IB Plant and Microbial Sciences

Mineral Nutrition

Prof. Julia Davies & Dr. Sonja Dunbar

Lecture 16 Overview
Lecture 17 Potassium
Lecture 18 Nitrogen
Lecture 19 Phosphate
Lecture 20 Micronutrients: Iron and Boron

Plants need a range of nutrients to complete their life cycles. Some nutrients are needed in
large quantities (e.g. N, P, K) and others in smaller quantities (the micronutrients). This
lecture series will look at how plants maintain adequate nutrition by employing a range of
transporters for uptake, and how plants respond to nutrient deprivation. We will also consider
the role of distribution and storage mechanisms within the plant.
These mechanisms have key implications for agriculture, ecology and human health.
1B PMS Nutrition Lecture 16 Overview Julia Davies jmd322cam.ac.uk
1 Introduction – the importance of plant nutrition
Diversity Ability to access nutrients from soil/water determines species presence (e.g.,
Ericaceae on low N acid heathland soils). This will also be affected by presence of
mineralising microbes and symbionts (Rhizobia/mycorrhizal fungi).
Human health Entry point for nutrients into food webs. Human health depends on plant
uptake however cereals are low in key minerals (Fe and Zn) leaving millions worldwide
suffering from nutrient deficiencies. Biofortification through fertilisers could alleviate some
cereal deficiencies (e.g., Se in British wheat) or we produce plants with more efficient uptake
systems.
Sustainability NPK fertiliser production contributes to greenhouse gas emissions and run-
off can pollute. P reserves are running out. There is an urgent need to produce more efficient
crops.
Bioremediation Hyperaccumulators have evolved to tolerate otherwise toxic concentrations
of metals (Cd, Cu, Zn…). These plants could be deployed/exploited in land clean-up.
2 Soil – the basics
Soil horizons Available minerals will depend on bedrock, subsequent
weathering/depositions and vegetation history. The greater the plant colonisation, the
greater the humic (organic) content of the topsoil as plants decay.
Cation Exchange Capacity (CEC) Soil particles are negatively charged and can bind
cations that are exchangeable with soil water. CEC is affected by particle size distribution,
humic content and pH. Clayey soils have higher CEC than sandy as clay particles are larger
but have a problem with tendency to waterlog and hypoxia. High humic content results in
high CEC and better drainage. Maintaining high humic content is therefore beneficial for
productivity.
Soil mineral content will therefore vary but it will determine plant yield.
This fundamental tenet of plant nutrition is exemplified by the results shown below for barley.
As available K+ in the soil increases, so does the K+ concentration in leaf cells (left graph)
and as the latter parameter increases, so does yield (right graph).

At the end of this lecture block, try defending the statement “Mineral availability will
ultimately determine yield”.

3 Nutrient requirements; uptake and distribution

Nutrient requirements Plants require a range of minerals to complete their lifecycles;
2
 macronutrients are required at 1000-1500 mg/kg DW. Examples of macro and micronutrients
are given below.

Elements Functions

N, S Assimilated into organic compounds

P, B Energy storage and structural integrity

K, Ca, Mg, Cl, Mn Remain as ions. Osmotic relations, signalling, enzyme cofactors

Fe, Zn, Cu, Ni, Mo Redox reactions

There is a dose-response relationship for

each mineral; the “adequate zone” supports
maximum growth. To either side of this lies
deficiency or toxicity that can impair growth
and cause lesions (e.g., cork spot Ca
deficiency, leaf tip necrosis B toxicity). The
transition zone between the two is the
range in which fertiliser application should
be considered.

Can you see that the shape of this graph is the same as for barley K + content as a
function of soil availability?

Mineral uptake and content vary with species and reflects the habitat the species has
evolved in.

This can be illustrated by growing a plant under controlled

conditions and varying nutrient supply. Avenella has
evolved to grow on nutrient poor soil. It is better able to
grow on low P than the ruderal Urtica. But increase P
supply and Avenella’s growth response saturates before
Urtica’s. Avenella lacks the “machinery” to cope. Can we
exploit plants such as Urtica? Evaluating mineral
composition across species is already proving to be a
valuable resource for breeding programmes, for example
improving selenium content of bread wheat.

3
Uptake and distribution

Minerals reach the root xylem by apoplastic flow (cell wall aqueous phase), symplastic flow
(protoplast to protoplast via plasmodesmata) and the coupled trans-cellular pathway
(transport protein-mediated efflux from one cell allowing transport protein-mediated uptake
by a neighbour).

Symplastic
Apoplastic

Roots can control plasmodesmatal aperture, transporter expression and activity to regulate
uptake. Anatomical adaptations regulate apoplastic flow. The exodermis is an outer cortical
layer, a mixture of passage cells and suberinized cells. The Casparian strip “plugs” the
endodermis which in turn undergoes adaptive suberin deposition controlled by ABA (+) and
ethylene (-). The overall picture that emerges is that the root apex will be a key site for uptake
using all routes but that the older regions will exert greater control of pathways as a function
of environmental conditions, particularly water availability. To what extent do you think
excluding the apoplastic pathway is to minimise efflux?

At membrane protein level, low and high affinity transporters (LATS) and (HATS) enable
uptake and distribution. For example, see blow, this is how they would line up in the root
epidermal plasma membrane (PM).

