The Plant Cell, December 2014, www.plantcell.org ã 2014 American Society of Plant Biologists. All rights reserved.
TEACHING TOOLS IN PLANT BIOLOGY™: LECTURE NOTES
Plant Nutrition 2: Macronutrients (N, P, K, S, Mg, and Ca)
The study of plant nutrition requires an experimental approach;
little can be learned merely by direct observation. From the time
of the Greeks until the 17th century, it was assumed based on
observations that plants were composed mainly of water, and
the role of mineral nutrients was poorly understood. A key study
published in 1699 changed that, when John Woodward showed
that plants grew better in water drawn from the Thames River
(with its obvious “mineral matter” that he describes in some
detail) compared with those grown in much purer and clearer
springwater or rainwater. From these studies, Woodward correctly
concluded that “Earth, and not water, is the matter that constitutes
vegetables.”
From Woodward’s time to the present, countless scientists
have contributed to our understanding of plant nutrition. Marschner
(2012) is an excellent textbook and source for historical studies.
An older but equally significant textbook is Justus von Liebig’s
1841 “Die Organische Chemie in ihre Anwendung auf Agrikultur
und Physiologie” (published in English as “Organic Chemistry in
its Applications to Agriculture and Physiology”). Von Liebig
described the importance of N, P, and S for plant nutrition and
also popularized the idea, based on Carl Sprengle’s work, that
a plant’s growth is limited by the nutrient in shortest supply. This
idea is known as the Law of the Minimum, and examples of it at
work will be found in this article.
From 1843 onwards, Joseph Gilbert and John Lawes experi-
mented extensively on the nutrient requirements of plants on
Lawes’ estate, which is now known as Rothamsted Research
Station (UK) and is the longest running agricultural research
station in the world. Their collected works were published in
1895 as “The Rothamsted Experiments.” One of their major
findings was that phosphates in bones and minerals could be
made more available for uptake by plants by acid treatment.
Their experiments were largely funded by sales of the resulting
“superphosphate,” the first commercially produced artificial
fertilizer.
By the start of the 20th century it was well established that the
six elements we know as macronutrients, N, P, K, S, Mg, and Ca,
are each indispensable for growth. Macronutrients are required
in relatively large amounts: 1 to 20 mg/g fresh weight, compared
with micronutrients that are required on the order of mg/g fresh
weight (and which are discussed in a separate article). Here, we
examine how macronutrients contribute to plant growth. Specif-
ically, we look at (1) the availability of nutrients in the soil along with
the effects of soil microbes and physical properties on their
availability; (2) nutrient uptake from the external environment,
across plasma membranes and into plant cells; (3) in some cases,
the assimilation of the nutrient into organic molecules; (4) the
distribution and redistribution of nutrients throughout the plant;
and (5) regulation of these processes. In parallel, we will examine
www.plantcell.org/cgi/doi/10.1105/tpc.114.tt1214
the genetic basis of a plant’s nutrient use efficiency (NUE) and
evaluate strategies by which to replenish the nutrients that growing
plants extract from soil.
NUTRIENT USE EFFICIENCY
NUE is usually defined as the amount of grain or other harvested
material divided by the amount of nutrient supplied to the
plant (in other words, yield per unit input), but there are several
other ways to calculate NUE depending on the purpose (see
Good et al., 2004). As an example, only ;30 to 50% of the
nitrogen applied to a field ends up in the harvested grain, and
this value can be much lower. Because of the high costs of
nutrient fertilizers, considerable effort goes toward improving
crop plant NUEs through breeding and agronomic practices.
NUE varies by species but also by variety. High NUE varieties
can be particularly important for farmers growing in resource-
poor environments, where large inputs of fertilizers are not
feasible.
NUE is determined by a very large number of genes and
processes and can be difficult to select for directly, but as more
is learned about the factors involved, it becomes increasingly
feasible to identify and select for improvements. NUE depends
on the physiology, anatomy, and morphology of the plant as well
as the nutrient fertilization regime. It is frequently considered to
be the product of two separate processes: nutrient uptake
efficiency and nutrient utilization efficiency.
Macronutrients are usually taken up as inorganic ions from the
soil or surrounding environment, which in vascular plants takes
place largely through roots. Root systems have a large surface
area to maximize contact with the soil. Nutrient uptake is
determined by root system architecture and morphology, root
exudations, mycorrhizas, and the activities of various transporters,
as well as the availability and distribution of nutrients in the soil.
Nutrient utilization usually involves the assimilation of the
nutrient into organic forms and the recycling and remobilization
of the nutrient to maximize plant productivity within the context
of finite resources. Across the plant’s lifespan, NUE is affected
by how the nutrient is used when it is abundant and how the
plant responds when it is limiting. Responses to nutrient limitation
involve conserving and optimizing nutrient use through decreased
growth rate, metabolic shifts that minimize requirements for the
nutrient, or remobilization of the nutrient from less critical to more
critical tissues (e.g., seeds and growing tissues). For years,
modeling approaches have been used to understand this complex
“trait” and judge when and how to apply supplemental nutrients.
With modern sensors and assays, -omics approaches, and
systems biology methods, these models are becoming ever
more sophisticated and are helping to reveal the molecular
parameters involved, which will be useful for breeding efforts.
2 The Plant Cell
SOIL-NUTRIENT HETEROGENEITY
Although our topic focuses primarily on the processes controlled
by the plant, the understanding of plant nutrition also requires an
understanding of soil properties and the roles of soil-dwelling
microbes. The physical, chemical, and biological properties of
soils are tremendously heterogeneous and dynamic. Soils are
composed of crushed rocks from various geological eras, oceanic
and aquatic sediments, volcanic debris, and decomposing organic
materials. The science of soils incorporates an immensely broad
and specialized vocabulary with which to describe these diverse
soil types (for example, see IUSS Working Group WRB, 2014).
Young soils such as those found at sites of volcanic activity (e.g.,
Hawaii) are often rich in P but poor in N. As soils age, P leaches
away and becomes less abundant, whereas N levels increase due
to microbial N fixation. Beyond simply aging, soil nutrient
availability changes with human or natural activities. Soils can be
rejuvenated through the addition of new materials as a conse-
quence of glaciation or dust borne by wind, and soils can lose
nutrients as a consequence of erosion. Ice age glaciations
contributed to the relatively rich soils in the northern hemi-
sphere. By contrast, parts of Australia, South America, and
southern Africa have geologically ancient soils that are frequently
P deficient. Many species endemic to these regions have evolved
special adaptations for survival on nutrient-poor soils.
Soil pH is an important consideration for the availability of
nutrients, particularly those that are taken up as positively or
negatively charged ions. Soil pH can affect the electrochemical
gradient across plant membranes, as well as the ionic form of
some nutrients. Many ions form insoluble complexes with other
minerals in a pH-dependent manner. All things considered, for
most plants, a neutral or slightly acidic soil pH is optimal.
Finally, soil microbes have direct and indirect effects on plant
nutrition. Nitrogen-fixing bacteria and mycorrhizal fungi are
important contributors to nutrient uptake for some or most
plants, respectively, and are discussed in detail in Teaching
Tools in Plant Biology 19: Plants and Their Microsymbionts.
Numerous and incredibly diverse soil microbes alter soil nutrients
in a variety of ways ranging from the enrichment of soil through
decomposition and recycling of organic matter, to the removal of
fixed nitrogen by denitrifying bacteria. Many of the contributions
of soil microbes to plant nutrition, as well as the influences of
plants on soil microbial communities, are just being described
with the advent of large-scale metagenomic sequencing projects
of the soil microbiota.
