Regulation of phosphate starvation responses in higher plants
Xiao Juan Yang1,2and Patrick M. Finnegan1,*
1School of Plant Biology, University of Western Australia, 35 Stirling Highway, Crawley WA 6009, Australia and2Department of
Plant Nutrition, College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China
* For correspondence. E-mail email@example.com
Received: 12 October 2009Returned for revision: 12 November 2009 Accepted: 5 January 2010 Published electronically: 24 February 2010
†Background Phosphorus (P) is often a limiting mineral nutrient for plant growth. Many soils worldwide are
deficient in soluble inorganic phosphate (Pi), the form of P most readily absorbed and utilized by plants. A
network of elaborate developmental and biochemical adaptations has evolved in plants to enhance Piacquisition
and avoid starvation.
†Scope Controlling the deployment of adaptations used by plants to avoid Pistarvation requires a sophisticated
sensing and regulatory system that can integrate external and internal information regarding Piavailability. In this
review, the current knowledge of the regulatory mechanisms that control Pistarvation responses and the local and
long-distance signals that may trigger Pistarvation responses are discussed. Uncharacterized mutants that have Pi-
related phenotypes and their potential to give us additional insights into regulatory pathways and Pistarvation-
induced signalling are also highlighted and assessed.
†Conclusions An impressive list of factors that regulate Pistarvation responses is now available, as is a good deal
of knowledge regarding the local and long-distance signals that allow a plant to sense and respond to Piavail-
ability. However, we are only beginning to understand how these factors and signals are integrated with one
another in a regulatory web able to control the range of responses demonstrated by plants grown in low Pi
environments. Much more knowledge is needed in this agronomically important area before real gains can be
made in improving Piacquisition in crop plants.
Key words: Phosphate signal, phosphate regulon, transcription factor, non-coding RNAs, phosphate starvation
Phosphorus (P), in the form of phosphate (Pi), is an essential
macronutrient for all living organisms, including plants. It is
a structural element in nucleic acids and in the phospholipids
that make up biomembranes. Many phosphoesters have an
indispensible role in metabolic reactions, particularly those
that involve energy transfer. Pi, through protein phosphoryl-
ation and dephosphorylation, is also a key component of the
numerous signal transduction cascades that establish adaptive
patterns of gene expression.
A ‘phosphorus paradox’ in relation to plant nutrition has
been pointed out by numerous authors (Bieleski, 1973;
Marschner, 1995). Although the total P content of soil is gen-
erally high, P availability is frequently a limiting factor for
plant growth and productivity. This paradox arises because
the concentration of available Piin the soil solution averages
about 1 mM and seldom exceeds 10 mM (Bieleski, 1973). A
number of morphological, physiological, biochemical and
molecular responses have evolved in plants that allow them
to grow and prosper in the presence of such low levels of avail-
able Pi. These responses include the development of lateral
roots and root hairs, as well as more dramatic root structures
such as proteoid and dauciform roots, the secretion from
roots of phosphatases and organic acids, and the induction of
high-affinity and some low-affinity Pitransporters (Lambers
et al., 2006; Ai et al., 2009; Fang et al., 2009). Many plants
also establish symbiotic associations with mycorrhizal fungi
that aid Piacquisition (Burleigh et al., 2002).
The application of P fertilizer can compensate for low Pi
availability in cropping systems, but high Piinput can cause
severe environmental problems such as eutrophication. In
addition, the global source of rock P is non-renewable and is
being rapidly depleted. It is predicted that easily accessed
global P reserves may be depleted in 50–100 years (Steen,
1998; Cordell et al., 2009). A fuller understanding of the strat-
egies used by plants to acquire and utilize Piefficiently, there-
fore, is vitally important for the rational breeding and
engineering of crop plants with greater capacity to acquire,
store and recycle soil Pi. In this review, recent advances in
our understanding of the molecular aspects of plant responses
to Pistarvation will be discussed. The regulation of Pitranspor-
ters, while centrally important, is not covered here, but has
been reviewed elsewhere (Raghothama and Karthikeyan,
2005; Bucher, 2007; Lin et al., 2009).
COMPONENTS OF THE PiREGULON IN PLANTS
Avariety of adaptive strategies have evolved in plants that alle-
viate or help them cope with Pideficiency. The implemen-
tation of these strategies requires changes in the expression
profiles of hundreds of genes, as demonstrated by transcrip-
tome analyses in Arabidopsis thaliana (arabidopsis), Oryza
sativa (rice), Lupinus albus (white lupin) and Phaseolus vul-
garis (common bean) (Hammond et al., 2003; Uhde-Stone
et al., 2003; Wasaki et al., 2003a; Wu et al., 2003; Misson
et al., 2005; Herna ´ndez et al., 2007). The extent and
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Annals of Botany 105: 513–526, 2010
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complexity of the network of regulatory genes necessary to
sense Pistatus and regulate the deployment of these adaptive
strategies is now being revealed. The network components
identified so far include transcription factors, SPX sub-family
proteins, non-coding RNAs and protein modifiers, including
proteins involved in SUMOylation, phosphorylation, depho-
sphorylation and protein translocation (Fig. 1).
Transcription factors bind to specific DNA sequences and
regulate gene expression by altering the ability of RNA poly-
merase to bind to a target promoter sequence. These proteins
have vital roles in many plant responses to stress conditions,
including Pideprivation. Transcription factors themselves are
quite heavily regulated in response to Pi. In an experiment
involving 6172 arabidopsis genes, the transcript abundance
of about 30 % of the genes is changed .2-fold within 72 h
of Pi deprivation (Wu et al., 2003). Among these were
one-third of the 333 transcription factor genes assessed.
At-PHR1 from arabidopsis was the first transcription factor
identified to be involved in the control of the Pistarvation
response in a vascular plant (Rubio et al., 2001). The gene
was identified in a genetic screen set up using a transgenic
line harbouring an At-IPS1::GUS chimeric reporter gene.
The At-IPS1 promoter is specifically responsive to Pi
deprivation. Screening an ethylmethane sulfonate (EMS)-
mutagenized M2population of the reporter line identified a
mutant, subsequently named phr1, that had a reduced response
of the At-IPS1::GUS reporter gene to Pideprivation. Analysis
of the mutant revealed that other well-known Pistarvation
responses, such as the accumulation of transcripts from a
sub-set of Pi-responsive genes and the accumulation of antho-
cyanin, were also impaired (Rubio et al., 2001). In addition,
the response of 56 of 64 Pi-responsive genes was attenuated
in At-phr1 mutants (Bari et al., 2006). The latter result,
together with the knowledge that the Pi responsiveness of
Low-Pi-induced root morphology
Non-coding RNAs: At4,
At4.1, At4.4, At-IPS1
Overlapping subsets of genes induced by low Pi
FIG. 1. A model for the plant Piregulon. For simplicity, the interactions of genes that are induced (heavily outlined boxes), do not respond (lightly outlined
boxes) or are repressed (dashed boxes) by Pideficiency in arabidopsis are shown. The activities of genes from other plants are discussed in the text. The
target genes and functions are under either positive (arrowheads) or negative (T-junctions) regulation by the indicated factors. Genes that have been experimen-
tally determined to be influenced by At-SIZ1 are indicated by heavy arrows. Convergent box arrows show gene products that physically interact with one another
as part of a complex. Question marks indicate possible signalling pathways that are not currently supported by experimental evidence.