4
 Extracellular space

Channels are LATS (remember passive transport from 1A Cells and PoO). Antiporters and
symporters are HATS (active transporters) and at the PM symporters would be able to
accumulate a nutrient from a low external concentration. Note the critical involvement of the
H+-ATPase. Transport protein complement and activity will change to enable uptake and
distribution. Nutrient starved plants show higher uptake rates than nutrient replete ones.

Left, uptake rates as a function of

nutrient availability. Lower panels,
roots and upper panels shoots.
How might rate change be
achieved?

Once in the xylem, transpiration is the driver for distribution to aerial tissues – nutrient and
water relations are inextricably linked. Mobilisation is via the phloem…nutrients are moved out
from leaves and this is especially important for seed filling. We know that minerals are not
distributed homogeneously either in organs, tissues or cells. For example in leaves, K+
content of the epidermis is higher than the mesophyll. We need to understand how this is
effected and its significance. It may relate to pathogen defence. We also know that mineral
content varies within the cell, for example see below for K+ distribution between the cytosol
and vacuole in barley root epidermis.

5
 Once again we see that distinctive relationship
between availability and content but this time for
the cytosol. It too has deficiency and adequate
zones. We will see in a later lecture how the
vacuole is involved in this.

We know that at maximum growth (with nitrate as

an example, storage in vacuoles is triggered. They
are an immensely useful cellular resource.

Hyperaccumulators These plants (such as Thlaspi, Alyssum and Macadamia) can

survive on otherwise toxic levels of mineral nutrients or non-nutrient metals. It’s estimated
that there are around 720 species globally and some are under threat of extinction due to
habitat loss caused by mineral mining. Accumulation can facultative or obligate, it can be in
cell types (such as leaf mesophyll) that can appear counter-intuitive to us. Potentially they
are useful for bioremediation but tend to be dwarf and slow growing. Understanding their
transporters (including distribution patterns) may help in the production of faster growing
plants capable of withstanding toxicity.

4 Root adaptations
There is now a drive to produce crops with more extensive root systems, capable of better
soil penetration and nutrient/water uptake. Plants already invest heavily in generating a
ramifying root system architecture (RSA). Under nutrient depletion, assimilates are invested
in roots. Roots system architecture adapts to nutrient supply and we can observe
proliferation of lateral roots in response to localised supply of many minerals (see below).
We can expect different complements of transport proteins in roots that have grown in
response to nutritional availability. These growth changes will involve changes in auxin
distribution. Root hair length and number are also important determinants of nutrient
uptake, often increasing under deprivation. Again, this will involve auxin (link this to
Development lectures in Lent term).

6
 5 Summary

Understanding nutrition is central to ecology, sustainable crop production and human health.
Maximum growth requires levels of each nutrient to be in adequate zones.

Nutrient distribution is heterogeneous at organ, tissue and cell level; mediated by a range of
transporters. We need to identify them and understand their regulation if we are to improve
crops (particularly seeds) and bioremediation.

Root system architecture and transport adapt to maintain adequate nutrition. Understanding
developmental processes is central to nutritional studies.

6 Reading
Try the nutrition chapters of

• L. Taiz and E. Zeiger Plant Physiology, DA 261

• F.B. Salisbury and C.W. Ross Plant Physiology DA 244

The mineral nutrition masterpiece is

• H. Marschner Mineral Nutrition of Plants, DF 68

7
1B PMS Nutrition Lecture 17 Potassium

1 Introduction

There is rarely enough phytoavailable K in soils to permit rapid crop growth; millimolar levels
are desirable. K is needed for ribosome function, metabolism (pyruvate kinase as a key
example) and osmotic relations. K-stressed plants are less pathogen resistant. In this lecture
we will examine mechanisms of K uptake and distribution plus changes in root architecture
as functions of K depletion.

2 Basic mechanisms

If we think about how a plant gets K+ from the soil to the leaves then we can invoke
apoplastic, symplastic and trans cellular pathways in the root to start off with. We must also
be mindful that every cell along the way will need its own K + supply and every vacuole will fill
up. That’s a lot of transport proteins! Measurements of K+ (typically using an ion-selective
microelectode) allow us to predict whether active or passive transporters would be
necessary for uptake and distribution. These are the predictions for barley root epidermis as
a function of available K+.

The plasma membrane (PM) would have to be able to express an active transporter for K +
uptake (probably a K+-H+ symporter) under K+ deprivation but a K+ channel could do the job
under K+ replete conditions.

8
 The plasma membrane H+-ATPase is the cell’s
powerhouse for K+ uptake. With 5 functional domains, it
sets the pH gradient to drive symporters and the
membrane voltage to open channels. Its autoinhibitory
R domain is displaced by phosphorylation and 14-3-3
protein to activate H+ extrusion. This is a great example
of the importance of post-translational modification in
nutrition.
H+-ATPase expression is high at hot spots of
nutrient transfer such as the root epidermis and xylem
parenchyma.

3 Uptake under K+ depletion

Plant genomes contain multiple genes for K+ transporters, all predicted to be membrane-
spanning integral proteins. Starving Arabidopsis of K+ leads to upregulation of HAK5 (High
Affinity K transporter 5). Expression is repressed by K+ re-supply (we will see this is a
common theme). Cereals also contain HAK genes so this is a good example of translational
research.