REPLENISHING NUTRIENTS EXTRACTED FROM SOIL
In agricultural systems with low productivity, soil can be
replenished naturally, by lying fallow or by growing legume
rotation crops that enrich soil. In agricultural lands, soil nutrients
that are depleted by high-yielding crops must be replaced by
some sort of fertilizer, which can be composed of composts and
animal manures, blends of inorganic salts, or a combination of
these. The three major elements found in most fertilizers are
N, P, and K, and we will examine the sources of these fertilizer
compounds as we discuss them individually. Suffice it to say
that the global trade in fertilizers is a multi-billion-dollar business
annually and a considerable contributor to food prices. Their
costs, their sometimes limited availability, and the environmental
impacts associated with their use make it imperative that
fertilizer application be managed appropriately. Furthermore, it
is important to note that the health of the humans and animals
that eat plants is affected by the nutrient status of the plant, and
it is common for animals that eat nutrient-deficient plants to
themselves experience poor nutritional health.
NITROGEN: THE MOST ABUNDANT MINERAL ELEMENT
OF A PLANT
After C, H, and O, nitrogen is the most abundant element in
a plant or animal body, and under some conditions (e.g., post-
glacial North America and Europe) nitrogen is the mineral
nutrient that most limits plant growth. For many crops, optimal
growth demands the application of large amounts of nitrogen,
on the order of 20 to 50 g per kg harvested. Nitrogen is an
essential component of amino acids for protein synthesis,
nucleic acids for DNA/RNA synthesis, chlorophyll, and a large
number of other nitrogen-containing compounds that are
required for defense and other functions. There are four routes
to N assimilation: uptake as nitrate; uptake as ammonium;
uptake as organic molecules such as amino acids, peptides,
proteins, and nucleic acids; and fixation of gaseous N2. N2
fixation only occurs in association with microbes and is
discussed in Teaching Tools in Plant Biology 19: Plants and
Their Microsymbionts.
The Global Nitrogen Cycle
Nitrogen is one of the most abundant elements on Earth, but it is
largely inaccessible to life because although more than three-
quarters of the Earth’s atmosphere is nitrogen, it is found in the
very stable triple-bonded form of dinitrogen gas (N2). Nitrogen
enters the biosphere when it is “fixed” (reduced), which occurs
by the process of biological nitrogen fixation (by free-living or
symbiotic nitrogen-fixing prokaryotes), by physical processes
such as lightning or by industrial nitrogen fixation. Currently, 120
million tons of N2 are fixed annually to produce ammonium for
fertilizer; this accounts for roughly half of terrestrial nitrogen
fixation and accounts for ;2% of the world’s fossil fuel
consumption. The annual production of industrial fixed ammo-
nium has increased 9-fold since 1960 as a consequence of
increasing population growth and increasing meat consumption;
without the input of nitrogen-containing fertilizers, it would
be impossible to produce enough food for today’s human
population.
Only 30 to 50% of nitrogen in fertilizers is taken up by plants.
The remainder is lost back to the atmosphere as N2 or nitrogen
oxides (which are significant greenhouse gases) or washed
away to contaminate waterways. The energy used to produce
fixed nitrogen and its downstream effects are serious environ-
mental concerns that drive our need to understand how nitrogen
is taken up and used by plants as well as our need to improve
the efficiency of these processes.

Nitrogen Uptake and Transporters
Nitrogen in soil varies in its form as well as its quantity. Industrial
or biological nitrogen fixation produces a highly reduced form
of nitrogen, ammonium (NH41), which in solution is in a pH-
dependent equilibrium with ammonia (NH3). Nitrogen fertilizers
can be applied to soils as urea [CO(NH3)2], NH41, and/or NO32.
Plant residue or animal waste can be in organic forms, including
proteins and peptides, amino acids, or urea, or as their degradation
product NH41. As a highly reduced, energy-storing molecule,
NH41 can be oxidized to NO32 by soil-dwelling nitrifying
bacteria. Therefore, in soils containing oxygen, NO32 is the
major form of nitrogen available to and acquired by plants,
although plants generally fare best when both ionic forms are
present. Some plants, particularly those adapted to acid or
waterlogged soil, preferentially take up NH41. When NH41 levels
are elevated, for example, in flooded soils in which anaerobic
conditions prevent nitrification, plants can experience ammo-
nium toxicity, which occurs due to futile cycling of NH41 and
NH3 across cell membranes.
As charged molecules, NH41 and NO32 require transporters
to move across membranes. Many transporter proteins have
been identified that can transport NO32 or NH41 with varying
affinities and degrees of specificity. For example, both low- and
high-affinity transporters have been identified for each, enabling
nitrogen to be taken up under a wide range of soil conditions.
Some transporters are localized in the plasma membrane and
function in uptake from the soil. Others are located in the vacuolar
membrane and transport ions into or out of this important storage
compartment. Still others are efflux transporters that move ions
outward from the cytosol to the apoplast; these are critical for
moving ions into the xylem stream for long-distance transport and
for the remobilization of nutrients during periods of starvation or
organ senescence.
Several different families of nitrate transporters have been
identified. To accommodate the tremendous range of concen-
trations at which nitrate can be found in soil, some of these are
specialized for very high affinity uptake (at low nitrate concen-
trations of ;1 µM to 1 mM) and some for low affinity uptake (at
high nitrate concentrations .1 mM).
The NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER
FAMILY is a large gene family of nitrate/peptide transporters,
some of which can transport other molecules including the
hormones indole-3-acetic acid or abscisic acid. Most members
of this large gene family have not been functionally character-
ized. NPF6.3 (also known as NRT1.1 or CHL1) was the first
nitrate transporter to be identified, through a screen for chlorate
resistance. Chlorate is a nitrate analog that can be taken up into
a plant and reduced to chlorite, which is toxic. CHL1/NRT1.
1/NPF6.3 is particularly interesting for several reasons. It is
expressed in the root epidermis, cortex, and endodermis and is
involved in nitrate uptake from the soil. It is a dual-affinity
transporter, meaning that it can take up nitrate with both low and
high affinity depending on its phosphorylation status. Furthermore,
it has been identified as a nitrate sensor, as will be discussed
further below. Another family of nitrate uptake transporters is the
nitrate-induced, high-affinity transporters encoded by the NRT2
genes, of which there are seven in Arabidopsis thaliana. NRT2
December 2014 3
transporters were first identified by homology with nitrate trans-
porters in fungi and algae.
Nitrate transport out of cells and into subcellular compart-
ments is just as important as its import into the cytosol. Another
member of the NPF family (NRT1.5; new name NPF7.3) pumps
nitrate from xylem parenchyma into xylem for transport to the
shoot. The energy for nitrogen assimilation comes from photo-
synthesis, so much of the nitrogen acquired from the soil is carried
to the shoot as inorganic ions. In the shoot, it can be stored in the
vacuole (pumped in by chloride-channel transporters CLCa and
CLCb) or assimilated in the cytoplasm. As an anion, nitrate can be
transported by some nonselective anion channels as well as some
in the chloride channel family. Nitrate also can be transported by
some members of the slow anion channel-associated 1 homolog 3
SLAC/SLAH family.
Plants also have specific and passive transporters for other
nitrogen-containing compounds, including transporters for urea,
amino acids, and ammonium; see Nacry et al. (2013) for more
information on these transporter families.
Primary Nitrogen Assimilation
Ammonium is a reduced form of nitrogen ready for assimilation
into amino acids, whereas nitrate must first be reduced to
ammonium, either in the root or after transport to the shoot;
where these processes take place depends on the species and
on various environmental conditions. NO32 is reduced by the
sequential action of nitrate reductase in the cytosol and nitrite
reductase in the plastid. Nitrate reductase is a large enzyme with
a complex catalytic scheme; electrons from the electron donor
are passed to FAD then heme, with the final transfer to NO32
involving an essential covalently attached molybdenum co-
factor. Nitrite reductase uses ferredoxin, an electron acceptor in
the light-harvesting photosynthetic reactions, as an electron
donor. Genes encoding theses enzyme are generally upregu-
lated by increasing tissue nitrate concentration.