Yang & Finnegan — Regulation of phosphate starvation responses in higher plants 514
many of those genes examined was fully reversible within 3 h
of Piresupply, led to the conclusion that At-PHR1 may be a
starvation-induced genes (Fig. 1). The At-PHR1 gene was
mapped to locus At4g28610 and encodes a member of the
MYB transcription factor superfamily. The At-PHR1 protein
sequence has high similarity to the sequence of the
PHOSPHORUS STARVATION RESPONSE1 (PSR1) gene
product from Chlamydomonas reinhardii (Wykoff et al.,
1999).Both At-PHR1 and
DNA-binding domain and a coiled-coil domain, indicating
the potential for protein–protein interactions. At-PHR1 binds
DNA as a dimer to an imperfect palindromic sequence
(GNATATNC) present in the promoters of many Pistarvation-
responsive genes (Rubio et al., 2001; Franco-Zorrilla et al.,
2004). Thus, At-PHR1 acts downstream in the Pistarvation
signalling pathway. The At-PHR1 gene is not itself particularly
Piresponsive and the protein is located in the nucleus indepen-
dently of the Pistatus of the plant, indicating that induction of
At-PHR1 activity does not require transcription (Rubio et al.,
Two rice genes, Os-PHR1 and Os-PHR2, orthologous to
At-PHR1 and with functions in the Pi stress signalling
network similar to At-PHR1 (Fig. 1), have recently been
characterized (Zhou et al., 2008). Transgenic plants with
reduced expression levels of either Os-PHR1 or Os-PHR2
had lower transcript amounts for several Pistarvation response
genes. These same Pi-responsive genes had increased
expression in transgenic plants that over-expressed Os-PHR1
and Os-PHR2. Of the two rice PHR genes, only Os-PHR2
over-expression led to an increase of Pi in shoots under
Pi-sufficient conditions, phenocopying over-expression of
At-PHR1 in both wild-type and At-phr1 arabidopsis. Thus,
Os-PHR2 is probably a functional homologue of At-PHR1,
although it remains possible that Os-PHR1 and Os-PHR2
share the regulatory duties encompassed by At-PHR1.
At-MYB62 encodes another MYB transcription factor
involved in the Pi deprivation response of arabidopsis.
Unlike At-PHR1, At-MYB62 expression is induced by Pistar-
vation (Misson et al., 2005), but only in leaves of young seed-
lings (Devaiah et al., 2009). The response to low Piis not
mimicked by potassium, iron or nitrogen deficiencies.
Expression in roots is apparently always low. At-MYB62
represses at least some Pi-inducible genes when over-
expressed. Thus, At-MYB62, itself induced by Pideprivation,
is likelyto bea negative
starvation-inducible genes, and may moderate their activity
during Pistarvation. The At-MYB62 protein, like At-PHR1,
is localized to the nucleus irrespective of the Pistatus of the
At-MYB62 transcripts upon Piresupply and its role in the regu-
lation of genes involved in Pisignalling, high-affinity Pitrans-
port and mobilization, suggests a global role for At-MYB62
during Pi deficiency. Over-expression of At-MYB62 under
Pi-sufficient conditions induced responses reminiscent of Pi
reduced primary root length and increased root acid phospha-
tase activity. Despite increases in Piuptake and root Piconcen-
tration that probably arose from changes in root architecture,
the shoot Pi concentration of over-expressing plants was
most, but not all,Pi
Cr-PSR1 havea MYB
lower than that of the wild type (Devaiah et al., 2009). The
partial rescue of the phenotype by exogenous gibberellic
acid and the suppression to varying degrees of the expression
of all five genes involved in gibberellic acid biosynthesis
suggests that MYB62 may have a role in regulating biosyn-
thesis of the hormone. Thus, MYB62 may regulate the Pistar-
The Os-PTF1 gene encodes a protein with a basic helix–
loop–helix (bHLH) motif and was identified by differential
screening of rice plants grown under normal and Pistarvation
conditions (Yi et al., 2005). Expression of Os-PTF1 is induced
in roots during Pideprivation, while expression is constitutive
in shoots. Like At-PHR1 and At-MYB62, Os-PTF1 is located
in the nucleus independently of Pistatus. Under Pi-deficient
conditions, both in the field and in hydroponics, plants over-
expressing Os-PTF1 have greater tillering, higher root and
shoot biomass, and a 25 % higher P content than wild-type
plants. Microarray analysis showed
abundance of 158 genes is altered by .2-fold in plants over-
expressing Os-PTF1 (Fig. 1). The induced genes encode pro-
teins such as nutrient transporters, transcription factors and
ATP-binding proteins. The promoters of the induced genes
generally have E-box elements (Massari and Murre, 2000),
with about 20 % of the genes having at least one copy of the
G-box element (Atchley et al., 1999). Further analysis of the
responsive genes will enhance our understanding of the func-
tion of Os-PTF1 in the regulation of Piresponse pathways.
Microarray analysis revealed that At-ZAT6 (At5g04340),
encoding a cysteine-2/histidine-2 (C2H2) zinc finger transcrip-
tion factor, is strongly induced by Pistarvation (Hammond
et al., 2003). Green fluorescent protein (GFP) fusion exper-
iments showed that At-ZAT6 is located in the nucleus.
Suppression of At-ZAT6 expression through RNA interference
(RNAi) is lethal (Devaiah et al., 2007b). Over-expression of
the gene retards primary root growth independently of the Pi
status of the plant and reduces Piuptake. Over-expression of
At-ZAT6 reducesthe expression
starvation-induced genes, confirming its role in regulating Pi
homeostasis (Fig. 1). Hence, At-ZAT6 appears to be a repres-
sor of primary root growth, regulating Pihomeostasis through
the control of root architecture.
At-WRKY75 is another
induced by Pi deficiency in arabidopsis (Misson et al.,
2005). The At-WRKY75 protein is located in the nucleus
and its transcripts increase in abundance, but to various
extents, in different parts of the plant during Pideprivation.
RNAi suppression of At-WRKY75 expression causes anthocya-
nin accumulation, indicating that the mutant plants are more
susceptible to Pistress than wild-type plants. In At-WRKY75
RNAi plants deprived of Pi, the expression of several Pi
starvation-inducible genes is reduced. In contrast, lateral root
length and number, as well as root hair number, significantly
increase under both Pi-deficient and Pi-sufficient conditions.
Thus, At-WRKY75 has a role in the Pistarvation response
as well as in root development (Fig. 1) (Devaiah et al., 2007a).
Microarray experiments indicate that the expression of
At-BHLH32 (At3g25710) is induced in both leaves and roots
after 48 h of Pistarvation (Wu et al., 2003). In an At-bhlh32
T-DNA insertion mutant grown under Pi-sufficient conditions,
Yang & Finnegan — Regulation of phosphate starvation responses in higher plants515
there is a significant increase in the expression of the Pi
starvation-inducible genes At-PPCK1 and At-PPCK2 (encod-
ing isoforms of phosphoenolpyruvate carboxykinase), as well
as increases in the accumulation of anthocyanins, the for-
mation of root hairs and the total Picontent compared with
wild-type plants (Chen et al., 2007). These results indicate
that At-BHLH32 acts as a negative regulator in the Pistar-
vation response (Fig. 1). During Pideprivation, mutations in
At-TTG1, At-GL3 and At-EGL3, genes that take part in root
hair formation (Bernhardt et al., 2005), lead to the decrease
in transcript abundance of At-PPCK1 and At-PPCK2 com-
pared with wild-type plants. These results indicate that the
three genes are positive regulators in the Pistarvation response
pathway (Fig. 1). Saccharomyces cerevisiae (yeast) two-hybrid
experiments showed that At-BHLH32 can physically interact
with At-TTG1 and At-GL3. Thus, At-BHLH32 was inferred
to interfere with the function of TTG1-containing complexes
(Fig. 1) and consequently influence the biochemical and mor-
phological processes that respond to Pistatus (Chen et al.,
SPX domain-containing proteins
The SPX domain appears at the N-terminus of various pro-
teins, especially those involved in signal transduction
(Barabote et al., 2006). In yeast, proteins containing the SPX
domain are involved in Pi transport and sensing, or the
sorting of proteins to endomembranes (Wang et al., 2004).