9
 Heterologous expression in yeast is a useful tool for
demonstrating transport activity. AtHAK5 in yeast
permits accumulation of Rb (used as a tracer for K).
Altering pH provides evidence for HAK5’s being a K +-H+
symporter, so that’s a win for energetic predictions.

Mutant analysis shows that HAK5 supports a significant fraction of root growth under K+
deprivation. However, it is Cs permeable and so puts plants at risk of Cs toxicity. That’s a note
of caution in transporter studies; we need to know about selectivity before we get too excited.
But could this be useful for decontamination of radioactive sites?

4 Uptake from K-replete soils

Energetics suggest that uptake at the root PM under K + replete conditions could occur
through a channel. Again, Arabidopsis has been the lab rat for discovering the channels
involved. Within that extensive family of predicted K + transporters lies a group of what look
like channel subunits. A channel would need 4 subunits each containing a pore region or
2 subunits if each contained 2 pore regions.

10
Distribution of AKT1 in Arabidopsis is consistent with function in K+ absorption (root
epidermis and cortex). Patch clamp electrophysiology is used to test for channel function.

The patch electrode allows

membrane voltage to be
controlled. If channels open, ions
flow through and current is
detected. Negative current is
cation flow (in this case K+) into
the cytosol. AtAKT1 expressed in
insect cells (bottom left) supports
K+ influx into the cytosol at
negative membrane voltage (that
would occur in a plant cell). It has
voltage sensing residues to
permit this. akt1 mutant lacks the
K+ influx current (bottom right).
So it all adds up nicely and even
better, AKT1 is present in
cereals.

5 Loading the xylem and unloading from the phloem

Energetics also point to the need for a K+ efflux channel to load the xylem. The SKOR
(Stelar K Outward Rectifier) channel lets K+ efflux across the plasma membrane of stelar
cells into the xylem. Expressing AtSKOR in Xenopus oocytes (below left) gives the
characteristic current-voltage relationship of an outward rectifier. The Atskor mutant doesn’t
accumulate as much shoot K+ as wild type (below right), showing the importance of this
channel in xylem loading. K+ is important for loading sucrose into phloem. More than 50% of
K+ moving to shoots comes back down to the roots in phloem. Analysis of channel mutants
reveals that another channel AKT2 is probably involved in unloading K + from the phloem in
roots.

11
 6 Vacuolar K+ transport - accumulation

Vacuolar K+ accumulation is critical for osmotic relations. Under K + replete conditions,

active transport is predicted to sequester K+ in vacuoles. There are two H+-pumps in the
tonoplast to set up a H+-motive force to drive K+ uptake by a H+- antiporter – the V-
ATPase and the V-PPase. The latter is stimulated by cytosolic K+ and is likely to be linked
to K+ sequestration. In Arabidopsis, NHX1 and 2 act as K+- H+ antiporters to fill the
vacuole. They can also transport Na+ and are involved in regulating stomatal movement.

Try drawing NHX on the cartoon of the tonoplast below.

7 Vacuolar K+ transport - release

Vacuolar K+ release can “top up” the cytosol; this can be achieved by channels in all but
starvation conditions. Patch clamping has identified several such channels and these are now
being identified at the genetic level. In Arabidopsis, TPK1 is activated by Ca2+ and 14-3-3
proteins to release K+ to the cytosol. Channels would not be able to release K + to the cytosol
during starvation – that would require an active transport system.

Can you put all of the relevant transport proteins in a diagram now?

12
 8 Adaptive growth

How low K+ is sensed remains subject to debate but it triggers ethylene production. This
inhibits elongation of primary and lateral roots but also causes root hair elongation. Ethylene

also triggers HAK5 expression. K+ deprivation leads to increased suberin deposition in the
endodermis and downregulation of SKOR transcription, classic ABA responses linking the
need to retain K+ in the roots under water deprivation with nutrition need.

9 Summary

• H+ pumps are essential

• HAK5 mediates uptake under K+depletion but puts plant at risk of Cs toxicity
• AKT1 voltage-sensitive K+ inward rectifier channel for uptake at higher external K+
• SKOR for delivery to xylem; AKT2 for retrieval from phloem
• NHX and TPK as uptake and release pathways in vacuole
• Ethylene involved in RSA responses

10 Consolidation ideas

Annotate a diagram of a root epidermal cell showing the key transport proteins for K +
relations.

Why do plants have such large gene families for K + transporters?

If you could engineer a Cs hyperaccumulator, what sort of transporters would you make it
have?

Why are K+ transporters important for water relations?

11 Advanced reading
This review takes you beyond 1B and into Part II: Cherel et al., (2014) Molecular
mechanisms involved in plant adaptation to low K + availability. Journal of Experimental
Botany 65 833.

13
IB PMS Nutrition Dr Sonja Dunbar (sdd29)

Lecture 18: Nitrogen

Overview:
- Context and importance of nitrogen in agriculture
- Uptake routes for nitrogen
- Recap of nitrogen assimilation
- Nitrogen transport, storage and remobilization
- Links to carbon metabolism

18.1 Importance of Nitrogen

Nitrogen is the most abundant mineral nutrient in plants.