NH41 produced by the action of nitrate reductase/nitrite
reductase is assimilated into the amino acids glutamine and
glutamate by the action of glutamine synthase (GS) and glutamine-
2-oxoglutarate aminotransferase (GOGAT). This energy-demanding
step requires an ample supply of inorganic nitrogen and available
carbon skeletons, and it is highly regulated. In angiosperms, GS is
encoded by multiple genes, with GS1 genes encoding cytosolic
enzymes and the single GS2 gene encoding a plastid-localized
enzyme. Different isoforms have slightly different physiological
roles that can include primary assimilation as well as reassimilation
during senescence and photorespiration. The regulation of these
enzymes, including their posttranscriptional control, has been
studied extensively because the overall nitrogen use efficiency
of a plant is determined to a large extent by GS activity. Once
assimilated into organic form, nitrogen is transported throughout
the plant and used in the synthesis of other nitrogen-containing
metabolites.
Nitrogen Remobilization
Plants have evolved several strategies by which to optimize their
allocation of assimilated nitrogen. For example, after its assimilation,
4 The Plant Cell
a nitrogen atom may be incorporated into the carbon-fixing enzyme
Rubisco, which can constitute as much as 25% of the protein in
a leaf. As the leaf ages and becomes less photosynthetically
efficient, Rubisco is degraded into amino acids, some of which
release their amino group for reuse. The free NH41 is then
reassimilated by the action of GS, primarily in the cytosol.
Remobilized nitrogen can be translocated to younger leaves as
NH41 or amino acids and then incorporated into other com-
pounds. This process occurs repeatedly as the plant manages
its N reserves. In long-lived plants, leaf N reserves are withdrawn
prior to leaf senescence and shedding. Annual crop plants
remobilize some or most of the N reserves from leaves and
vegetative tissues into their seeds.
Photorespiration is another source of recycled NH41. During
photorespiration, the oxygenase activity of Rubisco produces
the two-carbon compound 2-phosphoglycolate, which is con-
verted to glycine. NH41 is removed from glycine by the action of
glycine decarboxylase and reassimilated by GS in the plastids.
Numerous forward and reverse genetic studies highlight the role
of these GS-catalyzed remobilization and recycling processes
as a major determinant of the overall nitrogen use efficiency of
a plant. In other words, in terms of nutrient use, it’s not just how
much you “earn,” but how wisely you “spend.”
Regulation of Nitrogen Uptake and Assimilation
Plants are very responsive to their nitrogen status as well as to
the availability of nitrogen in their environment. This sensitivity is
evident from studies of transcriptional changes associated with
changes in N status as well as changes in root growth pattern.
Under conditions of N deficit, these changes contribute to an
enhancement of N uptake, for example, by transcriptional induction
of certain N transporters, and changes in N utilization, for example,
the acceleration of senescence and accompanying remobilization
of nitrogen to younger leaves or reproductive tissues.
In addition to downstream N-containing metabolites and
hormones, NO32 itself is a signal that regulates diverse responses
such as lateral root development, leaf expansion, and the tran-
scription of nitrate-regulated genes. A nitrate reductase mutant
allows responses to nitrate to be distinguished from responses to
downstream metabolites. Nitrate sensing occurs by way of the
nitrate transporter/receptor (transceptor) CHL1/NRT1.1/NPF6.3,
and a mutation has been identified in this protein that affects its
nitrate-transport capacity but not its nitrate-sensing capacity,
greatly facilitating the study of this unique protein.
Metabolic and Transcriptional Responses to Nitrogen
Plants respond to nitrogen deficiency by a dramatic reprogram-
ming of transcription; up to 10% of the genome is responsive to
nitrogen resupply after N deprivation. Genes transcriptionally
regulated by N include N transporters and enzymes involved in
N assimilation, such as nitrate reductase and glutamine synthase,
as well as genes involved in carbon metabolism (demonstrating
the close link between N and C metabolism). Beyond transcrip-
tion, many components of the nitrogen uptake and metabolism
pathways are further regulated by posttranscriptional controls,
including the actions of microRNAs, phosphorylation, and
subcellular localization. Genetic studies show some but not all of
the N responses are mediated by NO32 by way of CHL1/
NRT1.1/NPF6.3.
The plant’s responses to nitrogen are also sensitive to the
availability of other metabolites such as sucrose and organic
nitrogen. These metabolites indicate how effectively N can be
assimilated, as well as how great the demand is. Nitric oxide
also contributes to the regulation of nitrogen uptake and
assimilation. Hormones can be long-distance signals of nitrogen
status, particularly cytokinins (CKs), the accumulation of which
generally correlate with nitrogen availability. For example, the
current model proposes that when nitrogen-starved roots are
resupplied with nitrate, CK synthesis is increased in the roots,
CKs are translocated to the shoot in the xylem sap, and
nitrogen-responsive genes are induced in the shoot.
Root Responses to Local and Systemic Signals
It is well established that both local and systemic signals
contribute to root as well as transcriptional responses to nitrogen.
In a landmark study by Drew (1975), when plants were grown in
chambers with different segments of the primary root exposed to
different concentrations of NO32, lateral roots on the root segment
in the region of higher NO32 grew much longer as compared
with those on the lower-NO32 root segment. This study shows
that roots respond strategically to N-rich patches by stimulating
growth.
Split root systems have proven to be effective models in which
to investigate local versus systemic signals. For example, when
a plant is grown with one-half of the root system subjected to
low-nitrogen stress, the other half grown with KNO3 grew more
vigorously than a control plant in which both halves were grown
with KNO3. This study suggests that the roots in the low-N
environment broadcast an N demand signal to which the other
roots respond by proliferation. When the split-root plants are
decapitated, this effect is not observed, nor is it observed in
plants that are deficient in the production of cytokinin. Analysis
of this system also provides support for a systemic N repletion
signal. Other studies have identified small peptides and their
cognate receptors as well as a transcription factor involved in
systemic N signaling.
In a locally mediated lateral root outgrowth response, high
NO32 perception by the CHL1/NRT1.1/NPF6.3 transceptor
activates a transcription factor that promotes lateral root outgrowth.
This protein also acts as an auxin transporter that is oriented in such
a way that it can transport auxin away from a developing lateral root
tip and its auxin transport activity is suppressed by nitrate. When
nitrate levels are low, auxin is transported efficiently and lateral root
outgrowth is minimized. When nitrate levels are high, the auxin
transport activity is suppressed, and auxin accumulation in the
developing lateral root supports its growth.
Strategies to Mitigate the Environmental Consequences
of N Fertilizers
High yields demand the use of fertilizers, but much of the nitrogen
applied to soils eventually ends up as pollutants of water and air.

Nitrate in groundwater can reach levels that can be detrimental to
human health. When N-enriched runoff enters waterways, it
contributes to algal and cyanobacterial blooms that are toxic to
humans and animals and that contribute to deadly hypoxic
conditions. As a recent example, during the summer of 2014, the
water supply for the city of Toledo, Ohio (US) was deemed
undrinkable due to contamination by cyanobacterial toxins result-
ing from fertilizer contamination of nearby Lake Erie. Similarly, the
region of the Gulf of Mexico that the Mississippi River drains into
regularly becomes oxygen-deprived and unable to support marine
life due to algal blooms. Nitrogen from fertilizers additionally
harms terrestrial ecosystems; many invasive plant species out-
compete native plants on inadvertently fertilized soils. Finally,
N-containing fertilizers contribute to air pollution and green-
house gas formation when nitrate is oxidized to nitrogen oxides
(NOx), including nitrous oxide (N2O), which is a more potent
greenhouse gas than CO2.
