Journal of Experimental Botany, Vol. 60, No. 5, pp.

14271438, 2009
Perspectives on Plant Development Special Issue
doi:10.1093/jxb/ern303 Advance Access publication 23 January, 2009

REVIEW PAPER

Molecular regulators of phosphate homeostasis in plants

Wei-Yi Lin1,2,3,*, Shu-I Lin1,4,* and Tzyy-Jen Chiou1,2,3,4,
1
Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC
2
Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica,
Taipei 115, Taiwan, ROC
3
Department of Life Sciences, National Chung-Hsing University, Taichung 402, Taiwan, ROC
4
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan, ROC

Received 1 September 2008; Revised 5 November 2008; Accepted 10 November 2008

Abstract
An appropriate cellular phosphate (Pi) concentration is indispensable for essential physiological and biochemical
processes. To maintain cellular Pi homeostasis, plants have developed a series of adaptive responses to
facilitate external Pi acquisition and to limit Pi consumption and to adjust Pi recycling internally when the Pi
supply is inadequate. Over the past decade, significant progress has been made toward understanding such
regulation at the molecular level. In this review, the focus is on the molecular regulators that mediate cellular Pi
concentrations. The regulators are introduced and organized according to their original identification
procedures, by the forward genetic approach of mutant screening or by reverse genetic analysis. These genes
are involved in Pi uptake, allocation or remobilization or are upstream regulators, such as transcriptional factors
or signalling molecules. In the future, integration of current knowledge and exploration of new technology is
expected to offer new insights into molecular mechanisms that maintain Pi homeostasis.

Key words: Phosphorus, phosphate homeostasis, forward genetics, reverse genetics.

Introduction
Phosphorus (P), a plant macronutrient, is involved in the
regulation of many biochemical and physiological processes
and is an essential building block of cell components such
as nucleic acids, membranes, and energy sources. Plants
acquire P as the inorganic phosphate ion (Pi) through Pi
transporters in the roots (Marschner, 1995). Because of the
binding and precipitation by other cations and conversion
into organic compounds in the soil, the availability of Pi is
often limited, despite a large amount of total P in the soil
(Raghothama, 1999; Poirier and Bucher, 2002). To cope
with low Pi availability, plants have evolved a series of
adaptive responses through the integration of external
signals with internal factors to maintain steady cellular Pi
concentrations. The maintenance of Pi homeostasis under
inadequate Pi supply is co-ordinated by enhanced acquisi-
tion of external Pi and by conservation and remobilization
of internal Pi (Raghothama, 1999; Poirier and Bucher,
2002). Enhanced Pi acquisition is achieved by the induction
of high-affinity Pi transport systems, secretion of root
exudates to release Pi from unavailable soil P, the alteration
of root morphology and architecture by reduced primary
root length and increased length and density of branch
roots and root hairs, and symbiotic association with
mycorrhizal fungi. Conservation and remobilization of
internal Pi is facilitated by the alteration of metabolic
pathways and by the reallocation and recycling of
internal Pi among different organs or cellular compart-
ments. Recent study has uncovered several genes involved in
regulating Pi homeostasis, which has begun to shed light on
the mechanism of such regulation. In this review, we shall
mainly concentrate on these genes that contribute to
Pi homeostasis in plants (Table 1). They were identified by
forward or reverse genetic approaches, with loss-of-function
mutants or overexpressing transgenic plants showing altered

* These authors contributed equally to this work.

y
To whom correspondence should be addressed: E-mail: tjchiou@gate.sinica.edu.tw
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 1428 | Lin et al.