Most SPX-domain proteins with known functions in plants
are involved in the regulation of either nutritional homeostasis
or the response to environmental cues. The arabidopsis
genome contains 20 genes encoding SPX-domain proteins.
These proteins have been grouped into four sub-families
based on sequence similarity.
Members of three of the four SPX protein sub-families in
arabidopsis, a total of 16 proteins, possess an EXS domain
in addition to the SPX domain. The At-PHO1 gene
(At3g23430) encodes one such protein that is involved in
the regulation of Pihomeostasis (Wang et al., 2004). The
N-terminal half of At-PHO1 is mainly hydrophilic, while the
C-terminalhalf hassix potential
domains. The At-pho1 mutant of arabidopsis contains
approx. 95 % less Piand 50–75 % less total P in shoots than
wild-type plants (Poirier et al., 1991). The At-pho1 mutant is
deficient in the transfer of Pifrom root epidermal and cortical
cells to the xylem, but the role of At-PHO1 in Piloading to the
xylem is still unclear. The gene is expressed mainly in roots
and is slightly upregulated during Pideficiency. There are 11
At-PHO1 homologues in arabidopsis (Hamburger et al.,
2002). Among these, only At-PHO1 and At-PHO1;H1
(At1g68740) are able to complement the At-pho1 mutant
(Stefanovic et al., 2007). At-PHO1;H1, like At-PHO1, is
involved in the Pitransport pathway; however, its response
to Pistatus is through a separate signal transduction pathway.
The transcript abundance of At-PHO1;H1 is upregulated by
the At-PHR1 transcription factor under Pistarvation conditions
(Fig. 1) and suppressed by the Pianalogue phosphite, while
At-PHO1 expression is independent of both At-PHR1 and
phosphite (Stefanovic et al., 2007).
The fourth sub-family of SPX-domain proteins does not
contain an EXS domain. There are four proteins of this
type in arabidopsis, encoded by At-SPX1 (At5g20150),
At-SPX2 (At2g26660), At-SPX3 (At2g45130) and At-SPX4
(At5g15330). In response to Pi starvation, At-SPX1 and
At-SPX3 are strongly induced, while At-SPX2 is weakly
induced and At-SPX4 is repressed in both shoots and roots
(Duan et al., 2008). Of the six rice homologues in this sub-
family, Os-SPX1–Os-SPX6, five are induced by Pistarvation
in the roots and/or shoots, while no members are repressed
(Z. Wang et al., 2009). The SPX1 and SPX2 isoforms from
both arabidopsis and rice are targeted to the nucleus, while
other the forms are located elsewhere in the cell (Duan
et al., 2008; Z. Wang et al., 2009). The induction by Pi, the
diversity of cellular locations, coupled with a variety of cell
type-dependent transcription patterns suggest that that these
SPX proteins may be involved in the Pisignalling networks
that regulate the expression of Pi-responsive genes and that
each SPX protein has a unique physiological function (Fig. 1).
The strong repression of At-SPX1 and At-SPX2 in At-siz1 (a
SUMO E3 ligase gene, see below) and At-phr1 mutants indi-
cates that both genes are positively regulated by the
At-PHR1 regulon (Duan et al., 2008). At-SPX3 is also strongly
repressed in the At-phr1 mutant but only weakly repressed in
the At-siz1 mutant, suggesting that At-SPX3 acts downstream
of At-PHR1. The point of action for At-SPX4 is unclear,
because its expression is repressed about 50 % in both
At-siz1 and At-phr1 mutants, which may mean that At-SPX4
is regulated by At-PHR1 in combination with other factors,
or independently of At-PHR1.
Over-expression of At-SPX1 increases the transcript abun-
dance of three Pistarvation-induced genes, more significantly
under Pi-sufficient than Pi-deficient conditions, indicating that
At-SPX1 is a positive regulator in the Pisignalling pathway
(Duan et al., 2008). Partial RNAi repression of At-SPX3
leads to primary root growth retardation, increased Pitranspor-
tation from roots to shoots and enhanced expression of a
sub-set of Pistarvation-responsive genes, including At-SPX1.
These observations suggest that At-SPX3 provides negative
feedback in the At-SPX1 response to Pi starvation (Duan
et al., 2008). The functions of At-SPX2 and At-SPX4 require
further exploration to determine the level of their involvement
in the Pistarvation response.
Much more work is needed before strong inferences can be
drawn about the functional equivalence, if any, among the
SPX isoforms between rice and arabidopsis. Overlapping
expression patterns and nuclear localization suggest that
Os-SPX1 might be functionally equivalent to At-SPX1. Also,
Os-SPX1 and At-SPX1 are each positively regulated by
PHR1 orthologues, Os-PHR2 (C. Wang et al., 2009) and
At-PHR1, respectively. However, Os-SPX1 is also functionally
dissimilar to At-SPX1. Os-SPX1 and At-SPX1 are negative and
positive regulators, respectively, of different sets of Pi
starvation-induced genes. Unfortunately, until overlapping
sets of target genes are assessed, it remains possible that the
rice and arabidopsis SPX1 proteins act as both positive and
negative regulators, targeting similar gene sets in similar
ways. Another difference is that Os-SPX1 regulates the
expression of the other five genes in this rice SPX sub-family
in various ways depending on the tissue and Pistatus (Z. Wang
Yang & Finnegan — Regulation of phosphate starvation responses in higher plants 516
et al., 2009), a function that has not been ascribed to At-SPX1.
However, At-SPX3 has been shown to be a negative regulator
of At-SPX1, highlighting functional similarities between
At-SPX3 and Os-SPX1. RNAi suppression of Os-SPX1 in
rice also led to an increase in leaf Piconcentration (C. Wang
et al., 2009), as seen in arabidopsis where At-SPX3 is sup-
pressed by RNAi (Duan et al., 2008). The transcript abun-
dance of certain PHT-type Pitransporters is enhanced in the
roots of SPX1-suppressed lines of both rice and arabidopsis,
suggesting a mechanism for the enhanced Piabundance in
the leaves. Taken together, these studies show that SPX pro-
teins have complex, and as yet unclear, roles in the response
Small ubiquitin-related modifier (SUMO) proteins are small
polypeptide tags showing greatest primary sequence similarity
to ubiquitin. Like ubiquitin, SUMO proteins function through
conjugation with other proteins, a post-translational modifi-
cation that is involved in various cellular processes (Colby
et al., 2006). While the ubiquitin system typically tags proteins
for proteosome degradation, SUMO conjugation can stabilize
the target proteins and alter their sub-cellular localization, as
well as indirectly influence ubiquitination and protein degra-
dation (Colby et al., 2006). At-SIZ1 functions as a SUMO E3
ligase in the arabidopsis Piregulon (Fig. 1), as well as in other
cellular processes, including abscisic acid signalling (Miura
et al., 2007, 2009). The At-SIZ1 gene (At5g60410) was orig-
inally identified in a genetic screen designed to isolate genes
that confer NaCl tolerance (Miura et al., 2005). An At-siz1
T-DNA insertion mutant showed retarded primary root growth
compared with wild-type plants when the nutrient supply was
restricted. Wild-type root growth was restored by the addition
of Pi, but not other nutrients. The original At-siz1-1 mutant dis-
played a similar set of symptomstowild-type plants in response
to Pideprivation, but to a more severe degree, despite similar
intracellular Pilevels. Under Pi-deficient conditions, the abun-
dance of transcripts from several Pi starvation responsive
genes is similar in the At-siz1-1 mutant and wild-type plants,
but under Pi-sufficient conditions these transcripts are more
abundant in the At-siz1-1 mutant. Additionally, other genes
are induced more slowly by Pi limitation in the At-siz1-1
mutant. At-SIZ1 was localized to the nucleus and can replace
the yeast Sc-Siz2 SUMO E3 ligase in the in vitro
SUMOylation of an Sc-Cdc3 substrate (Miura et al., 2005),
indicating that At-SIZ1 is a SUMO E3 ligase. In vitro exper-
iments demonstrated that
SUMOylation of At-PHR1, indicating a role for At-SIZ1 in
the Pi deficiency response pathway (Fig. 1) (Miura et al.,
2005). In this regard, it is noteworthy that MYB62 contains
two functionally untested sites for potential SUMOylation
(Devaiah et al., 2009).