Field-Based Practices to Minimize the Effects of
N Fertilizers
The simplest and cheapest strategy to minimize the effects of
N fertilizers is to avoid using them. The traditional method
for resupplying soil with N is to use nitrogen-fixing crops as
companion or rotation crops; this approach was used widely
until chemically synthesized fertilizers became affordable in the
mid-20th century and continues to be used in regions where
fertilizer costs are prohibitive. Traditional methods mean that the
land produces less grain or is less amenable to mechanization,
so they are not widely used in the most productive grain-growing
regions.
The second best strategy to minimize N fertilizer effects is to
use them only when and where they are needed. Toward this
end, there are several methods by which plant or soil nitrogen
status can be determined. Some methods measure chlorophyll
concentration, which is closely correlated with nitrogen status.
Transmission methods measure the ratio of red light (absorbed
by chlorophyll) to infrared light (not absorbed) that passes
through a leaf. Other sensors measure various wavelengths of
reflected or fluoresced light and can be used at the plant or field
scale. Plant or soil nitrate levels can be determined by nitrate-
selective electrodes or dipsticks. These methods can inform
a farmer when the plant needs nitrogen. An alternative strategy
can be to use slow-release or deeply buried fertilizers that
prolong the time that the fertilizer remains in the soil. Each of
these strategies adds cost or labor to food production, but also
brings environmental benefits.
Nitrifying bacteria oxidize soil NH41 to NO32, which is more
prone to leaching away from the root zone. Nitrification inhibitors
are compounds that interfere with this process and maintain soil
N availability. Some tropical grasses such as Brachiaria humidicola
exude nitrification inhibitors, in this case brachialactone. Studies
are ongoing to determine the feasibility of extending this trait to
crops; already sorghum (Sorghum bicolor) genotypes and some
species related to commonly grown crop plants have been
identified that inhibit nitrification.
Finally, the damage caused when NO32 washes away from
fertilized fields can be mitigated by actively cultivating soil
December 2014 5
denitrifying bacteria. Denitrifying bioreactors are simple struc-
tures that filter field runoff through a bed of bacteria that capture
NO32 and convert it to harmless N2.
Breeding for Enhanced Nitrogen Use Efficiency
The environmental and economic costs of food production can
be lowered by increasing plant NUE. To accomplish this, we
have to understand where the bottlenecks to nitrogen uptake
and physiological utilization are and identify how to make these
reactions occur more efficiently. To a first approximation, we
can surmise that a crop plant optimized for NUE would have
a high rate of uptake of scarce N from soil, a high rate of
N incorporation into organic forms, and a high efficiency of N use,
recycling, and remobilization into grain.
These assumptions have been largely borne out. Key genes
have been identified through forward and reverse genetic
approaches as well as modern -omics methods and experi-
mentally validated using transgenic plants. Through these
approaches, two of the key NUE-modulating enzymes have
been identified as GS and GOGAT, which are involved in
primary N assimilation as well as remobilization. In fact, it has
been observed that each nitrogen atom passes through the
catalytic cycle of GS/GOGAT many times between its primary
assimilation and its ultimate departure from the plant, so it is
reasonable that small changes in these activities can add up to
large NUE effects. Promising results also come from expres-
sion of the enzyme alanine amino transferase, which re-
generates 2-oxoglutarate by transferring an amino group from
glutamate to pyruvate, producing alanine. Alanine can serve as
a storage form of nitrogen, and transgenic plants overexpress-
ing alanine amino transferase have been shown to have in-
creased NUE.
Other identified genes affecting NUE include various trans-
porters, transcription factors, regulatory proteins, and metabolic
enzymes. As an example, a recent report that started with a
quantitative trait mapping study in rice (Oryza sativa) identified
a single amino acid substitution in a plant-specific component of
a heterotrimeric G protein as contributing to NUE, opening a new
avenue for research. Several other studies indicate that root
systems that are better able to assimilate N from soil contribute
to enhanced NUE.
Transgenic strategies have had mixed results (for a compre-
hensive summary, see McAllister et al., 2012). Several candidate
genes have been investigated, and although these studies often
show some positive effects in laboratory settings, their results
seem to be variable in the field. The confounding effects of the
plants’ regulatory mechanisms, including the important con-
tributions of posttranscriptional regulatory controls, may be
responsible for this observation or the fact that more than
one gene may need to be altered to obtain a significant in-
crease in NUE. Ongoing studies are addressing both of these
possibilities. Considering that the global market in chemically
synthesized nitrogen fertilizers is worth $100 billion annually,
as well as its well-documented downstream consequences,
a small increase in plant nitrogen use efficiency could have big
payoffs.
6 The Plant Cell
PHOSPHORUS: THE MOST DIVERSE SET OF FUNCTIONS
In many soils, particularly younger soils, phosphorus (P) is the
second most limiting nutrient for plant growth after nitrogen; in
older soils, such as those found in parts of Australia and South
America, P can be the most limiting nutrient. In biological
systems, it is found in a large number of organic forms as well as
in the form of inorganic phosphate (Pi), an oxidized cation that is
generally written as PO432. The exact formula of inorganic
phosphate is pH dependent, ranging from H3PO4 in very acidic
conditions through H2PO42, HPO422, and PO432 with increasing
pH. Of all the mineral nutrients, P has the most diverse set of
cellular functions. It is involved in structure (e.g., phospholipid
membranes), energy storage (ATP), information storage (DNA),
and information transfer (RNA, protein kinase, and phosphorelay
cascades).
P Reserves Are Limited and Being Used Quickly
Globally, 40 to 60% of arable land crop yields are limited by P
availability. In some cases, soil P levels are absolutely low, but in
many others, P is present but in an unavailable form (see below).
Crop yields can be enhanced by the application of Pi fertilizer
that is mined from rock deposits, but there are economic and
ecological costs associated with this practice. The term “peak
phosphorus” has been used to describe the present situation
because P reserves are finite resources. Estimates for when P
reserves will be depleted vary and range from 50 to 400 years.
The depletion of easily accessible P reserves will drive up costs
for the extraction process and ultimately food. Serious environ-
mental problems occur as a consequence of phosphate runoff,
which can threaten the biodiversity of natural environments and
in aquatic environments can lead to harmful algal or cyanobac-
terial blooms. For these reasons, it is important to learn about
the uptake and use of P by plants, to develop plants with better
P usage efficiencies, and to develop methods for improved P
management, including methods to reclaim it from agricultural
and waste sources.
Phosphate Acquisition
Phosphate Mining: Root Exudates and
Mycorrhizal Symbioses
Most of the P in soil is found either as insoluble phosphate
sorbed (adsorbed or absorbed) onto soil particles or as organic
compounds. Only a tiny amount of the P is present in the soil
solution as Pi, the only form that can be taken up by plants.
Therefore, plants have evolved strategies by which to “scav-
enge” or “mine” phosphorus, including the production of root
exudates and the establishment of symbioses with mycorrhizal
fungi.
Root exudates act on inaccessible phosphorus to increase its
availability; estimates vary but as much as 30% of the carbon
fixed by the plant might be exuded into the rhizosphere. Low
molecular weight organic acids (e.g., citrate, malate, and oxalate)
displace and replace phosphate from insoluble complexes, such
as calcium phosphate and phosphates of aluminum and iron
oxides and hydroxides. Soil can be very rich in P in the form of
organic molecules, which are substrates for exuded enzymes
such as phosphatases and phytases (phytate is inositol hexa-
phosphate). These enzymes release phosphate in a form suitable
for uptake by the plant.