Table 1. Genes involved in the regulation of Pi homeostasis in plants

Gene/mutant Description of function Reference

Locus no.
(transcriptional
regulation by low Pi)

PHR1 d a MYB transcription factor Rubio et al., 2001

At4g28610 d activates a subset of Pi starvation-induced genes by the binding to P1BS element Nilsson et al., 2007
(unchanged) d phr1 shows reduced Pi concentration Zhou et al., 2008

SIZ1 d SUMO E3 ligase Miura et al., 2005

At5g60410 d facilitates the sumoylation of PHR1
(up) d regulates the expression of several Pi starvation-responsive genes
d siz1 shows higher shoot Pi concentration

PHF1 d a ER-located SEC12-related protein Gonzalez et al., 2005

At3g52190 d facilitates the trafficking of PHT1 proteins to plasma membrane
(up) d regulated by PHR1
d phf1 shows reduced Pi content

PHO1 d a protein with SPX and EXS domains Poirier et al., 1991
At3g23430 d involved in Pi loading into the xylem Hamburger et al., 2002
(up) d pho1 displays Pi starvation symptoms with reduced shoot Pi Stefanovic et al., 2007

PHO2 (UBC24) d a ubiquitin E2 conjugase Delhaize and Randall, 1995

At2g33770 d regulates Pi uptake, allocation and remobilization Dong et al., 1998
(down) d a target gene of miR399s Aung et al., 2006
d pho2 displays Pi toxicity with excessive shoot Pi Bari et al., 2006

MiR399a,b,c,d,e,f d a microRNA Fujii et al., 2005

At1g29265, At1g63005, d negatively regulates PHO2 Chiou et al., 2006
At5g62162, At2g34202, d serves as a shoot-derived long-distance signal Bari et al., 2006
At2g34204, At2g34208 d regulated by PHR1 Lin et al., 2008
(up) d overexpression of miR399 mimics the Pi toxic phenotype of pho2 Pant et al., 2008;

PHO3 (SUC2) d a sucrose/H+ symporter Zakhleniuk et al., 2001

At1g22710 d regulates Pi starvation responses Lloyd and Zakhleniuk, 2004
d pho3 shows reduced total P concentration

pup1 d pup mutants show aberrant APase activity and less accumulation of Trull and Deikman, 1998
pup3 Pi in organic phosphate-containing medium Tomscha et al., 2004

pdr2 d pdr2 cannot utilize organic phosphate, leading to less accumulation of P Ticconi et al., 2004
d pdr2 mutant is oversensitive to Pi starvation and has higher
Pi content under P deficient conditions
d likely to be involved in Pi signalling or sensing

PHT1;1(AtPT1)
At5g43350 d high affinity Pi transporters involved in Pi acquisition
(up) d pht1;1 mutant shows reduced Pi uptake and shoot Pi content Mitsukawa et al., 1997
d increased Pi uptake by overexpression of PHT1;1 in tobacco cells Misson et al., 2004
PHT1;4, (AtPT2) d pht1;1/pht1;4 double mutant shows significant decrease Shin et al., 2004
At2g38940 in Pi uptake, shoot Pi content and fresh weight
(up)

PHT2;1 d a low-affinity Pi transporter in chloroplast Daram et al., 1999

At3g26570 d involved in Pi allocation between roots and shoots and Versaw and Harrison, 2002
(unchanged) Pi remobilization between old and young leaves.

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 Molecular regulators of Pi homeostasis | 1429

Table 1. Continued

Gene/mutant Description of function Reference

Locus no.
(transcriptional
regulation by low Pi)

At4
At5g03545
d non-coding RNAs
(up)
d regulates Pi allocation between roots and shoots Shin et al., 2006
d inhibits the cleavage of miR399 to PHO2 mRNA by mimicking Franco-Zorrilla et al., 2007
IPS1
the target sequence of miR399
At3g09922
(up)

OsPTF1 d a bHLH transcription factor Yi et al., 2005

Os06g09370 d regulates the expression of several Pi starvation responsive genes
(up) d overexpression of OsPTF1 increases Pi content, root growth and tiller number

WRKY 75 d a WRKY transcription factor Devaiah et al., 2007a

At5g13080 d positively regulates Pi starvation responses but negatively
(up) regulates lateral root and root hair growth

ZAT6 d a C2H2 zinc finger transcription factor Devaiah et al., 2007b

At5g04340 d regulates several Pi starvation-responsive genes
(up) d controls root architecture, Pi uptake and Pi accumulation

BHLH32 d a bHLH transcription factor Chen et al., 2007

At3g25710 d a negative regulator of Pi starvation responses.
(up) d bhlh32 has hypersensitive Pi starvation responses and
increased Pi concentration