At-SIZ1 maywork in
At-UBC24 in the SUMOylating pathway (Fujii et al., 2005).
The At-UBC24 gene (At2g33770) encodes a putative E2
ubiquitin-conjugase (Aung et al., 2006; Bari et al., 2006). It
is responsible for the Pi misallocation phenotype in the
At-pho2 mutant. The At-pho2 mutant accumulates up to
3-fold more total P in leaves, mostly as Pi, than wild-type
At-SIZ1 canmediate the
plants (Delhaize and Randall, 1995). Like the At-pho2
increased uptake and translocation of Pifrom roots to shoots
and reduced Pi remobilization within leaves (Aung et al.,
2006). In the At-pho2 mutant, transcripts from a number of
Pi-replete conditions, in contrast to wild-type plants where
transcripts from these genes are repressed by high Pisupply
(Shin et al., 2004; Bari et al., 2006). At-PHO2 and At-PHR1
evidently share a number of downstream targets. A study on
the expression of 64 Pi-responsive genes in At-pho2 and
At-phr1 mutants revealed 21 genes with an altered Piresponse
in both backgrounds (supplementary data in Bari et al., 2006).
That is, the repression of these genes by Pi-replete conditions
was impaired in the At-pho2 mutant, while their induction by
Pi-deficient conditions was weakened in the At-phr1 mutant.
This same study found that the abundance of primary tran-
scripts from all five At-MIRNA399 genes in a At-pho2
mutant grown under Pi-replete conditions was the same as in
wild-type plants, but they were not fully induced in the
At-phr1 background. These results and the location of putative
At-PHR1-binding sites 160–270 nucleotides (nt) upstream of
several At-MIRNA399 genes place At-PHO2/At-UBC24 down-
stream of At-PHR1 in the plant Pisignalling pathway (Fig. 1)
(Bari et al., 2006). While the interactions of At-PHO2/
At-UBC24 with downstream elements of the Pi signalling
pathway remain unclear, the At-SPX genes are likely candi-
dates, as Os-SPX1 functions downstream of Os-PHO2
(C. Wang et al., 2009).
Protein phosphorylation and dephosphorylation
Protein phosphorylation by kinases and dephosphorylation
by phosphatases is a potent binary switch involved in the regu-
lation of most cellular activities and processes (Luan, 2003).
Microarray analysis revealed differential regulation of several
protein phosphatases at the onset of Pideprivation in arabidop-
sis (Wu et al., 2003). However, Le-PS2 from Lycopersicon
esculentum (tomato) is the only protein phosphatase induced
by Pistarvation that has been studied in any detail (Baldwin
et al., 2001, 2008). The ability of recombinant Le-PS2
expressed in bacteria to dephosphorylate a phosphopeptide
substrate, an activity that is suppressed by okadaic acid,
demonstrates that Le-PS2 is a phosphoprotein Ser/Thr phos-
phatase (Baldwin et al., 2008). However, its target proteins
are not known. Given the central importance of reversible
protein phosphorylation in other cellular processes and signal
transduction pathways, it is surprising that its involvement in
the Pideprivation response has received so little attention.
One crucial determinant for the activity of Pitransporters is
their delivery through the secretory pathway to the plasma
membrane. At-PHF1, the first Pistarvation-responsive com-
ponent of the trafficking pathway to be isolated, enables the
high-affinity Pitransporters to exit from the endoplasmic reti-
culum (ER). The At-phf1 mutant was isolated (Gonza ´lez et al.,
2005) using a reporter gene screen similar to that used to
isolate the At-phr1 mutant (Martı ´n et al., 2000; Rubio et al.,
Yang & Finnegan — Regulation of phosphate starvation responses in higher plants517
2001) and described above. The At-phf1 mutation causes the
retention of the At-PHT1;1 Pi transporter in the ER and
leads to a decrease in the concentration of Pi in whole
plants. The lesion in the At-phf1 mutant was positionally
cloned to At3g52190, which encodes a SEC12-related
protein specific to plants. The At-PHF1 gene is expressed
widely in the plant, but mainly in roots, flowers and senescing
leaves. At-PHF1 has a promoter motif that conforms to the
core binding sequence of At-PHR1 (Rubio et al., 2001). The
decrease in At-PHF1 transcript abundance in the At-phr1
mutant suggests that At-PHF1 is indeed under direct transcrip-
tional control of At-PHR1 (Fig. 1). The mechanism by which
At-PHF1 regulates the intracellular transport of proteins and
determines sub-cellular localization is still unclear. The possi-
bility of a separate trafficking apparatus for an individual
protein, or a group of proteins involved in a single process,
is intriguing, and further investigations may provide new
ideas on how plant cells deliver proteins to specific intracellu-
lar locations, especially during acclimation to physiological
Non-coding RNA genes produce functional RNA molecules
rather than proteins. They play key roles in chromosomal silen-
cing, transcriptional regulation, translational repression, devel-
opmental control and responses to stress (Axtell et al., 2007).
Non-coding RNAs are derived predominantly from introns and
intergenic regions, but also from the opposite strand of protein-
coding genes (Storz, 2002). MicroRNAs (miRNAs) are non-
coding RNAs 20–24 nt in length. They are derived from the
stem of hairpin-like precursors of about 75 nt that are in turn
derived from longer pri-miRNAs (Zhu, 2008). miRNAs
silence genes with complementary or partly complementary
sequences by aiding mRNA cleavage or translational repres-
sion (Carrington et al., 2003; Bartel, 2004; He and Hannon,
2004). A screen of miRNAs regulated by Pideprivation led
to the identification of miR399 (Sunkar and Zhu, 2004).
There are six MIRNA loci in arabidopsis that encode miR399
species a–f. In arabidopsis, At-PHO2/At-UBC24 was con-
firmed to be a target gene for At-miR399 (Fig. 1); it has five
miR399 target sites in the 50-untranslated region (UTR) of
its transcripts (Sunkar and Zhu, 2004; Allen et al., 2005).
At-miR399 accumulation is induced by Pideprivation, a con-
dition that suppresses the At-PHO2/At-UBC24 target gene
(Fujii et al., 2005). Experiments with transgenic plants that
produce artificial At-PHO2/At-UBC24 mRNAs with or
without the 50UTR indicated that miR399 downregulates
At-PHO2/At-UBC24 mRNA accumulation by targeting the
50UTR (Fujii et al., 2005).