Around 80% of the nitrogen in leaves is concentrated in the
chloroplast, with 30% of leaf nitrogen in Rubisco alone.

Needed for:
• amino acids
• nucleotides
• secondary metabolites (including defence compounds)

Nitrogen deprivation leads to:

• stunted growth
• photosynthetic capacity decreased
• chlorosis
• early senescence and leaf loss
• poor quality grain

Plants try to avoid this by foraging for nitrogen with their roots
(see Development lectures, Lent term).

18.2 Step 1 – Nitrogen fixation

Performed by diazotrophs with nitrogenase, found in a range

of bacterial taxonomic groups.

Some diazotrophs live in symbioses with plants e.g.

• Rhizobia that form root nodules in legumes and some
woody shrubs.
• Anabaena (cyanobacteria) in the leaves of the water
fern Azolla. Agriculturally important for the cultivation of
rice in paddy fields. (see Beneficial Plant-Microbe
Interactions lectures, Lent term).

1
A majority of diazotrophs are free living in the soil, benefiting
from carbon compounds exuded from the roots of nearby
plants, and will enrich the soil.

18.3 Step 2 – Uptake of nitrogen

Form of nitrogen taken up from the soil depends on pH:

• Acidic soils = ammonium and amino acids (plus some
carnivorous plants).
• Higher pH and aerobic = nitrate.
• Arctic and tundra soils = amino acids.

In some environments urea will also be a key source.

18.3.1 Nitrate uptake

Via proton symporters. Constitutive low affinity as well as

constitutive and inducible high affinity.

NRT1 family (53 genes in At) = low affinity transporters (LATs)

at different locations throughout plant, e.g.
• root epidermis (NRT1.2)
• loading of xylem (NRT1.5)
• epidermal plasma membrane LAT that can convert to a
HAT via phosphorylation under nitrogen deprivation
(NRT1.1)

NRT2 family = high affinity transporters (HATs)

• lateral root epidermal plasma membrane (NRT2.4)
• epidermal plasma membrane HAT upregulated under
nitrogen deprivation by downregulation of a suppressing
microRNA, miR169. Note, is also a two-component
system that requires an accessory protein, NAR2.1, to
form a heterotetramer (NRT2.1)

18.3.2 Ammonium uptake

Ammonium is toxic to plants:

- dissipates proton gradients
- affects uptake of other positively charged ions e.g. K+

AMT family (6 genes in At), including high and low affinity

transporters. High affinity ammonium transporters found in root
hairs and the epidermis. Predominantly low affinity transporters
found further in the tissues of the plant, such as the
endodermis.

2
18.3.3 Amino acid uptake

Low and high affinity transporters in three main gene families

e.g. lysine-histidine transporters, charged amino acid
transporters, amino acid permeases. Diversity reflects
properties of amino acids for transport.

18.3.4 Urea uptake

Urea major N source in animal waste-based fertiliser.

Alternatively, from the breakdown of soil bacteria.

Proton symporters e.g. DUR3, in the root plasma membrane in

Arabidopsis, rice and maize.

DUR3 function in urea uptake was confirmed via heterologous

expression in Xenopus eggs. Further evidence comes from the
expression of the rice gene for this transporter in Arabidopsis.
of the rice urea-proton symporter in these plants increased the
growth of the Arabidopsis seedlings grown on urea as sole N
source.

18.4 Step 3 – Transport

Assimilation in roots or shoots. Pea and radish have over 80%

of nitrogen in the sap in the form of organic nitrogen =
assimilation in the roots. Arabidopsis and pumpkin have only
2% organic nitrogen in the sap = assimilation in shoots. Many
tropical species opt for assimilation in the shoots, while more
temperate plants opt for assimilation in the roots.

Transport of nitrate via xylem. Loaded by low or high affinity

dedicated transporters e.g. NRT2.3 in Oryza sativa. Located in
xylem parenchyma (living cells adjacent to the vessels of the
xylem which load the xylem). Mutants retain nitrate in the roots
and have a resulting decrease in the amount in the shoots.

18.5 Step 4 – Assimilation

Proceeds: Nitrate → nitrite → ammonium.

Ammonium + glutamate → glutamine.

Labelling patterns using 15N (detected via mass spectrometry)

show same pathway whether plants fed with nitrate or nitrite.

Note – nitrite is toxic due to chelating metal ions.

3
high affinity nitrate and nitrite reductases
18.5.1 Nitrate reductase

Dimeric enzyme found in the cytosol. It facilitates the transfer of

electrons from NADPH or NADH to nitrate to reduce it to nitrite.
These electrons are funnelled through a self contained electron
transport chain comprised of the three key domains:
• FAD
• Cytochrome b557 (haem-containing)
• Molybdopterin (Mo-containing)

Tightly regulated control point between N and C.

Factors that signal availability of carbon-based acceptors for

toxic ammonium promote transcription e.g. circadian clock,
light, sucrose. Factors that suggest assimilation pathway
backed up inhibit transcription e.g. glutamine.

Individual enzyme activity controlled by phosphorylation by a

dedicated kinase, creating a site at which an inhibitory 14-3-3
protein can bind and inactivate the enzyme. Triose and hexose
phosphates inhibit the activity of nitrate reductase kinase.