SQD2 d involved in sulfolipid biosynthesis Yu et al., 2002

At5g01220 d sqd2 mutant shows reduced growth under Pi deficient conditions
(up)

PLDz1
At3g16785
Cruz-Ramirez et al., 2006
(up) d involved in phospholipid degradation and synthesis of galactolipid in roots
Li et al., 2006a
d regulates root architecture in response to Pi starvation
Li et al., 2006b
PLDz2
At3g05630 (up)

NPC5 d involved in phospholipid degradation and synthesis of galactolipid in leaves Gaude et al., 2008
At3g03540
(up)

CAX1
At1g08960
d tonoplast Ca2+/H+ antiporters Cheng et al., 2003
d cax1/cax3 double mutant has increased shoot Pi content Cheng et al., 2005
CAX3
At3g51860

IPK1 d an inositol polyphosphate kinase Stevenson-Paulik et al., 2005

At5g42810 d reduced phytate but increased Pi concentration in ipk1 mutant seeds
d ipk1 mutant has high leaf Pi concentration

SPX1
At5g20150
(up) d SPX domain-containing proteins Duan et al., 2008
d regulates the expression of several Pi starvation-responsive
SPX3 genes involved in Pi uptake, allocation and remobilization
At2g45130
(up)

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 1430 | Lin et al.
cellular Pi concentrations. Some other genes also involved in
adaptive responses will not be discussed here because changes
in cellular Pi have not been reported following alterations of
their gene expression.
Forward genetic approaches
Several Arabidopsis mutants were discovered from screening
Pi concentration, phosphatase or nuclease activities, or
reporter activity specifically responsive to Pi starvation.
Characterization of these mutants and subsequent
identification of genes has yielded valuable information
about the molecular mechanisms regulating Pi homeostasis.
Mutants isolated by the AtIPS1 promoter::GUS reporter
By screening the activity of a b-glucuronidase (GUS) re-
porter gene driven by IPS1 (induction by Pi starvation 1)
promoter, several genes involved in the crucial regulatory
steps of Pi homeostasis have been isolated. The expression
of IPS1 is barely detected under Pi-sufficient conditions but
highly induced by low Pi (Martn et al., 2000). The clear-cut
and vigorous transcriptional induction of IPS1 by low Pi
renders it an excellent device for mutant screening. The
GUS reporter gene controlled by the IPS1 promoter
introduced into wild-type Arabidopsis (IPS1::GUS) shows
very low GUS staining under high Pi conditions but strong
GUS staining in low Pi medium. Mutants with abnormal
GUS activity were isolated from the EMS-mutagenized
IPS1::GUS transgenic plants. By this means, phr1 (phos-
phate starvation response 1) was first identified by the
reduction in GUS activity under low Pi conditions (Rubio
et al., 2001). PHR1, encoding a member of the MYB family
of DNA-binding proteins, regulates a number of Pi
starvation-induced genes through binding a P1BS (PHR1
specific binding sequence) cis-element (GNATATNC)
(Rubio et al., 2001). In agreement with this finding,
genome-wide expression profiling of Pi-responsive genes
also revealed the over-representation of the P1BS cis-
element in the promoter regions of Pi starvation-induced
genes encoding Pi transporters, phosphatases or translation-
related proteins (Hammond et al., 2003; Misson et al.,
2005). The phr1 mutant displays a reduced concentration of
Pi under both Pi-sufficient and Pi-deficient conditions and
minimal induction of anthocyanin accumulation in response
to Pi deprivation (Rubio et al., 2001). Further character-
izations involved the analysis of T-DNA knockout phr1 and
PHR1-overexpressing Arabidopsis plants (Nilsson et al.,
2007). Overexpression of PHR1 leads to an increased
concentration of Pi in the shoots, together with the elevated
expression of a range of Pi starvation-induced genes that