The degradation of At-PHO2/At-UBC24 transcripts by
miR399 in the Piregulon is itself regulated by non-coding
RNAs from the Mt4/TPSI1 gene family (Fig. 1). These non-
coding RNAs originally came to prominence because of
their strong induction in Pi-starved plants (Burleigh and
Harrison, 1997, 1998; Liu et al., 1997). Members of the
Mt4/TPSI1 gene family include Le-TPSI1 in tomato (Liu
et al., 1997), Mt4 in Medicago truncatula (Burleigh and
Harrison, 1997, 1998), an Mt4-like gene in Glycine max
(soybean) (Burleigh and Harrison, 1999), At4, At4.1, At4.2
and At-IPS1 in arabidopsis (Burleigh and Harrison, 1999;
Martı ´n et al., 2000; Shin et al., 2006) and Os-PI1 in rice
(Wasaki et al., 2003b). The transcripts of Mt4/TPSI1 genes
typically contain multiple short open reading frames that are
not conserved among the family members and are unlikely
to encode proteins. The lack of conserved open reading
frames originally suggested that the transcripts from Mt4/
TPSI1 were the active gene products. The overall sequence
identity across the family is quite low, except for a fairly
well conserved 23 nt motif in the central region of each
The Mt4/TPSI1 genes have a dramatic impact on Pidistri-
bution. For example, an At4 T-DNA insertion mutant does
not redistribute shoot Pito the roots. Instead, Piaccumulates
in the shoots, causing an increase in the shoot to root Pi
ratio compared with that in wild-type plants (Shin et al.,
2006). The first clue to the mechanism mediating the action
of the Mt4/TPSI1 RNAs came from the observation that the
23 nt sequence in the middle of At4 that is well conserved
among all Mt4/TPSI1 family members hybridizes to an
approx. 22 nt RNA expressed during Pi starvation (Shin
et al., 2006). It was also noted that the 23 nt conserved
sequence had extensive homology to miR399. However,
there are critical mismatches, including a bulge opposite
positions 10–11 of miR399, disrupting the base pairing
that is required for miR399-guided cleavage of mRNA
targets (Jones-Rhoades et al., 2006; Franco-Zorrilla et al.,
At-miR399 (Franco-Zorrilla et al., 2007). Moreover, At-IPS1
over-expression results in the accumulation of At-PHO2/
At-UBC24 mRNA, an miR399 target, demonstrating that
At-IPS1 represses miR399-dependent transcript cleavage.
Therefore, Mt4/TPSI1 RNAs may function as non-cleavable
miR399 substrate competitors, sequestering miR399 in a
state where it cannot act upon target gene transcripts
(Franco-Zorrilla et al., 2007). Interestingly, At-miR399b and
At-miR399c are less efficient in suppressing At-PHO2/
At-UBC24 than At-miR399f. This differential efficiency may
be due in part to differences in complementary between the
various At-miR399 isoforms and At4/At-IPS1 transcripts,
where higher complementarity may lead to more efficient
sequestration by the At4/At-IPS1 transcript (Doerner et al.,
2008). The differential efficiency of its members suggests
that the At-miR399 family is part of a fine-tuning mechanism
that allows the cell to respond subtly to the dynamics of Pi
Revealing the details of regulatory circuits such as the
miR399, Mt4/TPSI1 and PHO2/UBC24 system is only the
first step in understanding what is likely to be a complex invol-
vement for non-coding RNAs within the Pistarvation regulat-
ory network. Deep sequencing and other global RNA analysis
tools are revealing a rapidly growing number of small RNAs
whose abundance responds to Pistatus (Hsieh et al., 2009;
Pant et al., 2009). Several of these small RNAs are miRNAs
and are likely to be involved in regulating processes such as
Piuptake and translocation (i.e. At-miR399), anthocyanin bio-
synthesis, oxidative stress reduction, sulfate translocation and
nutrient recycling. While the expression of some of the
miRNAs seems to respond only to Pi, others respond not
Yang & Finnegan — Regulation of phosphate starvation responses in higher plants518
only to Pistatus, but also to the availability of other mineral
nutrients, such as N, K, S, Cu and Fe. Among the miRNAs
in this latter group, the regulatory response can be either in
the same or the opposite direction as the response to Pi
status. Another group of Pi-responsive small RNAs are
miRNA star strands, such as At-miR399*, indicating that
these molecules may have important biological functions.
The role of still other Pi-responsive small RNAs in the Pi
regulon is entirely unknown. Together, these molecules
provide the prospect of many exciting discoveries in the future.
PiSENSING IN HIGHER PLANTS
The local availability of Picontrols responses such as root hair
number and length, and primary root length. Pre-existing root
hairs of plants grown under Pi-replete conditions are shorter
than new root hairs that grow after plants are transferred to
Reciprocally, root hair growth is suppressed in plants trans-
ferred from Pi-deficient to Pi-replete conditions. Primary root
growth also slows dramatically when the root tip reaches
patches of growth medium that contain little Pi (Linkohr
et al., 2002). This Pideficiency-dependent arrest is not a nutri-
tional response but a response to a local Pisignal because
primary roots that detached from the growth medium and
entered the air phase did not stop growing despite the lack
of Piuptake by the growing tip (Svistoonoff et al., 2007).
Moreover, growth of these roots stopped immediately when
the tips encountered medium containing low concentrations
of Pi(Svistoonoff et al., 2007). Using a reporter gene approach
to identify dividing cells at the G2/M transition, it was shown
that the root tips growing through air divided normally, but
ceased to divide when the root tips encountered growth
medium containing low Pi(Svistoonoff et al., 2007).
Mutant analysis is revealing the mechanism of local Pi
sensing. The EMS-induced At-pdr2 mutant of arabidopsis
showed reduced primary root growth compared with wild-type
plants when DNA was the only source of Pi (Chen et al.,
2000). The mutant had reduced rates of root cell division
and elongation when grown in Pi-deficient conditions. The
mutant was not affected in the P concentration within the
root tip or in Piuptake rates, excluding a defect in high-affinity
Piacquisition. These results helped lead to the conclusion that
the At-pdr2 phenotype is caused by a defect in the sensing of
the local external Pi concentration (Ticconi et al., 2004).
At-PDR2 was recently identified by map-based cloning
(Ticconi et al., 2009) and found to encode the single P5-type
ATPase (At5g23630). At-PDR2 was localized to the ER and
found to regulate stem cell differentiation and meristem
activity through a pair of GRAS family transcription factors,
SCR and SHR (Ticconi et al., 2009) that are key regulators
of radial root patterning (Di Laurenzio et al., 1996;
Gallagher and Benfey, 2009).
At-PDR2 was found to interact genetically with At-LPR1 in
an ER-resident pathway (Ticconi et al., 2009). At-LPR1, a
quantitative trait locus (QTL) responsible for determining
root responses to Pistarvation in arabidopsis, was mapped to
the At1g23010 locus, which encodes a multicopper oxidase
required for low Pi-dependent growth arrest (Svistoonoff
et al., 2007). At-LPR1, like At-PRD2, is expressed in the
root tip, including the meristem and root cap. At-LPR1 may
be involved in the switching of primary root growth from inde-
terminate to determinate, moderating the activity and/or distri-
bution of a hormone-like compound (Svistoonoff et al., 2007).
At-LPR1 and At-PDR2 are thought to function together to
adjust meristem activity in response to external Pi status
(Ticconi et al., 2009). Elucidating the nature of this interaction
and uncovering other components of the ER-resident control
pathway will provide new insights into the adjustment of
root characteristics to local Pisignals.