The symbiotic association with mycorrhizal fungi is a critical
strategy for Pi uptake used by most plants (;80% of species,
with Arabidopsis being a familiar exception). In this association,
the fungal partner scavenges Pi, which is then taken up into the
fungal cell and transferred to the plant (the fungus in exchange
is provided with carbon). The fungus greatly increases the
effective surface area of the root as well as the volume of soil
from which Pi can be assimilated. This is particularly important
because Pi has an extremely low mobility in soils; the zone
immediately surrounding a root quickly becomes depleted of
Pi, but the fungus extends beyond this zone to forage more
effectively. The intricate signaling choreography upon which the
mycorrhizal symbiosis depends is described in detail in Teaching
Tools in Plant Biology 19: Plants and Their Microsymbionts.
Phosphate Foraging: Root System Architecture
Because of Pi’s immobility in soil, root growth patterns are
extremely Pi sensitive. Phosphate reserves can be more abundant
in the topsoil, so plants growing in low-P soil often show decreased
primary root elongation and enhanced lateral root outgrowth,
enhanced production of adventitious roots (produced by the shoot),
as well as a change in root angle so that the lateral roots spread in
a more horizontal pattern. Root hairs tend to become elongated and
form more densely, increasing the area of the root/soil interface in
low-P soils. Because investing in root growth can be metabolically
expensive, some roots remain slender and do not expand radially,
whereas others produce air-filled cavities known as aerenchyma.
Many members of the predominantly Southern Hemisphere
plant family Proteaceae have adapted to low-P soils and reveal
mechanisms for survival with severe P limitation. One common
adaptation is the production of cluster roots that exude copious
amounts of carboxylates or phosphatases to enhance Pi assimi-
lation. The balance between investing in root growth versus root
exudation may be determined by the absolute versus available
levels of phosphorus. White lupin (Lupinus albus), a legume native
to Mediterranean regions, also makes cluster roots and provides
a genetically tractable model for studies of cluster-root develop-
ment and function.
Phosphate Transporters
Pi uptake into the negatively charged cytosol is electrically
unfavorable and requires the cotransport of two to four protons,
so it is energetically expensive compared with cation uptake.
Transport into the plant occurs by way of the PHT1 family of
plant Pi transporters that were identified by homology and
functional similarity to fungal Pi transporters. The PHT1 family
has nine members in Arabidopsis and 13 in rice. Different family
members have different affinities for Pi and expression patterns,
although most are expressed in roots, particularly the root hairs
and cortical cells, and in mycorrhizal species some are expressed
at the fungal-plant interface. In general, their transcription level is

determined by Pi availability, and the transcription factor PHR1
(PHOSPHATE STARVATION RESPONSE1) is involved in their
regulation. Other types of Pi transporters have been identified that
reside in the mitochondrial, plastid, or Golgi membranes. Once
phosphate enters the cell, it is part of a large pool of free
phosphate that is incorporated into diverse organic compounds
by the action of hundreds of enzymes.
Metabolic Adjustments to Low Phosphate
Plants can acclimate to mild Pi deficiency through a variety of
metabolic adjustments. One set collectively reallocates Pi to
high-priority targets, for example, by mobilizing Pi from vacuolar
reserves, though as yet the molecular identity of vacuolar Pi
transporters remains obscure. Other metabolic reallocation
responses include the activation of RNases, acid phosphatases,
and Pi transporters in older or senescing tissues and the trans-
location of released Pi to younger tissues. PHO1 is a putative Pi
efflux carrier that has been implicated in long-distance Pi transport
from source to sink by exporting it to the apoplasm for transport in
the xylem. Phosphate starvation also leads to increased production
of root-exuded carboxylates and phosphatases to enhance Pi
uptake.
Longer term Pi limitation leads to global metabolic changes
that enable the plant to function in spite of the less than optimal
availability of Pi. For example, phospholipids in membranes can
be replaced by sulfolipids and galactolipids, and the number of
ribosomes can be lowered (rRNAs represent the largest pool of
organic phosphorus). Decreasing ribosomal pools can make
plants slower to grow or respond but can be an effective
strategy for survival when P is limiting. Accelerated leaf
senescence and anthocyanin accumulation are also signs of Pi
deficiency.
An extreme example of ribosome rationing can be seen in
some Proteaceae that maintain many fewer ribosomes than
other plants. Although mature leaves operate with ;20% of the
rRNA of other plants, they have similar rates of photosynthesis.
Furthermore, through a developmental strategy known as
delayed greening in which leaf expansion precedes chloroplast
maturation, growth is temporally separated from photosynthe-
sis; cellular phosphate reserves can be employed first in cytosolic
rRNAs (during growth) and subsequently in chloroplastic rRNAs
(during greening).
Regulatory Controls of P Uptake
The metabolic and transcriptional shifts in response to P
starvation are under tight control. A core group of P starvation-
induced (PSI) genes have been identified that include genes
involved in P uptake and remobilization as well as genes
encoding phosphatases that can increase Pi availability. PHR1
and its orthologs are transcription factors that bind to a conserved
DNA element (P1BS) found in the PSI genes, ensuring that
they are downregulated when P is abundant and inducible by
P starvation. Recently, the interaction between PHR1 and this
DNA element was shown to be inhibited in the presence Pi
through the action of proteins of the SPX family. When Pi is
December 2014 7
present, SPX proteins bind to PHRs and prevent them from
activating the transcription of PSI genes. This interaction is
particularly interesting because in yeast, proteins related to
SPX are known to act as Pi sensors; this interaction therefore
may indicate how plants sense Pi availability.
In the regulation of P nutrition, it is similarly important to
maintain the Pi assimilation pathways in a downregulated state
when this nutrient is not limiting. An example of this comes from
the study of PHO2. When Pi levels are sufficient, PHO2 mRNA is
translated to produce PHO2, a ubiquitin ligase that targets PHT1
and PHO transporters for degradation. Under P starvation,
miR399 is expressed, which is a small RNA that targets PHO2
mRNA for degradation; thus, the transporters are stable and P is
taken up and translocated to the shoot. Interestingly, a target
mimicry that interferes with miR399 action is also upregulated by
Pi deficiency, providing a fine-tuning mechanism. PHT1 trans-
porter activity is further regulated by endoplasmic reticulum
retention and phosphorylation.
Several mutants with disruptions in Pi regulatory controls
show Pi hyperaccumulation in the shoot and a corresponding
decrease in growth rate, suggesting that in terms of Pi accumulation,
plants have mechanisms in place to assimilate not too little and not
too much but just the right amount.
In addition to miR399 and other miRNAs, many hormones
have been implicated in Pi starvation responses, including auxin,
ethylene, gibberellins, cytokinins, and particularly strigolactones,
the synthesis of which is stimulated by Pi starvation. Strigolac-
tones promote associations with arbuscular mycorrhizal fungi,
alter root system architecture, and are translocated to the shoot
where they suppress branching and so lower the plant’s demand
for Pi by slowing the growth rate.
Breeding Crops for Improved Phosphate Use Efficiency
The identification of genes that are involved in root exudation and
architecture, Pi uptake and remobilization, and regulatory controls
of these processes opens the door to genetic approaches to
improve P use efficiency. Several strategies have been examined
in lab and field studies, including the overexpression of Pi regulatory
transcription factors and transporters and increased exudation of
carboxylates, phosphatases, and phytases to the rhizosphere. The
results have had mixed success. For example, in nutrient solution,
overexpression of a PHT1 transporter leads to plants with increased
Pi content but smaller stature, possibly because Pi accumulates to
inhibitory levels; whether this approach contributes to Pi uptake in
crops growing in P-limited soils remains to be seen. The fact that
the regulatory machinery seems to be as important in avoiding
excess Pi uptake as it is in ensuring adequate Pi uptake suggests
that efforts to enhance Pi uptake will require a subtle approach.