encode a Pi transporter, phosphatase and RNase (Nilsson
et al., 2007). Taken together, PHR1 is a key transcriptional
activator in controlling Pi uptake and allocation, anthocya-
nin accumulation, and carbon metabolism. As in Arabidop-
sis, two homologues of PHR1, OsPHR1 and OsPHR2, were
identified to be involved in regulating several Pi starvation-
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induced genes in rice (Zhou et al., 2008). However, only
overexpression of OsPHR2 results in increased shoot Pi
accumulation in transgenic rice (Zhou et al., 2008). Over-
expression of OsPHR2 also enhances root elongation and
root hair proliferation, which was not observed in Arabidop-
sis with altered expression of PHR1 (Rubio et al., 2001;
Nilsson et al., 2007).
Psr1 (Phosphate-stress response 1), a PHR1 homologue in
green algae (Chlamydomonas reinhartii), is up-regulated by
Pi starvation (Wykoff et al., 1999). However, both the
transcript level of Arabidopsis PHR1 and nuclear localiza-
tion of PHR1 protein are not altered by Pi status (Rubio
et al., 2001). Therefore, post-translational modification was
hypothesized to be involved in PHR1 activity. In support of
this notion, PHR1 was later revealed to be sumoylated by
SIZ1, a SUMO E3 ligase (Miura et al., 2005). A siz1 mutant
showing a suppression phenotype of a salt-sensitive mutant
was initially isolated (Miura et al., 2005). Surprisingly, the
siz1 mutant exhibits hypersensitive responses to Pi starva-
tion, including changes in root architecture with reduced
primary root growth and the massive formation of lateral
roots and root hairs, an increased ratio of root-to-shoot
mass and greater anthocyanin accumulation than Pi-starved
wild-type plants (Miura et al., 2005). The intracellular Pi
concentration is higher in shoots of siz1 than in wild-type
plants grown under Pi-sufficient conditions, possibly be-
cause of enhanced expression of the PHT1;4 Pi transporter.
From expression analysis of several Pi-starvation induced
genes in the siz1 mutant, SIZ1 may act as a negative as well
as a positive regulator (Miura et al., 2005). The expression of
these genes is likely to be modulated positively or negatively
by sumoylation, either directly or indirectly through PHR1 or
other regulators. In addition to Pi starvation responses, SIZ1-
mediated sumoylation is involved in the response to other
oxidative stresses, such as drought and low temperature
(Catala et al., 2007; Miura et al., 2007). It will be of interest
to know whether this SIZ1-mediated oxidative stress is
associated with the generation of reactive oxygen species by
Pi and other nutrient stresses (Shin and Schachtman, 2004;
Shin et al., 2005).
An approach similar to that for the isolation of phr1
identified a phf1 (phosphate transporter traffic facilitator1)
mutant with constitutive expression of GUS regardless of
whether the plant grew under Pi-sufficient or -deficient
conditions, because of low internal Pi concentrations
(Gonzalez et al., 2005). In Pi-sufficient medium, the phf1
mutant is impaired in Pi uptake and thus displays Pi
starvation symptoms. The reduced Pi uptake activity is
caused by a defect in targeting the PHT1;1 Pi transporter to
the plasma membrane (Gonzalez et al., 2005). PHF1 encodes
a plant-specific protein that functions as an endoplasmic
reticulum (ER)-located facilitator to assist the trafficking of
PHT1 family proteins. Its structure is similar to proteins of
the SEC12 family that are involved in vesicle exit from the
ER to the secretary system (Chardin and Callebaut, 2002;
Gonzalez et al., 2005). Expression of PHF1 is up-regulated
by Pi deficiency, which is mediated, in part, by the
transcriptional activation of PHR1.