Piitself can function as a local signal. Both Piand phos-
phite, a supposedly metabolically inert analogue of Pi, could
rescue the root meristematic activity of the At-pdr2 mutant
under Pi-limiting conditions (Ticconi et al., 2004). The
appearance within 1 h of Pi limitation of transcripts from
genes that are Pistarvation inducible and the decrease in abun-
dance of these transcripts within 30 min of Piresupply, while
shoot Piand carbohydrate status did not change, has also been
taken as evidence that local Piconcentration acts as a signal
(Wang et al., 2002; Muller et al., 2004).
the roots and transport them to shoots. The balance between
shoot demand and root supply for any particular mineral nutri-
status in the various tissues (Lin et al., 2008). The existence of
long-distance signals that report on Pistatus in the tissues has
been demonstrated in split-root experiments, where the roots
of Pi-starved plants were divided, with one part exposed to
Pi-replete medium and the other part exposed to Pi-deficient
medium. In tomato, genes that were normally induced in
response to Pistarvation were systemically repressed in the
portion of the root system exposed to Pi-deficient medium
when the remaining roots were exposed to Pi-sufficient
medium (Liu et al., 1998; Baldwin et al., 2001). Similarly,
lateral root elongation is also controlled by shoot Pistatus
(Linkohr et al., 2002; Shane and Lambers, 2006; Shane et al.,
2008). Long-distance signals are also important for the regu-
lation of resource allocation during leaf development, flowering
and pathogen defence. These long-distance signals move
through the phloem, which contains proteins, sugars, organic
acids, amino acids, phytohormones, miRNAs and other types
of small RNAs, all of which are potential signal molecules
(Lough and Lucas, 2006).
Sugars have been suggested to be a systemic signal for the Pi
starvation response in plants, in addition to their more estab-
lished roles as signals in other plant adaptive responses
(Koch et al., 2000; Grigston et al., 2008), substrates for the
biosynthesis of complex carbohydrates and in energy metab-
olism, and in driving phloem transport and delivery of C skel-
etons to sink tissues for growth and development. Sugars are
required for Pistarvation responses, including the stimulation
of lateral root growth and the induction of Pi starvation-
responsive genes (Karthikeyan et al., 2007). Sucrose induces
the accumulation of At-Pht1;1 and At4 transcripts in plants
grown under Pi-sufficient conditions, and its absence under
Yang & Finnegan — Regulation of phosphate starvation responses in higher plants 519
Pi-deficient conditions represses transcript accumulation from
these genes. Since plants grown under Pisufficiency contain
significantly more Pithan those grown under Pideficiency,
the effect of sucrose on the expression of Pi starvation-
responsive genes is not merely the result of sequestration of
Piin sugar–phosphates. Instead, it appears that sucrose acts
(Karthikeyan et al., 2007; Hammond and White, 2008).
The mapping of the At-pho3 mutation to At-SUC2, which
encodes a sucrose–proton symporter that is important for
phloem loading of sucrose, provides further evidence that
sugar acts as a signal during Pi starvation (Lloyd and
Zakhleniuk, 2004). The At-pho3 mutant has reduced root
acid phosphatase activity when grown under Pi-deficient con-
ditions (Zakhleniuk et al., 2001). When grown under
Pi-sufficient conditions, the total P content is much lower in
the shoots and roots of 11-d-old At-pho3 mutant seedlings
than in wild-type seedlings. The At-pho3 mutant also accumu-
lates much more anthocyanin and starch than wild-type seed-
lings, clear indicators of a Pistarvation response (Zakhleniuk
et al., 2001). While suggesting that sugar acts as a systemic
signal in the plant Pistarvation response, these experiments
cannot exclude the possibility that sugar is only being used
as a substrate. More research needs to be done to understand
fully the role of sugars as components of the Pistarvation
Small non-coding RNAs are likely to be another important
class of long-distance signalling molecules. miR399 has been
detected in the phloem sap of Cucurbita maxima (Yoo et al.,
2004) and Brassica napus (Buhtz et al., 2008). The high Pi
deficiency-dependent abundance of miR399 in the roots was
dwarfed by the abundance of miR399 in phloem sap (Pant
et al., 2008). This led to the demonstration through grafting
experiments that miR399 is transported from the shoots to
the roots through the phloem in both arabidopsis and
Nicotiana benthamiana (Lin et al., 2008; Pant et al., 2008).
When shoots over-expressing miR399 due to the presence of
a miR399 transgene were grafted onto the wild-type rootstocks
lacking miR399, the roots accumulated mature miR399 to very
high levels, while the corresponding primary transcripts were
virtually absent. The grafted plants also accumulated 5-fold
more Piin the shoots than the wild-type plants did. Thus,
the phloem transport of miR399 from the shoots to the roots
apparently systemically controls the maintenance of plant Pi
homeostasis (Lin et al., 2008; Pant et al., 2008).
The At-pho1 mutant may give important insights into how
miR399 exerts this systemic control. Under Pi-sufficient con-
ditions, At-pho1 plants have decreased amounts of Piin the
shoots, despite wild-type amounts of Piin the roots. The Pi
deficiency in the At-pho1 shoots results in the accumulation
in this tissue of both primary and mature miR399 transcripts.
The amount of primary miR399 in roots is also increased. It
is not yet clear whether the increased abundance of miR399
in roots is the result of increased expression of miR399 in
the roots or the translocation of miR399 from the shoots
(Lin et al., 2008). Distinguishing between these two possibili-
ties will give us a greater appreciation of the mechanism
through which miR399 controls Pihomeostasis.
Phloem sap contains a large variety of RNA molecules,
ranging in size from miRNAs to mRNAs (summarized in
Zhang et al., 2009), and including miRNA star strand
sequences (Buhtz et al., 2008; Pant et al., 2009). Among
these RNAs are a group of small RNAs with sequences
related to nuclear or organelle genes encoding rRNA and
tRNA, as well as nuclear genes for small nucleosomal RNA,
processing-related small RNA and signal recognition particle
RNA (Zhang et al., 2009). The recent finding that several
RNA fragments falling into these classes, as well as several
miRNA star sequences, accumulate specifically in Pi-starved
plants (Buhtz et al., 2008; Hsieh et al., 2009; Pant et al.,
2009) suggests that these classes of RNA may have a biologi-
cal function. Moreover, their presence in phloem sap (Buhtz
et al., 2008; Pant et al., 2009; Zhang et al., 2009) suggests
that some may have systemic regulatory functions. This possi-
bility adds a potentially exciting new dimension to the
RNA-based regulatory components of the Pi starvation
Global integration of Pisignalling
Many of the factors described above are components of a
complex regulatory web that dynamically assesses and
responds to the availability of Piat the cellular or whole-plant
level. The establishment and maintenance of a mineral nutrient
balance that allows optimal growth under the prevailing
environmental conditions and within the specific developmen-
tal stage of the plant requires the integration of these
Pi-specific responses with other signalling networks operating
at the whole-plant level. Such signals include information on
the availability of other mineral nutrients, such as iron and
zinc, and long-distance and local signals embedded within
molecules such as phytohormones. A thorough understanding
of the integration of the relevant signals and responses is
central to obtaining a holistic view of plant nutrient acqui-
sition. Aspects of this integration have been discussed in
detail elsewhere (Ward et al., 2008; Rubio et al., 2009;
Santner et al., 2009).
The interaction of P with iron is a long-known example
where the availability of one nutrient affects the activity of
another. The chemical interaction of P and iron in the
growth medium, at the root surface or within the plant itself
can lead to the formation of complexes that lower the bioavail-
ability of both nutrients. The inhibition of primary root
elongation that is commonly observed in arabidopsis upon Pi
deprivation is not typical of all plants and was recently
linked to iron toxicity (Ward et al., 2008). When plants were
grown in media containing decreased iron, the removal of Pi
had no effect on primary root elongation. This observation
goes some way to explain why a number of iron-responsive
genes were found among the genes that respond to Pi
deficiency in tomato and arabidopsis (Wang et al., 2002;
Misson et al., 2005; Zheng et al., 2009). These genes were
likely to be responding to an increase in available iron under
low Piconditions. Also in arabidopsis, increases in plant iron
concentration and modifications to iron storage under low Pi
conditions are related to enhanced iron availability in the
plant and the external medium (Hirsch et al., 2006).