Breeding strategies that alter patterns of root growth architec-
ture have been somewhat more promising. New phenotyping
methods make it possible to select directly for root architecture in
breeding programs. Already varieties with root system architec-
tures favorable for Pi uptake have been developed and shown to
be more efficient at Pi uptake.
Interestingly, a strategy that started with a rice variety with
enhanced Pi uptake ability has led to the discovery of a gene
8 The Plant Cell
involved in regulating root system architecture. Analysis of the
stress-tolerant aus rice variety grown in regions with very low soil
P availability led to the identification of a major quantitative trait
locus Pup1 (Phosphate Uptake 1) that confers enhanced growth
in P-limiting conditions. This locus proved difficult to work with,
but after some effort the gene PSTOL1 (Phosphate starvation
tolerance 1), encoding a protein kinase, was identified. Although
the direct targets for PSTOL1 are not yet known, overexpression
of this protein causes an increase in grain yield on P-deficient
soils, due at least in part to an enhancement of root growth.
Strategies to Mitigate the Environmental Consequences
of P Fertilizers
As discussed previously, there are compelling environmental
and economic reasons to employ greater precision when
applying fertilizers. Monitoring soil and plants for signs of P
limitation can ensure that it is applied at the appropriate rate,
time, location, and chemical form. In particular, the very low
mobility of Pi in the soil means that it should be applied where
the roots are. As Pi reserves become more limiting, price
increases are likely to drive a greater precision in Pi applications.
At the other end of the chain, it is important to try to decrease the
amount of P that ends up in waterways as a consequence of fertilizer
runoff, contamination from farmed animal excreta, and urban water
treatments. At the farm level, greater care in the timing and amount of
Pi fertilizer applications can help, as can the adoption of no-till
practices that decrease soil erosion and nutrient runoff. Human
urine is a rich source of Pi that can be collected separately from
solid waste through special two-compartment toilets and
used as fertilizer. Pi can be reclaimed from sewage and farm
wastewater through chemical methods such as the selective
precipitation of magnesium-phosphate or other salts, or adsorption
or ion exchange methods. Biological methods take advantage of
the ability of microbes or algae to extract and concentrate Pi from
dilute systems, reclaiming it for reuse as well as cleaning the water
prior to discharge.
Phytate accumulates in seeds but is indigestible to mono-
gastric animals (e.g., pigs, as opposed to ruminant animals such
as cows that have four-chambered stomachs); monogastric
animals excrete P-rich manure that is an environmental pollutant.
Furthermore, micronutrients such as zinc and iron are chelated by
phytate and rendered inaccessible. For more efficient Pi and
micronutrient assimilation, low-phytate plant varieties have been
developed through conventional and genetic engineering methods,
including through the overexpression of bacterial or fungal
phytase genes. Another approach is to apply the enzyme phytase
directly to animal foods. Finally, genetically engineered pigs that
produce phytase in their saliva have been developed, which
although effective at phytate metabolism still await regulatory
approval.
POTASSIUM: POTASH, FROM THE ASHES IN THE POT
Potassium Reserves
Potassium is the seventh most abundant element in the Earth’s
crust and is usually found as large underground deposits
resulting from past evaporations of bodies of water. Potassium
is essentially always found in the K1 form. In association with
several different anions, K1 can form soluble salts including KCl
(potassium chloride, also known as sylvite), K2SO4 (potassium
sulfate), K2CO3 (potassium carbonate), and as insoluble rocks of
various forms. Potash is a traditional term that encompasses
any form of soluble potassium salt and literally is derived from
the potassium-rich ashes left in the pot after burning plant
matter. The term potash also refers to any of several forms of
potassium that are used commercially as fertilizers.
Globally, more than 30 billion kilograms of potash are used as
agricultural fertilizers each year. The supply comes from mining
operations around the world, with Canada, Russia, and Belarus
being major producers. The demand for potash has been in-
creasing, particularly with increasing agricultural usage in China
and India. The small number of potash suppliers means that the
price can be manipulated by altering the supply, so potash prices
can be volatile; notably, between 2008 and 2011, the price more
than quadrupled from ,$200 to .$800 per metric ton (1000 kg),
largely as a result of a decrease in production. Since then, the
price has returned to the $300 to $400 range. The demand for
potassium is mainly a consequence of its essential role in plant
growth and so food production. One estimate indicates that 1 kg
of K2O is required to produce 44 kg of rice or to produce enough
maize and soybeans to produce 10 kg of pork meat. As there is no
substitute for potassium in plant or animal cells, food production
remains dependent on the continual mining and distributing of
potash.
Potassium Is an Essential Macronutrient
Potassium is an essential nutrient in plants and in all living cells.
It is not incorporated into other molecules but instead functions
as an unconjugated ion. Although not covalently attached, it has
essential roles in stabilizing the structures of many proteins and
macromolecules. Potassium also has essential roles in water
and ionic balance. Plant movements, including phototropism,
the rapid movements of the sensitive plant (Mimosa pudica), and
the guard cell volume changes that regulate transpiration, are
mediated by the transport of potassium across cell membranes
and the accompanying movement of water. Additionally, potas-
sium is a cofactor for some enzymes, including pyruvate kinase,
starch synthase, and plasma membrane H1-ATPase, and through
this and other functions, potassium contributes to diverse metabolic
processes. Potassium deficiency is particularly associated with
small stature, wilting, decreased rates of photosynthesis and
growth, and increased susceptibility to stress.
Potassium Uptake and Remobilization
Plants take up potassium from soils against a steep concentra-
tion gradient. Plant cells maintain a cytosolic K1 concentration
of ;100 mM, but soil concentrations are typically 0.1 to 1 mM,
100- to 1000-fold lower. More than 50 years ago, classic studies
by Epstein and colleagues identified a biphasic potassium
uptake response. Using a wide range of K1 concentrations, they
measured the rate of uptake of radioactively labeled potassium.

Their results showed different kinetic properties at low and high
external [K1], which they interpreted to mean that different
mechanisms are involved. When potassium is scarce, a high-
affinity system is required for uptake, whereas when potassium
is abundant a low-affinity system is sufficient. We now know
that, to a first approximation, potassium/proton cotransporters
that consume the energy equivalent of pumping two protons
across the membrane are primarily involved in high-affinity
uptake, and potassium channels that consume the energy
equivalent of pumping one proton are primarily involved in low-
affinity uptake. The channels and carriers involved in potassium
uptake as well as recycling and remobilization strategies are
described in more detail in Teaching Tools in Plant Biology 29:
Membrane Transport and Energetics, Potassium Nutrition, and
Sodium Toxicity.
SULFUR: CLEAN AIR LEADS TO DEFICIENT PLANTS
Sulfur in Global Cycles and Cells
Sulfur can be found in many redox forms: elemental sulfur (S),
reduced forms including sulfide (H2S) and organic sulfur (R-SH),
and oxidized forms including sulfate (SO422), sulfite (SO32), and
sulfur dioxide (SO2). Plants assimilate sulfur primarily from soil as
sulfate and to a lesser extent from the atmosphere as SO2 or
H2S. Until recently, sulfur was not considered limiting for plant
growth in most soils, but since air pollution control measures
were effected in the 1980s, sulfur deposition into soil from the
atmosphere has decreased dramatically and uncovered the fact
that some plants benefit from sulfur fertilizer.
There are several similarities between the nitrogen and sulfur
cycles. Both N and S are found in the atmosphere as well as the
soil, and both are subject to oxidative and reductive reactions by
soil microbes. In the soil, a reduced form of each (NH41 or H2S)
is readily oxidized to a form assimilated by plants (NO32 or
SO422), which then must be reduced by the plant for incor-
poration into organic molecules.
In plants, sulfur makes up ;1 mg/g dry tissue mass, which is
small compared with carbon (450 mg/g) or nitrogen (15 mg/g).