18.5.2 Nitrite reductase

Monomer, located in plastids. Contains a siroheme prosthetic

group and a 4Fe4S cluster at the active site. It transfers
electrons from ferredoxin (photosynthetic cell) or oxidative
pentose phosphate pathway (non-photosynthetic cell) to reduce
nitrite into ammonium.

Evidence of important of enzyme in tobacco plants with

antisense for nitrite reductase. Plants grow poorly, are chlorotic
and accumulate toxic nitrite (~5x as much as WT).

4
18.5.3 GS-GOGAT cycle

Ammonium incorporated into amino acids. Glu, Gln, Ala, Asp

and Asn accumulate first (shown by tracking amino acids
accumulated after nitrogen starved plants have nitrogen
returned).

Experiments with 15N show that the amide group of glutamine is

labelled first when ammonia is combined with glutamate by
glutamine synthetase (Km ~20 µM). Then 15N is incorporated
into the amino group of glutamate when the amide transfers the
nitrogen and hydrogens to 2-oxoglutarate. This is performed by
glutamine-2-oxoglutarate aminotransferase or GOGAT.

The stoichiometry of the process means that the regenerated

acceptor can keep cycling, while another copy of the molecule
can be used to donate nitrogen to other compounds.

Transport out of the plastid removes glutamate from the GS-

GOGAT cycle. Then transaminases facilitate the transfer of
NH2 to an oxo acid. Depending on the identity of the oxo acid
any of the amino acids or nucleic acid bases can be formed.

18.6 Step 5 – Storage and remobilisation

Variety of formats for storage, depending on the intended

duration of storage.

18.6.1 Short term storage

Excess nitrate stored in the vacuole. Vacuole loading

transporters are proton antiporters.

5
18.6.2 Medium term storage

In the form of protein and non-protein amino acids e.g.

• Asparagine (major transport amino acid)
• Canavanine (also a defence compound)

18.6.3 Long term storage

High molecular weight storage proteins used for surviving

dormancy or in seeds e.g.
• Globulins in legumes and potatoes
• Prolamins in cereals

Key nutritional significance as they form a major protein source

for both humans and domesticated animals.

Prolamins have a high glutamine and proline content and are

repeat proteins with multiple repeats of a short 20 amino acid
motif. Excellent in baking and cooking due to their intrinsic
properties. Gluten is a composite of prolamins (glutenins and
gliadins). Within a dough you will find networks of gluten held
together by disulphide bonds. The tandem repeats allow
prolamins to have viscoelastic properties allowing them to
unfold repeat sections without breaking the protein and expand
around bubbles of carbon dioxide produced by yeast. Glutenins
provide this elasticity while gliadins provide viscosity.

Downside: wheat gliadins contain immunogenic peptide

sequences that can cause coeliac disease. Coeliac disease
results in an inflammation of the small intestine which affects
nutrient absorption and causes a range of symptoms. Affects
~1-2% of the human population.

Possible solution: hypoimmunogenic wheat. RNAi can reduce

expression of key gliadin families by 97%. The resulting wheat
did not stimulate an immune response triggered via T cells in
CD patients and also did not affect seed germination or dough
quality. It was subsequently shown that although around 20
gliadins showed reduced expression, the plant compensated
with increased use of alternative storage proteins.

However, we have a barrier to the use of these crops in Europe

- maybe it is time for a rethink of how these crops are judged.

More info in: Jouanin A., et al. (2018). Front. Plant Sci. 9:1523.
doi: 10.3389/fpls.2018.01523

6
18.6.4 Remobilisation

Plants have higher influx of nitrate during the vegetative stage

and this decreases during the reproductive stage.

As uptake is switched off, remobilisation is key to ensure

maximal use of( the nitrate acquired over the plants life.
Proteins dismantled back to amino acids and nitrate for
transport.

In an annual plant, where the entire plant dies off after setting
seed, the plant remobilises up to 90% of the nitrogen in its
leaves and sends it to the seeds. When deciduous trees lose
their leaves much of the nitrogen is scavenged back and is
stored in the tree trunk.

Dedicated members of the NRT1 and NRT2 families are key for
loading seeds.

18.6.5 Reassimilation

Some metabolic processes release toxic ammonia that must be

reassimilated.
1. Remobilisation - captured by a cytosolic form of
glutamine synthetase.
2. Photorespiration – when Rubisco adds O2 rather than
CO2 (~25% of reactions). Creates glycine leading to
ammonium release (10x rate of 1ry assimilation!):

Reassimilated by the chloroplast based glutamine

synthetase. Evidence from barley mutants that lack the
chloroplast enzyme. Mutants grow well in elevated CO2
(photorespiration is reduced), however we observe
elevated levels of ammonia in the mutants compared to
wild type after exposure to ambient CO2 concentrations.

18.7 Interplay of C and N

Multiple points of interaction between carbon and nitrogen

metabolism:
• Nitrate reductase regulation
• Availability of nitrogen itself impacts photosynthetic
capacity of a leaf
• Reassimilation due to photorespiration

Ultimately affect biomass of plants.

7
18.8 Agricultural perspectives

Many of the efforts to increase agricultural yields involved new

varieties that require the use of fertilisers including large
amounts of nitrates. These advances, during the Green
Revolution averted many hunger crises.