It has also been known for a long time that zinc deficiency
can cause high levels of Pito accumulate in plant tissues,
which can lead to toxicity when Pi is readily available
Yang & Finnegan — Regulation of phosphate starvation responses in higher plants520
(Cakmak and Marschner, 1986). In barley, the expression of
genes encoding the Hv-PT1 and Hv-PT2 high-affinity Pitrans-
porters was induced by zinc deficiency, independently of Pi
availability (Huang et al., 2000). The role of zinc in the regu-
lation of the high-affinity Pitransporter genes is specific, and
cannot be mimicked by manganese, another divalent cationic
micronutrient. From these results, it was suggested that zinc
may have a specific role in the signal transduction pathway
regulating the high-affinity Pi transporter genes (Huang
et al., 2000).
It is becoming clearer that phytohormones are involved in
the responses of plants to the availability of mineral nutrients,
including Pi(Rubio et al., 2009). However, this role is often
secondary to the initial response to low Pi and may only
affect specific components or segments of the Pi regulon.
Plant hormones are structurally unrelated small molecules
that have profound effects as growth regulators (Santner
et al., 2009). Generally acting at low concentrations, they
mediate plant responses to both biotic and abiotic stresses by
acting at a distance from the site of synthesis, and/or by
acting locally, at or near the site of synthesis. Hormone signal-
ling generally leads to major changes in transcript profiles.
Non-genomic hormonal responses are also likely to occur,
but these are not as well characterized. Recent work has
made it clear that regulated protein degradation mediated by
ubiquitin is a common theme in hormone signalling. Several
hormone receptors have now been identified as enzymes in
the ubiquitin–protein conjugation pathway, and the abundance
of key downstream signalling proteins has also been found to
be regulated by ubiquitin-dependent degradation (Santner
et al., 2009).
Cytokinins are negative regulators of Pistarvation responses
in arabidopsis. The expression of Pistarvation-induced genes,
such as At-PHT1;1, At-ACP5 and members of the Mt4/TPSI1
gene family, was repressed by the application of exogenous
cytokinin (Martı ´n et al., 2000; Shin et al., 2006). Mutation
of the cytokinin receptor genes At-CRE1/WOL/AHK4 and
At-AHK3 can attenuate the repression in the presence of
sugar (Franco-Zorrilla et al., 2002, 2005). Moreover, Pidepri-
vation repressed the accumulation of cytokinin and the
expression of At-CRE1, conditions that would be expected to
favouractivation of the
(Franco-Zorrilla et al., 2002). Evidence indicating that cytoki-
nins, generally acting through At-CRE1, also repress a number
of genes that respond to nitrogen, sulfur or iron deficiency
(Rubio et al., 2009) is a measure of the complexity of the regu-
latory network necessary to integrate the nutrient-dependent
signals within plants.
Several studies in arabidopsis have examined the role of
auxin as a mediator for the reduction in primary root growth
and the proliferation of lateral roots induced by Pistarvation.
Auxin-resistant mutants show largely wild-type primary root
growth responses under both Pi sufficiency and deficiency,
suggesting an auxin-independent mechanism for this response
(Williamson et al., 2001; Lo ´pez-Bucio et al., 2002; Al-Ghazi
et al., 2003). On the other hand, Pistarvation enhances the
responsiveness of the root system to the induction of lateral
root proliferation caused by the application of exogenous
auxin (Lo ´pez-Bucio et al., 2002). The accumulation of auxin
at the primary root apex, in pre-initiated lateral root primordia
and in young lateral roots suggested that auxin transport or bio-
synthesis was important (Nacry et al., 2005). However, plants
defective either in auxin sensing or in polar transport due to
mutation or inhibitors are still able to produce increased
lateral roots in response to Pistarvation (Williamson et al.,
2001; Lo ´pez-Bucio et al.,
Pe ´rez-Torres et al., 2008). Together, these results point to
increased auxin sensitivity, not increased auxin transport or
synthesis, as the mediator of proliferative lateral root growth.
The low Pi-induced increase in auxin sensitivity leading to
lateral root formation is likely to be mediated by the
At-TIR1 auxin receptor (Pe ´rez-Torres et al., 2008). Many
auxin-responsive genes are held in a repressed state by the
binding of an Aux/IAA protein to an auxin response factor
occupying the promoter (Santner et al., 2009). At-TIR1 is
induced in response to low Piand encodes an F-box protein
that binds directly with Aux/IAA, an interaction that is
enhanced by auxin. The combination of auxin, At-TIR1 and
Aux/IAA induces the ubiquitin-dependent degradation of
Aux/IAA by the 26S proteosome, allowing the auxin response
factor to proceed with transcription. The arabidopsis transcrip-
tion factor AUXIN RESPONSE FACTOR 19 (At-ARF19) has
been implicated in the increase in lateral root growth in
response to low Pi, and is a candidate participant in the
TIR1-mediated induction of lateral root formation in response
to low Pi(Pe ´rez-Torres et al., 2008).
An involvement of gibberellic acid in the Pi starvation
response is beginning to emerge. Gibberellic acid at least par-
tially represses low Pi-induced changes to arabidopsis root and
shoot growth and architecture, including the inhibition in
primary root growth, the increase in lateral root density and
the increase in root-to-shoot ratio (Jiang et al., 2007).
Moreover, over-expression of At-MYB62 led to symptoms of
gibberellic acid deficiency, which could be partially rescued
by the application of exogenous gibberellic acid (Devaiah
et al., 2009). The gibberellic acid deficiency arose from a
decrease in bioactive hormone brought about by associated
changes in the transcript abundance for genes involved in gib-
berellic acid metabolism.
At least some of the molecular events leading to low
Pi-induced changes in plant architecture and anthocyanin
accumulation are dependent on the gibberellic acid–DELLA
protein degradation (Jiang et al., 2007). Gibberellic acid
induces plant growth by promoting the destruction of the
growth-restraining DELLA proteins (there are five DELLA
proteins in arabidopsis). Similarly to the auxin signalling
pathway, the binding of gibberellic acid to a specific receptor
(GID1a, b or c) promotes binding of the hormone–receptor
complex to the DELLA proteins (Santner et al., 2009).
DELLA proteins are typically bound to, and inactivate,
various transcription factors. The binding of the gibberellic
acid–receptor complex to DELLA initiates the ubiquitin-
dependent destruction of DELLA by the 26S proteosome,
releasing the transcription factors to activate the gibberillin
response (Santner et al., 2009).
Other hormones seem to have only a limited role in regulat-
ing the Pistarvation response. Mutants with impaired abscisic
acid sensitivity (aba2-1) or biosynthesis (aba1) have a reduced
Pistarvation response, including reduced expression of some
2002;Jain etal., 2007;
Yang & Finnegan — Regulation of phosphate starvation responses in higher plants521
Pi-responsive genes and accumulation of anthocyanin (Trull
et al., 1997; Ciereszkoa and Kleczkowsk, 2002). However,
the mutants were not impaired in the production of Pi
starvation-induced phosphatases or in the ability to modulate
biomass allocation between roots and shoots. Application of
the hormone to the roots of Pi-deprived wild-type plants
decreased the transcript abundance in the roots for several
members of the Mt4/TPSI1 gene family (Shin et al., 2006).
It is interesting to note in this regard that At-miR399 homol-
ogues were identified in a population of arabidopsis plants sub-
jected separately to a number of stresses and abscisic acid
before pooling (Sunkar and Zhu, 2004). At-RAB18, which is
induced by Pi deprivation and sugar (Ciereszkoa and
Kleczkowsk, 2002), is also induced by abscisic acid. The
inductionis throughan At-ABI5-dependent
pathway, a process attenuated by the At-SIZ1-dependent
SUMOylation of At-ABI5, a transcription factor containing a
basic leucine-zipper domain (Miura et al., 2009).