Sulfur in plants is found mainly as methionine and cysteine in
proteins, with the third major form being glutathione, a small
molecule made from glutamate, cysteine, and glycine. Glutathi-
one is a redox agent that cycles between reduced and oxidized
forms and is involved in many enzymatic reactions as well as
protection from damaging reactive oxygen species. Sulfur is
also found in some defense compounds such as glucosinolates,
cysteine-rich proteins including defensins and metallothioneins
(metal binding proteins), and flavor compounds such as allium.
Sulfate Uptake
Sulfur uptake and transport is primarily mediated by the SULTR
family of sulfate transporters, which are divided into four sub-
families with varied tissue and membrane specificities. Much
of the primary uptake that occurs in roots occurs through
SULTR1;1 and SULTR1;2, which are expressed in the root
epidermal and cortical cell layers. Some of the other SULTR
December 2014 9
transporters have other functions, including efflux from the
vacuole to cytosol, or are involved in long-distance transport via
expression in the phloem or xylem parenchyma. In higher plants,
transport into plastids also appears to involve this family of
transporters.
Volatile sulfur compounds can be taken up by plants. These
include the gaseous H2S and SO2 from atmospheric sources
and compounds produced by plant growth-promoting bacteria.
Atmospheric SO2 enters the plant through stomata and is then
converted to sulfite (SO32) or oxidized to sulfate. Although H2S
and SO2 can serve as a nutrients, too much can be toxic.
Mosses and other bryophytes don’t have a protective cuticle
and are vulnerable to damage to elevated atmospheric sulfur
dioxide.
Assimilation into Organic Compounds
Once inside the cell, the first step in sulfate assimilation is
incorporation into adenosine 5#-phosphosulfate (APS) by ATP
sulfurylase (ATPS). APS is the entry point to two pathways. In the
primary metabolic pathway, APS reductase separates adeno-
sine monophosphate from the sulfur moiety, releasing sulfite
(SO32), which is further reduced to sulfide (H2S). In a two-step
reaction, sulfide replaces serine’s hydroxyl group to produce
cysteine. This reaction is catalyzed by a complex known as
cysteine synthase, which is composed of serine acetyltransferase
(SAT; also known as SERAT) and O-acetylserine(thiol)lyase
(OAS-TL). The cysteine synthase complex seems to act as a
metabolic sensor that fine-tunes the flux of the S assimilation
pathway. The other pathway downstream from APS involves
its phosphorylation to produce an activated form of sulfur
5#-phosphoadenosine 3#-phosphosulfate, which is subsequently
incorporated into defense and other compounds.
Regulation of Sulfur Uptake and Metabolism
Sulfate uptake is upregulated by sulfur deficiency and down-
regulated by reduced sulfur compounds in a form of negative
feedback regulation. Uptake is determined by transcriptional
and posttranslational controls of the transporters. For example,
low-S status induces transcription of several transporter genes.
Similarly, transcriptional and posttranslational mechanisms
contribute to the regulation of S assimilation. Expression of
genes encoding ATPS (the first step in S assimilation) is
regulated by miR395, which is induced under low-S conditions
and targets ATPS mRNA for degradation. The next enzyme in
the assimilatory pathway, APS reductase is thought to control
much of the assimilatory flux. Its expression is subject to both
positive and negative regulatory controls involving various
metabolites, but which of these act as direct versus indirect
signals remains a matter of debate. O-acetylserine (OAS) also
contributes to the regulatory control of the Cys synthase complex,
by destabilizing it and inactivating serine acetyltransferase when
S levels are low (with the net result being a switching off of the
pathway from Ser to Cys via OAS). Interestingly, the Cys synthase
complex functions in the cytosol, mitochondria, and plastids,
suggesting that the production of this amino acid occurs in the
compartment in which it is used.
10 The Plant Cell
Several additional contributors to the S regulatory network
have been identified, with a complex and not fully resolved
interaction network that modulates activity of uptake trans-
porters and assimilatory enzymes as well as root-to-shoot trans-
location. Ethylene, miR395, and several transcription factors,
including SULFUR LIMITATION (SLIM), contribute to this
network which under S deficiency promotes sulfur uptake
and translocation to the shoot as well as recycling of S-
containing compounds. S deficiency also affects root growth
by way of auxin. Specifically, primary root elongation is
promoted in low-sulfate conditions and is inhibited by
elevated cellular levels of cysteine.
Addressing S Deficiency in Plants
With less combustion-derived sulfur dioxide entering the
atmosphere, S deficiency in plants is becoming more common.
This condition is intensified in soils that are fertilized with NPK
fertilizers (recall the “law of the minimum”). S deficiency can
affect yields and quality of many crop plants, particularly wheat
(Triticum aestivum) and oilseed rape (Brassica napus), which has
a high S requirement. S demands also increase with increasing
demand for defense compounds or exposure to heavy metals,
which are chelated by S-containing metallothioneins. Sulfur
can be added to soils as ammonium sulfate fertilizer, as ele-
mental S, or as a liquid fertilizer containing ammonium thio-
sulfate; no mining is required as there is an ample supply of S
available as by-products of petroleum refining processes. The
possibility of increasing the uptake, assimilation, and remobi-
lization of S through breeding or genetic engineering is also
being explored.
MAGNESIUM: THE “FORGOTTEN ELEMENT”
Magnesium in Rocks and Cells
Magnesium, “the forgotten element,” has received less attention
than some of the other macronutrients. It is the eighth most
abundant element on Earth and (other than H and O) the third
most abundant element in seawater, after Na and Cl. Mg in soil
usually comes from weathered rock (dolomite, magnesite, and
serpentine are common Mg-containing rocks) or evaporated
seawater. The concentration and availability of Mg21 in soils
varies from ;0.1 to 8 mM, and it is sometimes present at higher
levels in soil than in the cytosol of plant cells. Therefore,
magnesium deficiency is not common, although it can occur due
to competition for uptake with other cations due to being leached
away from the root zone or in plants grown with fertilizers that do
not include Mg.
Cellular Mg21 has several roles. It interacts with the negative
charges on the phosphate groups of ATP and so acts as
a counter-ion, and through similar ionic interactions it is in-
dispensable for the proper structural assembly of ribosomes.
Mg21 is an essential activator for more enzymes than any other
mineral element, which leads to a pleiotropic set of deficiency
symptoms. Mg21 is required to activate the carbon-fixing en-
zymes Rubisco and PEP carboxylase, as well as many ATPases.
It also is found in the center of the chlorophyll molecule; thus,
Mg21 deficiency directly limits photosynthesis and causes leaf
chlorosis that is aggravated by high light due to the production
of reactive oxygen species.
Interestingly, in many species, Mg21 deficiency causes a
dramatic increase in the shoot to root ratio; this is opposite to the
effect of most mineral nutrient deficiencies, which preferentially
restrict shoot growth. The restriction on root growth in Mg-
deficient plants is thought to be caused by a deficiency of
sucrose loading into the phloem in photosynthesizing source leaves
that causes the leaves to accumulate sucrose and effectively
starves the root system; sugar signaling may also be involved.
Uptake and Assimilation
A family of Mg21 transporters known in plants as MRS or MGT
has been identified by homology to Mg21 transporters in yeast,
and there is evidence that these proteins support Mg 21
transport in plants. These transporters are distinct from other
cation transporters but are conserved among the domains of
life. Like other mineral elements, excess Mg21 is stored in the
vacuole, and tonoplast members of the MRS/MGT family have
been identified as well as MHX1, a tonoplast-localized Mg/
proton exchanger.