But there are issues with extensive fertilizer use:

1. Pollution. Nitrates are very mobile in the soil and are
applied in excess. Over 50% of applied nitrates leach
out into surrounding aquatic ecosystems and cause
algal blooms, eutrophication and stagnation of the
ecosystem.
2. Energy use. Production of ammonia, via the Haber-
Bosch process, uses ~1-2% of the world's energy
supply.

The challenge is how to reduce fertiliser use while maintaining

or even increasing yields.

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

A quiz/revision aid on nitrogen uptake and assimilation is

available on Moodle alongside these lecture notes.

8
1B PMS Mineral Nutrition Lecture 19 Phosphate

1 Introduction – Pi

Pi is central to energy metabolism and is a plant macronutrient. Although 90% of

angiosperms have evolved symbiotic relationships with fungi to enable acquisition, our crops
require Pi input for acceptable productivity. Rock Pi reserves for fertiliser production are
being exhausted, leaving us with a potential crisis. Understanding how plants adapt to low Pi
is therefore central to Food Security research.

2 Nutritional requirements

Pi has structural and metabolic roles:

• Energy donors ATP, PPi

• Phospholipids
• DNA RNA
• Starch/sucrose synthesis
Protein modification…..phosphorylation

It is absorbed as its oxidised anion and incorporated by phosphate esters with CBH (C-O-P),
pyrophosphate bonds (P-P) or diester bridges (C-P-C). Storage is as polyphosphates in the
vacuole (phytate in seed) and remobilisation is as an anion.

3 Deprivation

• Low metabolism
• Sulpholipids replace phospholipids
• Stunted growth, chlorosis, anthocyanins
• Delayed flowering
• Poor seed quality (low phytate)
• Leaf drop
• Poor frost resistance (suboptimal membranes)

4 P availability and fertiliser application

Pi is usually a limiting factor for growth. Pi is fixed in soils, bound to Fe, Al or Ca. Release is
pH dependent, with very narrow soil “windows” of availability. Acid and alkaline soils are
particularly bad for Pi release and chemical intervention risks increasing Al availability.

9
Microbial phosphatase activity is important for release from organic and inorganic sources,
hence soil “health” in the form of microbial populations is essential. Fertiliser application is
wasteful, with less than 10% taken up. More than 31 M tonnes manufactured phosphates
are used worldwide every year.

5 Pi acquisition mechanisms

Available Pi is typically 1-10 μM but is needed at mM level. Low mobility means that roots
easily set up a “depletion zone”. Around 90% of angiosperms rely on mycorrhizal (myc)
fungal symbioses for Pi acquisition; ectomycorrhizal or vesicular-arbuscular. Love these
more in the Lent term’s lectures.

Intensive agriculture has resulted in spore depletion, so Pi fertilisers are applied to crops. In
forestry, there are instances of spore inoculation of saplings to aid establishment.

Adapting to low Pi involves changes in root system architecture (RSA). Cluster roots form in
several families in response to deprivation in the absence of myc. These are tertiary laterals
forming on secondary lateral roots. White lupin is a useful model. Cluster roots secrete organic
acids to lower soil pH and help mobilise Pi and will also secrete phosphatases. Export of
10
organic acids requires adjustment of respiration and C allocation. Many members of the
Proteaceae (Southern hemisphere, growing on poor soil) have cluster (or “proteoid”) roots
(and don’t form myc symbioses). These will take up Pi in the winter for use in spring growth.

Many plants will produce basal roots to exploit topsoil Pi but this puts them at risk of
droughting.

Brassicas (Arabidopsis) are an excellent model system for exploring Pi deprivation

responses as they never form myc symbioses. How low Pi is sensed is not fully understood
but involves the LPR multicopper oxidases in the root cap ER. Their activation contributes to
cessation of primary root growth. There is an increased lateral root production, root hair
numbers and lengths increase. In Arabidopsis there is normally alternation of epidermal cells
for hair production but under Pi deprivation non-hair cells will reprogramme to produce hairs.
These changes in RSA are commanded by auxin. Root hairs increase absorptive surface
area and contribute to uptake. Mutants defective in root hair growth can’t respond well and
these will help us to understand growth regulation.

11
Over 300 At gene transcripts are upregulated in response to Pi deprivation, 33% are
transcription factors - AtPHR1 (PHosphate starvation Response) is the first. AtPHR1 -
upregulates root PM high affinity H symporters AtPht1;1 and AtPht1;4. There are 9 PHT1
genes in Arabidopsis. AtPht1;1 and AtPht1;4 are in root hairs; AtPHF1 (PHosphate
transporter traffic Facilitator) aids transport of AtPht1;1 and AtPht1;4 from ER to PM.

PHT1s are conserved in rice, where OsPHT1,9 and 1,10 support Pi uptake under
deprivation. Overexpression can significantly increase Pi content.

To maintain shoot levels, AtPHO1 is upregulated to aid AtPHT1,2 load the xylem.
PHOs are membrane proteins and it is unclear if they transport Pi. AtPHO2 is a negative
regulator; it is silenced by microRNAs induced by AtPHR1.

12
 all upregulated by master regulator
Within the plant, Pi distribution is enabled by families of transporters:
PHT2 family – plastid carriers
PHT3 family – mitochondria
PHT4 family- mostly plastid envelope
pPT family –plastidic phosphate translocators, inner envelope.