Ethylene was found to inhibit root elongation in Pi-sufficient
plants, but stimulated it in Pi-deficient plants (Borch et al.,
1999; Ma et al., 2003). Conversely, the inhibition of ethylene
production or action inhibited root elongation in Pi-deficient
plants while stimulating it in Pi-sufficient plants. However,
the mechanism mediating the ethylene-dependent response
of root elongation to Pideficiency remains unclear.
It is likely that there is a complex interplay among Pi, hor-
mones and sugar signalling. Mutation of the cytokinin receptor
genes At-CRE1/WOL/AHK4 and At-AHK3 can attenuate the
repression in the presence of sugar (Franco-Zorrilla et al.,
2002, 2005). The abscisic acid and Pi starvation-inducible
At-RAB18 gene is also induced by sugar (Ciereszkoa and
UNIDENTIFIED MUTANT GENES RELEVANT TO
PiNUTRITION IN HIGHER PLANTS
Many of the genes described above that are central to the Pi
starvation response network were identified by mutant analy-
sis, clearly reiterating the power of using mutants to determine
gene function and dissecting the genetic pathways. There are a
number of interesting mutants available that are relevant to Pi
nutrition where identification of the mutated gene and func-
tional analysis of the gene product will give insights into the
details of the Pistarvation response network.
The At-lpi mutants
The reduction in primary root elongation is a conspicuous
root developmental change that occurs during Pistarvation
(Williamson et al., 2001). The At-lpi mutants, representing
EMS-mutagenized population due to their ability to maintain
primary root growth during Pistarvation (Sa ´nchez-Caldero ´n
et al., 2006). The abundance of transcripts from a sub-set of
Pistarvation-induced genes is reduced during Pideprivation
in the At-lpi mutant compared with wild-type plants. The
At-lpi phenotype is indicative of a function that may be
central to the cross-talk between low Pistatus and the acti-
vation of Pi deficiency-responsive genes that control root
development. Isolation of the affected genes from the At-lpi
mutants may, therefore, identify major control points in the
Pi starvation-induced signalling network (Sa ´nchez-Caldero ´n
et al., 2006).
The At-psr1 mutant
Arabidopsis can grow on a medium containing DNA as the
main Pisupply (Chen et al., 2000). The At-psr1 mutant was
isolated from an EMS-mutagenized arabidopsis population
due to its inability to use exogenous DNA when Pi is
limited. The mutant grows well when Pi is supplied.
Biochemical analysis showed that RNase and acid phosphatase
activities in the At-psr1 mutant are generally reduced com-
pared with those in wild-type plants. Genetic analysis indi-
cated that the mutant phenotype is caused by a single
recessive allele, implying that At-PSR1 influences the
expression of a sub-set of genes encoding enzymes that
degrade exogenous organophosphate substrates, increasing
the ability of the plant to scavenge Pi (Chen et al., 2000).
Identifying the corresponding gene will give added insight
into how plants regulate the scavenging of Pi from the
The At-pup1 mutant
The At-pup1 mutant was isolated from a T-DNA-
mutagenized arabidopsis population due to reduced root stain-
ing for phosphatase activity when grown on a Pi-deficient
medium (Trull and Deikman, 1998). Analysis of the phospha-
tases produced by the mutant showed that a 160 kDa acid
phosphatase isoform is missing. The response of the At-pup1
mutant to Pideprivation, such as the accumulation of antho-
cyanin and the altered partitioning of P between root and
shoot, were the same as in wild-type plants, while the root
to shoot ratio was lower in the mutant under Pi-sufficient con-
ditions (Trull and Deikman, 1998). Identifying At-PUP1
within the 5 cM interval on chromosome 2 to which it has
been mapped (Trull and Deikman, 1998) will give us
another gene involved in scavenging Pifrom the rhizosphere,
providing further opportunities to dissect the regulatory path-
ways used to control the deployment of these strategies.
CONCLUSIONS AND FUTURE PERSPECTIVES
In the past few years, there has been rapid progress made in
understanding the ways that plants respond and acclimate to
Pi-deficient growth conditions. The PHR1 transcription
factors At-PHR1 and Os-PHR2 have emerged as central regu-
lators in the deployment of the adaptive strategies required to
cope with Pideficiency in arabidopsis and rice, respectively.
Their constitutive presence in the nucleus suggests they are
poised for action, awaiting the signal that Piconcentration is
insufficient. But what is the nature of that signal? In future,
it will be interesting to see if there are other central regulators,
and to determine the conservation of functions across evol-
utionary distances. For example, why does rice require a
second PHR-type transcription factor, Os-PHR1, to adapt to
Pideficiency? Does a transcription factor in arabidopsis have
a role analogous to that of Os-PHR1? Are the differences
seen between rice and arabidopsis simply an indication of
Yang & Finnegan — Regulation of phosphate starvation responses in higher plants 522
species diversity, or do they hint at something larger, the
divide between monocots and eudicots?
The PHR factors are supported by a host of other regulatory
functions, including the transcription factors BHLH32,
WRKY75, ZAT6 and MYB62. Some of these factors are posi-
tive regulators acting to induce the expression of genes necess-
ary for adaptation, while others are negative regulators. The
involvement of both positive and negative effectors illustrates
the dynamic process underlying the deployment and adjust-
ment of the Pi starvation response to maintain a balance
between nutrient availability and acquisition to satisfy the
nutritional demands imposed by the prevailing developmental
programme and growth requirements. A fuller understanding
of the interplay among factors such as ZAT6 and MYB62
that modulate the intensity of the low Piresponse and positive
regulators such as PHR1 will reveal the true complexity of
plant adaptive responses to limiting Pi.
Details are now rapidly emerging about the interactions of
the Piregulon with the availability of other nutrients and the
regulatory networks deployed to adapt to deficiencies in
these nutrients. In this regard, hormonal responses and small
RNA molecules have central roles that are just being documen-
ted. Both hormonal responses and pathways regulated by small
RNAs, such as miR399, highlight the importance of post-
SUMOylation, in the plant response to Piavailability. Future
discoveries on the targets of post-translational modification
and the regulation of the modification process in response to
Piavailability are areas needing much further work.
The recent finding of numerous small RNA molecules
whose abundance is responsive to Pi status (Hsieh et al.,
2009; Pant et al., 2009) is both exciting and sobering. The
involvement of miRNAs and other small RNAs in regulating
plant responses to Pi is clearly very poorly understood,
despite the enlightening work done on revealing the regulation
of PHO2/UBC24 by miR399. It will take much effort to reveal
the roles of small RNAs in the plant response to Pi. However,
the academic and practical rewards for this effort are likely to
be large. Especially intriguing is the role of small RNAs, as
well as hormones and sugars, as long-distance signals in the
Pistarvation response pathway. In the case of RNAs, what is
the mechanism of their loading into and unloading from the
phloem? What cells are responsible for their synthesis and
what cells receive and act upon the signals received?
There are many other intriguing questions yet to be
answered in regard to the Pistarvation response in plants.
Perhaps top among these is the nature and identity of the
sensor that sets the whole Pistarvation response in motion.
The finding that local Pi is sensed by an ER-localized
pathway involving PDR2 and LPR1 is an excellent step
forward, but what is the mechanism of action of these proteins
and how does this pathway interlink with the systemic signal-
ling pathways? Might it be something as simple as a protein
phosphorylation that is inhibited by the lack of Pias a substrate
or will it be something much more complex? Answering these
and many other questions will ultimately reveal the entire Pi
starvation regulatory network and provide the understanding
needed to develop new molecular genetic strategies for estab-
lishing crop plants with improved Piacquisition and Piuse
We thank Hans Lambers, Ricarda Jost and Weihua Chen for
their excellent comments on draft versions of the manuscript.
Thiswork was supported
Scholarship from the China Scholarship Council (to X.J.Y.)
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