Magnesium deficiency is common in soils that are acidic as
well as soils that are prone to leaching because these conditions
promote the elimination of Mg21 from upper soil profiles. High
levels of Al31 contribute to Mg21 deficiency through competition
for nonspecific binding in the apoplast as well as specific
competition for uptake transporters. Conversely, adequate soil
Mg21 or enhanced rate of Mg21 uptake by a plant can protect it
from the harmful effects of Al31 toxicity, which is a major
inhibitor of plant growth on acidic soils.
Humans require dietary consumption of Mg21 for bone con-
struction, and deficiencies can occur when their diet consists
mainly of Mg21-poor grains. Mg deficiency also can occur in
animals; cattle fed on young, rapidly growing spring grass can
develop a sometimes fatal ailment called grass tetany as a
consequence of inadequate Mg nutrition. Humans should en-
sure that their diet includes whole grains, nuts, and legumes as
well as leaves to ensure adequate Mg. Adding Mg21 fertilizers
to soils can enhance Mg uptake by the plant, and genetic
studies are underway to develop staple crop varieties with
enhanced Mg21 accumulation in the grain. Unlike some of the
other macronutrients, there appears to be no significant down-
stream environmental impact from using Mg21 as a fertilizer in
agriculture.
CALCIUM: LOW FREE CYTOSOLIC LEVELS
In its free-cation form, calcium is maintained at very low levels
in the cytosol, on the order of 0.1 to 0.2 mM. The overall
concentration of calcium in the cytosol can be in the millimolar
range, but most of it is not a free cation but instead buffered by
binding to organic compounds or proteins such as calmodulin.
In many plants, calcium is also found in the form of calcium
oxalate crystals that are formed by specialized cells called
idioblasts and accumulate in vacuoles and cell walls. Calcium

oxalate crystals can have defensive roles, contribute to heavy
metal tolerance, and serve to sequester excess calcium. Many
of calcium’s functions take place outside the cytosol: strength-
ening cell walls, stabilizing membranes, and serving as an
osmoticum in the vacuole. In the cytosol, Ca21 functions as
a second messenger that conveys information as transient
pulses or waves of variable frequency and amplitude that are
produced by the action of calcium channels and pumps. These
calcium signals are decoded by calcium binding proteins,
including calmodulins.
Calcium Uptake and Transport
Most plants generally are able to obtain sufficient calcium from
the soil, although this ability is affected by the presence of other
competing cations (Al 31 and Na 1) and soil pH. At the
membranes, the challenge is to maintain submicromolar free
Ca21 concentrations in the face of much greater free Ca21
concentrations in the apoplast, vacuole, and other organelles;
the Ca21 concentration may be thousands of times higher
outside the cells than in. Calcium transport across the plasma
membrane occurs by way of several highly regulated calcium
channels that control inward flux, calcium pumps (Ca21-
ATPases) that pump it outwards, and Ca21/ H1 antiporters.
Similar families of channels, pumps, and carriers are present in
the tonoplast membrane surrounding the vacuole where a siz-
able pool of Ca21 is sequestered.
Ca21 transport within a plant primarily occurs within the
apoplasm, where most of the free Ca21 is located. Because the
Casparian strip at the root endodermis or exodermis blocks
apoplastic transport, much of the uptake takes place at the tip of
the root where the Casparian strip is not yet developed. From
there, Ca21 can move into the xylem stream for transport
throughout the rest of the plant. Phloem transport takes place
via the symplast, so it presents a challenge to a plant’s ability to
remobilize Ca21 from older to younger tissues. Without the
ability to draw on internal stores, young growing tissues can be
damaged by even transient interruptions in their exogenous
supply of calcium.
Calcium’s Structural Role
Ca21 has structural roles in the cell wall and at the surface of
membranes. In membranes, it is thought to interact with the
negatively charged phosphate groups of membrane phospho-
lipids and to regulate numerous ion transporters. In the cell
wall, it stabilizes pectin, which is a polymer of galacturonic acids
with various modifications. Ca21 forms cross-bridges between
negative charges and so gels and stabilizes pectin. (This
function can be observed in molecular gastronomy in the
formation of “spherical,” in which a pectin-like alginate solution
is exposed to a Ca21-containing solution to form gel balls.)
Pectin forms the middle lamella between adjacent cells and
also the newly forming cell wall laid down during pollen tube
growth. Ca21 deficiency weakens the middle lamella layer
and allows cells to separate, which can result in unsightly
cracked skins of rapidly growing fruits, but also can result in
December 2014 11
cell death, in syndromes known as blossom end rot, tip burn,
and bitter tip.
Calcium signaling
Ca21 waves are secondary signals of many abiotic stresses,
light and gravity cues, and biotic interactions. The large Ca21
concentration difference between the cytosol and other cellular
compartments ensures that when Ca21 channels open, the ion
moves rapidly into the cytosol. Calcium is quickly removed from
the cytosol through the action of calcium/proton antiporters and
Ca21-ATPases that are related to plasma membrane H1-
ATPases. How these protein activities are coordinated to
produce Ca21 waves and how this oscillating signal is decoded
remains incompletely understood, but several constituents of
the signaling pathway have been identified including numerous
Ca21 binding proteins. Interestingly, long-distance spreading of
Ca21 signals have been identified, but these may be transduced
from cell to cell by a different signal than Ca21 itself.
PLANT MACRONUTRIENTS: SUMMARY AND
ONGOING RESEARCH
Aside from C, H, and O, the six elements most abundant in
a plant body are K, N, P, S, Mg, and Ca, each of which is usually
assimilated from soil as a charged molecule and transferred
across plant membranes by way of specialized transporter pro-
teins. The gene sequences of many nutrient transporters have
been identified, although much remains to be done to character-
ize their kinetic and regulatory properties in vivo.
N, P, and S are assimilated covalently into a large number of
organic molecules, whereas K, Mg, and Ca function by way of
ionic interactions; nevertheless each is completely indispens-
able. Uptake and assimilation are energetically demanding
and closely regulated to ensure that supply matches demand.
Current efforts examine how plants juggle their needs for each
of these nutrients; rarely is only one limiting and the others
replete.
Our understanding of plant nutrition has been established
largely through studies that focus on one nutrient at a time in
defined conditions, but increasingly it is clear that a broader
approach is necessary to identify how plants cope with the more
heterogeneous field environment. The growth of the root system
is dictated by local and systemic information about all required
nutrients (as well as water availability, physical properties of soil,
etc.). As an example, elongation of the apical root region is most
sensitive to inhibition by low Pi but is inhibited still further by
a limitation of another nutrient. Such an analysis can also identify
specific genes that respond to multiple nutrients, which will be
important in efforts to breed plants for success in nutrient-poor
soils. The effects of soil microbes in nutrient availability and the
effects of the plant on soil microbe activities are often over-
looked in laboratory-based studies.
The ultimate goal of much of the research described here
is the development of greener, more sustainable agricultural
systems. The enhancement of plant growth and yield by
application of fertilizers contributes one of the major impacts of
12 The Plant Cell
food production. Although the traditional methods of applying
complex organic (manure) fertilizers are less prone to leaching
and runoff than chemically synthesized fertilizers, high-
intensity crop farming areas are often not located close enough
to livestock farming regions to warrant the shipping costs. As
the environmental costs of overfertilizing have become more
evident, farmers are increasingly incorporating strategies to
mitigate these effects, often with the encouragement of local
governments, which can help to offset increased production
costs. In the coming decades, the tension between the demands
for higher crop yields and the desire for cleaner food production
will grow. It is a challenge for today’s students to work toward
innovative solutions to the challenge of feeding the plants that
feed us.
Mary Williams
mwilliams@aspb.org
Features Editor, The Plant Cell
American Society of Plant Biologists
c/o Laboratory of Plant Physiology and Biophysics
University of Glasgow
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(This is a representative list of sources to help the reader access
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