Studies must now address which are upregulated during deprivation. We are now starting to
understand how vacuoles accumulate and release Pi. Under Pi deprivation, Arabidopsis
increases expression of its 5 Glycerol-3-Pi transporter homologues which could be involved
in vacuolar Pi efflux. In rice, the VPE 1 and VPE2 transporters efflux Pi to the cytosol under
Pi deprivation. NMR shows vacuolar Pi retention in the double mutant (DM), which grows
poorly when Pi starved.
phosphate not mobile

Deprivation may be severe enough to trigger mobilisation from shoots to roots. Production of
cytokinins in roots is lowered so less reach the shoots; this helps promote this resource
resource allocation. Shoots are probably also capable of sensing low Pi and react
accordingly. Shoot Pi is sent to root; more sucrose sent too. This may be as a signal to
adapt or to fuel adaptive responses.

13
 Split root experiments are used to
explore nutrient remobilisation
and also water relations. In the
example opposite, high affinity
transporters were not induced in
the Pi-deprived root section
because the plant could absorb
Pi from the replete medium and
re-allocate it via the shoot

6 Interplay with S

14
S is also a macronutrient and in the past, crops have benefitted from foliar application from
industrial pollution. In Europe, some areas are now experiencing S deprivation as air quality
has increased and NPK fertiliser production no longer uses sulphuric acid. S deprivation can
worsen P deprivation because phospholipids can be replace with sulpholipids.

S is taken up as SO42- by low and high affinity H+-coupled symporters and stored in vacuoles
under replete conditions. High affinity symporters are induced in roots by starvation but
repressed when S supply returns. SO42- is transported into chloroplasts for assimilation into
cysteine. It is also used to make glutathione which is important in maintaining redox balance.
S can be stored and transported as glutathione.

7 Summary
We’ve seen that bioavailability of Pi is problematic. Myc symbiosis is a key adaptation but
without them, it’s RSA, secretions (that have an impact on C) and high affinity uptake. We’ve
seen a microRNA involved again, multigene families of transporters, “helper” proteins (PHF,

PHO) and signalling within the plant to direct resources to the root. We’ve also seen that
Text
limitation of a nutrient can be worsened if the substitute nutrient is limiting too.

8 Consolidation

Time to start spotting (and maybe tabulating) commonalities between nutrient deprivation
responses.

Why so many Pi transporters?

What don’t we know about Pi deprivation?

15
1B PMS Mineral Nutrition Lecture 20 Micronutrients

1 Introduction

In this lecture we will examine two critical micronutrients, Fe and B. In previous lectures
we have considered how plants cope with nutrient limitation but these two micronutrients
also show us how levels must be tightly controlled to avoid toxicity.

2 Fe – roles in the plant, storage and deprivation

Fe is an extremely valuable but potentially dangerous micronutrient. It can be present at up

to 11 mg per 100 g, making plants a useful Fe source in the human diet. However, it is not
readily bioavailable from cereal seeds due to chelation by phytate.

Fe is the most abundant transition metal in plants. In the plant, Fe is chelated by haems and
sulphur clusters (Fe-S) to act as an enzyme co-factor. As an efficient acceptor and donor of
electrons, the Fe in these co-factors acts in respiratory and photosynthetic electron
transport chains.

The bulk of leaf Fe is held in chloroplasts. Control of Fe bioavailability is important because

in its free form it is a Fenton catalyst that produces hydroxyl radicals. These are the most
potent reactive oxygen species and damage proteins, lipids and DNA. In some cases,

1
Fenton- generated radicals are used positively; for example in elongation growth they cause
wall weakening.

Excess Fe is captured by ferritin, a globular storage protein, to avoid toxicity in the cytosol,
mitochondria and chloroplasts.

Fe can be stored in the vacuole, loaded by VIT1 and FPN2 transporters. Fe is also stored in
the apoplast, bound to pectin.

Fe can be abundant in soils but not bioavailable and 30% of agricultural land worldwide is
Fe-limiting. In common with Pi, pH is the problem.

If plants can’t acquire sufficient Fe, chlorophyll synthesis is inhibited leading to extensive
inter-vein chlorosis and impaired growth/reproduction. N assimilation will be impaired.

2
3 Capturing Fe

Plants can be divided into Strategy 1 (dicots and non-grass monocots) and Strategy 2
(grasses).

In Strategy 1, the root plasma membrane (PM) H+-ATPase acidifies the rhizosphere to help
solubilise Fe3+ which is then captured by secreted chelators. A PM ferric-chelate reductase
reduces Fe3+ to Fe2+ and the latter is transported into the cell.

In Arabidopsis, AHA2 encodes the H+ pump, FRO2 encodes the ferric chelate reductase and
both are induced upon deprivation. The uptake transporters are encoded by IRT1/2 (which
can complement a yeast Fe2+ uptake mutant) and are also induced. IRT1 can also transport
Zn, Mn but also Cd (toxic).

The Fe sensor in roots has not been identified yet but early responses are well understood.
Over 600 genes are upregulated. bHLH transcription factors (including FIT) are upregulated
and bind to FRO2/IRT1 promoters. The mediator multi-protein complex links these to the
RNA polymerase Pol II to activate transcription.

