarity = dangerous because it
| | | can block up AA transporters and stop AA

CH2 Asparagine (major CH2 O

synthesis

| transport amino acid) | | and get incorporated into proteins causing

misfolding
CH.NH2 (CH2)2 (CH2)2
| | |
CH.NH2 CH.NH2
COOH
| |
COOH COOH

Canavanine
Arginine
(legumes)
Long term storage
• Storage proteins – key nutritional significance
• Globulins in legumes and potatoes
Cys-rich termini
• Prolamins in cereals
• High glutamine and proline content
• Multiple cysteines at N & C termini
not globular - built of
• Repeat proteins = viscoelastic modular blocks — highly stretchy
and quite fibery.
Repeats of a
20 aa motif

Networks of gliadins
and glutenins are
good for baking!
 Hypoimmunogenic wheat?
• Gliadins trigger Coeliac Disease in 1-2% of population
• RNAi has been used successfully to silence
immunogenic gliadins
• No effect on seed production or dough quality
• Plant compensated with other storage proteins

• GM regulations currently prevent this crop coming to

market in the EU (but not the US)
• Time for a rethink?

Jouanin A., et al. (2018) Development of Wheat With Hypoimmunogenic Gluten Obstructed
by the Gene Editing Policy in Europe. Front. Plant Sci. 9:1523. doi: 10.3389/fpls.2018.01523
Changes in the nitrogen
economy
• Arabidopsis lowers NO3- uptake at
reproductive stage
• Nitrogen is remobilised from take up less N
senescing leaves via autophagy
dismantling of chl.
Changes in the nitrogen
economy
• Remobilisation can be a
significant source of N:
• 90% of leaf N goes to the seed in
annuals
• Goes to the trunk in deciduous
trees

• Dedicated transporters for seed

loading: tend to be quite high affinity since generally against gradient
• AtNRT2.7 loads seed
• AtNRT1.6 loads embryo
Role of reassimilation
isoform of glutamine synthetase in the cytosol to capture ammonium

• NH3 released through metabolic processes needs to

be recaptured:
Released during photorespiration:
Released during remobilisation H2O + 2glycine à serine + NH4+ + CO2
so double bad..
or production of chlorophyll
Captured by GS in chloroplast

CO.NH2 COOH Barley

NH3 in detached leaves

| H2O NH3 | mutants
CH2 CH2
| |
CH.NH2 CH.NH2
| Asparaginase |
COOH COOH
WT

Captured by GS in cytosol
Mins illuminated in air
Interplay between C and N

+CO2
Photosynthesis Sugars
+O2
Proteins
Photorespiration Intermediates Biomass
-CO2
Amino acids +
NH4
Carbon skeletons Respiration
-
NAD(P)H NO3
N assimilation -CO2
Thoughts for agriculture Find out more
in Lent term!

• Nitrogenous fertilisers have

boosted yields and averted famines
• Lead to pollution
• Applied in excess and <50% ends up
in crops
• Are energy intensive to produce
• Uses ~1-2% of the world’s energy
supply!
• How can we use our understanding
to boost yields AND decrease
fertiliser use?
So… How does an atom of
nitrogen get from the
atmosphere to part of the
protein Rubisco?
• Made available by diazotrophs
• Uptake via variety of transporters
• Transport in the xylem
• Assimilation into amino acids

N N
PDB: IBXN