Mutants isolated by alteration of Pi concentration
The most direct approach used to isolate mutants involved
in the maintenance of Pi homeostasis is the measurement of
cellular Pi concentrations. Analysis of leaf Pi concentrations
revealed mutants with aberrant Pi accumulation in the
shoots. pho1 was isolated as a Pi-deficient mutant with leaf
Pi concentration of only 5% of that of the wild type but
with normal root Pi concentration (Poirier et al., 1991). The
normal Pi uptake and root Pi concentration but lower shoot
Pi concentration indicates that PHO1 may participate in Pi
loading into the xylem and the subsequent translocation of
Pi from the roots to the shoots. PHO1 protein was later
revealed to harbour a hydrophilic N-terminal SPX (SYG1/
Pho81/XPR1) tripartite domain followed by a six-trans-
membrane spanning domain of EXS at the C-terminus
(Hamburger et al., 2002; Wang et al., 2004). Of note,
proteins with the SPX domain in Saccharomyces cerevisiae
play important roles in Pi transport or sensing or in sorting
Pi into endomembranes (Lenburg and OShea, 1996; Wyk-
off and OShea, 2001). PHO1 is expressed predominantly in
the vascular cells of roots and may contribute to the xylem
loading of Pi (Hamburger et al., 2002), although the Pi
transport activity of PHO1 has not been demonstrated.
There are an additional 10 members (PHO1;H1 through
PHO1;H10) of the PHO1 gene family in the Arabidopsis
genome (Wang et al., 2004). The expression patterns of
these PHO1 family proteins in diverse tissues suggest that
they may function in Pi transport into other cells besides
vascular xylem. PHO1;H1 rescues the phenotype of the
pho1 mutant when its expression is under the regulation of
the PHO1 promoter (Stefanovic et al., 2007). The pho1/
pho1;h1 double mutant shows a more severe Pi starvation
phenotype than pho1 because of less Pi transported to the
shoots, which indicates that PHO1;H1 is also involved in Pi
loading into the xylem. Both PHO1 and PHO1;H1 are up-
regulated in response to Pi deficiency; however, PHO1;H1
but not PHO1 is regulated by PHR1 (Stefanovic et al.,
2007). Interestingly, PHO1 homologues have also been
identified in a moss (Physcomitrella patens), which lacks
a developed vascular system (Wang et al., 2008). Similar to
the expression of PHO1 in Arabidopsis, in P. patens,
expression of PHO1 and several other homologues of
Arabidopsis Pi starvation-induced genes are up-regulated by
Pi deficiency. P. patens and higher plants may share some
common features to adapt to Pi deficiency (Wang et al.,
2008).
In contrast to pho1, the pho2 mutant has a high
concentration of Pi in the shoots (Delhaize and Randall,
1995). The excessive accumulation of shoot Pi often results
in Pi toxicity with the initiation of chlorosis or necrosis at
the tip of old leaves, a consequence of enhanced Pi uptake
and translocation from the roots to the shoots and retention
of Pi in the old leaves (Dong et al., 1998; Aung et al., 2006).
The PHO2 gene was identified as UBC24, a member of the
E2 ubiquitin conjugase family, by two independent studies,
one involving map-based cloning (Bari et al., 2006) and the
other investigating the similarity of the Pi toxicity pheno-
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Molecular regulators of Pi homeostasis | 1431
type between pho2 and microRNA399 (miR399)-overex-
pressing plants (Aung et al., 2006). PHO2 is a target gene of
miR399s. The consequence of suppression of PHO2 by
overexpression of miR399 is comparable to that of pho2
loss-of-function. MiR399s are barely expressed under Pi-
sufficient conditions but are highly up-regulated by low
concentrations of Pi (Fujii et al., 2005; Bari et al., 2006;
Chiou et al., 2006; Chiou, 2007). MiR399s control Pi
homeostasis by regulating the expression of PHO2. Up-
regulating miR399s suppresses the expression of PHO2,
which is an adaptive response to Pi deficiency because a low
level of PHO2 activity enhances Pi acquisition and trans-
location. Moreover, the expression of miR399s is regulated
by PHR1 (Bari et al., 2006). A regulatory pathway
connecting PHR1 to PHO2 through the abundance of
miR399s was established. MiR399 expression is largely
reduced in phr1 (Bari et al., 2006), but increased with
overexpression of PHR1 (Nilsson et al., 2007). The tran-