3
Changes in RSA are subtle; root hair elongation increases and under moderate deprivation
there is some elongation of primary and lateral roots.

In Strategy 2 phytosiderophores are secreted. Phytosiderophores are nicotianamine (NA)

and its derivatives. These capture Fe3+ and are taken up by specific transporters such as
YS1 without reduction.

Here’s a really neat experiment that allows us to deduce that YS1 is taking up Fe3+ not
Fe2+. Express IRT1 or YS1 in the Fe transport-deficient yeast. Growth on a medium
containing DMA as the phytosiderophore and BPDS to chelate Fe 2+. IRT1 can’t support
yeast growth (there’s no Fe2+)…but YS1 can.

4
YS1 is a H+-symporter and a member of the oligopeptide transporter family (OPT). These
only occur in walled organisms and transport NA plus its derivatives.

4 Uptake by chloroplasts, mitochondria and remobilisation

Fe is transported in the xylem chelated with citrate. Entry into the chloroplast is by the CIP1
permease in Arabidopsis (a mitochondrial Fe uptake transporter has been identified in rice).

The plastids incorporate Fe into haem and Fe-S clusters. In chloroplasts these will be used
in, for example, ferredoxin. Mitochondria also manufacture Fe-S clusters; their assembly in
the cytosol requires release of an unknown substrate from mitochondria by the ABC
transporter ATM3.

5
How leaves sense low Fe supply is unknown but one possibility is through sensing the
availability of Fe for Fe-S synthesis by the organelles that then signal to the nucleus
(retrograde signalling). Fe can be retrieved from the apoplast under deprivation.
Remobilisation of Fe stored in vacuoles requires NRAMP transporters for Fe efflux. Fe
is redistributed in the phloem chelated to nicotianamine (NA). OPT transporters
retrieve the Fe- NA. The rice OsOPT3 is present in the leaf phloem and if mutated,
roots display a constitutive Fe deprivation response. This is because there is
insufficient Fe coming down from the shoots and the roots sense the reduced flux.
OPT transporters are also involved in loading seed with Fe. Unfortunately, they are
Cd-permeable; another example of how we need to know about transporter selectivity.

6 Interplay with S, Pi and ABA

Achieving a balance between Fe and S is critical for cluster assembly and to avoid Fe
toxicity. Under ++++Fe, S uptake is increased and vacuolar S efflux is increased by
upregulation of SULTR transporters. Under -S, phytosiderophore secretion and induction of
Fe uptake transporters can be suppressed. Pi deprivation can cause Fe overload; IRT1 is
downregulated and ferritin production is upregulated to avoid oxidative damage.

-Fe can induce production of ABA. Application of ABA can alleviate -Fe symptoms by
promoting uptake by roots and release from vacuoles.

7 Boron

B is easily limiting or toxic in soils, it has the narrowest range of all the minerals. It can be
deficient in high rainfall areas (SE Asia) and toxic in semi-arid regions that can still be used
for agriculture (Australia). It is present as electroneutral H(BO)3 and is taken up by plants in
this form. B is required for wall integrity, binding to pectins. Deficiency stunts growth and
causes male sterility (poor pollen grains and pollen tube growth). Toxicity causes leaf
necrosis and root growth inhibition, leading to 20% losses in Australia.

Uptake is by NIP channels (related to aquaporins). The Casparian strip forces symplastic
flow and BOR transporters efflux B to the xylem. NIPs and BORs are upregulated under B
deprivation (overexpression improves performance) then suppressed when B is replete.
BOR is ubiquinated to target it for endocytotic disposal. NIPs also operate in B distribution in
leaves. Redistribution is via the phloem.

6
To understand the basis of toxicity tolerance, a QTL analysis was undertaken of a cross
between a B-tolerant non-agronomic barley landrace (Sahara) and a B-sensitive Australian
commercial variety (Clipper). Sahara has a high B efflux rate from roots and low shoot B.
One of the genes identified by QTL was BOT1 (related to AtBOR1). Heterologous
expression showed this to be a B efflux transporter that is expressed in root tips. Sahara
BOT1 is a more efficient transporter than Clipper BOT 1 or AtBOR1; it differs by only 2
amino acids. Additionally, Sahara has more copies of the gene than Clipper. In a later
study, Sahara was found to be more salt tolerant than Clipper, even though it accumulates
more Na+ in its leaves. QTL should enable identification of genes conferring tolerance.

8 Summary

Rhizosphere modification is an important adaptation to low Fe availability, as is the chelating

systems for guarding against toxicity one Fe is acquired. Once again, multi-gene transporter
families are involved in uptake and (re)distribution. Signalling of deprivation may involve
retrograde signalling. Post-translational modification is also involved in transporter down-

7
regulation. Plants alter their nutrient capture to achieve balance between minerals. QTL is a
powerful approach for identifying critical components. We could improve plants by changing
just two amino acids.

9 Consolidation

Annotate a diagram that tells the story of Fe in a plant. Think “what if?” (“what if Fe and Pi are
limiting or Fe, P, S?” etc) ; jot down the chain of events/ annotate diagrams. Start collecting
examples of…post-translational modification…induction/suppression….Consolidate nutrient
transport at the vacuole in a diagram.