script level of two Pi transporter genes, PHT1;8 and
PHT1;9, is up-regulated in pho2 and miR399-overexpress-
ing plants, which implies that the genes could be down-
stream components of PHO2 (Aung et al., 2006; Bari et al.,
2006).
More recently, results from reciprocal grafts between
wild-type and miR399-overexpressing plants have demon-
strated the systemic regulation of PHO2 by long-distance
movement of miR399s from the shoot to the roots (Lin
et al., 2008; Pant et al., 2008). Upon Pi starvation, the early
induction of miR399s in the shoots serves as a long-distance
signal to down-regulate the expression of PHO2 in the
roots, which activates the Pi transport system and ulti-
mately controls the Pi homeostasis of whole plants (Lin
et al., 2008; Pant et al., 2008).
Mutants isolated by the measurement of phosphatase
or nuclease activity
One strategy by which plants utilize organic phosphate is to
liberate Pi by the hydrolysis of phosphate esters with
phosphatase or nuclease secreted from the roots. Goldstein
and colleagues first measured the acid phosphatase (APase)
activity by examining the blue staining of roots grown in Pi-
free medium containing 5-bromo-4-chloro-3-indolyl-phos-
phate (BCIP) (Goldstein et al., 1988). Several mutants with
less staining of root APase activity were identified. Root-
associated APase activity is decreased in pup1 (phosphatase-
underproducer 1) and pup3 mutants (Trull and Deikman,
1998; Tomscha et al., 2004). P concentration in pup1 and
pup3 is normal when Pi is the only source, whereas pup
mutants accumulate less total P than wild-type plants when
organic phosphate is the major P source. This finding
supports the notion that secreted phosphatase is important
for the utilization of organic phosphates in soil. Arabidopsis
contains five isoforms with APase activity secreted from
roots (Tomscha et al., 2004). pup1 and pup3 mutants are
defective in the activity of some isoforms. An isoform
corresponding to the low Pi-regulated purple acid phospha-
tase (AtPAP12) activity is reduced in the pup3 mutant, with
 1432 | Lin et al.
no change in the transcription level of AtPAP12. PUP3 may
be involved in the post-transcriptional modification of
APase. pup1 and pup3 are mapped to chromosomes 2 and 5,
respectively, and the genes responsible for these mutations
await identification.
A pho3 mutant shows aberrant APase induction in re-
sponse to Pi deficiency (Zakhleniuk et al., 2001). Under Pi-
sufficient conditions, pho3 has 30% less root APase activity
but about double the shoot APase activity of wild-type
plants. In contrast to pup1 and pup3, pho3 seedlings show
significantly reduced total P concentrations in the shoots and
roots, even under an adequate supply of Pi, which results in
a Pi starvation phenotype with increased levels of anthocya-
nin and starch (Zakhleniuk et al., 2001). The pho3 mutation
was later revealed to possess a malfunctioning SUC2 gene
encoding a sucrose proton symporter that is responsible for
sucrose loading into the phloem (Lloyd and Zakhleniuk,
2004). As a consequence of impaired sucrose loading, pho3
accumulates a large amount of sugar as well as starch in the
leaves. The association between P nutrient and sugar in
regulating gene expression has been well documented (Cier-
eszko et al., 2001; Franco-Zorrilla et al., 2005). Several Pi
starvation-induced genes are up-regulated by the exogenous
application of sucrose, which accelerates the development of
Pi starvation symptoms. Sucrose loading in the phloem may
provide a shoot-derived systemic signal to regulate the Pi
starvation responses of roots (Hammond and White, 2008).
From this assumption, the restricted translocation of sucrose
from shoots to roots as seen in pho3 may attenuate certain
responses of roots to Pi starvation, as seen in reduced Pi
starvation-induced APase activity.
In addition to the colorimetric or histochemical detection
of root enzyme activities, a conditional genetic screen was
used to isolate Arabidopsis mutants that fail to grow on
medium containing nucleic acid as the only P source, but can
then be recovered on Pi-containing medium (Chen et al.,
2000). This process was used to obtain several mutants
showing severe Pi starvation symptoms in nucleic acid-
containing medium. Because multiple enzymes are required
for the degradation of nucleic acid, the inability to utilize
nucleic acid implies impairment in many enzyme activities.
Therefore, these mutants are potentially defective in the
regulatory pathway to Pi limitation. One of mutants, pdr2
(phosphate deficiency response 2) was further characterized in
detail (Ticconi et al., 2004).
When grown on nucleic acid-containing medium, the roots
and shoots of pdr2 have 3050% less P concentration than
wild-type controls because of the defect in Pi acquisition
(Ticconi et al., 2004). The pdr2 mutant displays enhanced
sensitivity of Pi-starvation responses, such as the accumula-
tion of anthocyanin and starch or expression of Pi-responsive
genes. The enhanced sensitivity to low Pi may account for
the higher total P concentration in the Pi-starved pdr2
seedlings relative to wild-type plants. The most striking
phenotype of pdr2 is conditional short roots because of
reduced root meristem activity. This phenotype is specific for
Pi and is not induced by other nutrient deficiency. In-
terestingly, a recent finding suggests that the meristem
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activity determines the magnitude of Pi starvation responses
(Lai et al., 2007). PDR2 may function to modulate the root
meristem activity and root architecture in response to
external Pi availability in order to maximize Pi acquisition.
The observation of unaltered Pi uptake activity and total P
content in root tips of pdr2 and the rescue of root meristem
activity by phosphite, a non-metabolizable Pi analogue,
suggest that pdr2 is impaired in Pi sensing (Ticconi et al.,
2004). PDR2 is mapped to chromosome 5. Cloning and
molecular characterization of PDR2 will provide insights
into local Pi signalling and sensing mediated by PDR2.
Reverse genetic approaches
The early approaches of differential or subtractive hybrid-
ization to the recent genome-wide microarray analysis have
revealed the transcription level of several hundreds of genes
to be regulated by changes in external Pi concentration
(Hammond et al., 2003; Wasaki et al., 2003; Wu et al., 2003;
Misson et al., 2005; Morcuende et al., 2007; Calderon-
Vazquez et al., 2008). The functions of these genes are quite
diverse, underscoring the essential role of P in normal cell
functions. Among these genes, those with extreme up-
regulation by low Pi or with potential effects on Pi
acquisition, transcriptional regulation or signal transduc-
tion have been highlighted for further characterization.
Results from genetic manipulation of these genes via over-
expression, T-DNA or transposon knockout or RNAi
approaches have enhanced our understanding of their
regulatory roles in Pi homeostasis.
Genes involved in Pi acquisition and allocation
Arabidopsis contains four Pi transporter families, designated
PHT1, PHT2, PHT3, and PHT4 (Poirier and Bucher, 2002;
Guo et al., 2008). Members of the PHT1 family are high-
affinity Pi/H+ symporters located in the plasma membrane.
PHT1 transporters function to acquire Pi from the rhizo-
sphere because most of them are expressed in root
epidermal cells, whereas other PHT families are located in
the endomembrane systems. PHT2 is restricted to chlor-
oplasts (Versaw and Harrison, 2002), PHT3 is located in
mitochondria (Poirier and Bucher, 2002), and PHT4 is
located in non-photosynthetic plastids or the Golgi appara-
tus (Guo et al., 2008). To date, alterations in Pi concentra-
tion have been reported only in plants with mutations in
PHT1 or PHT2.
In Arabidopsis, the PHT1 gene family has nine members,
PHT1;1 to PHT1;9 (Mudge et al., 2002; Poirier and
Bucher, 2002), predominantly expressed in root tissues,
except for PHT1;6, which is expressed mainly in anthers
(Mudge et al., 2002). PHT1;1 and PHT1;4, previously
known as AtPT1 and AtPT2, respectively, have the highest
expression in Pi-starved roots. Overexpression of PHT1;1 in
tobacco culture cells increased Pi uptake and thus enhanced
cell growth when Pi was limited (Mitsukawa et al., 1997).
The Pi uptake rate of pht1;1 T-DNA knockout mutants is

reduced to ;80% of the wild-type level under low Pi
conditions and ;60% of the wild-type level under Pi-
sufficient conditions (Shin et al., 2004). The decrease in Pi
uptake results in lower Pi content in the shoots of pht1;1
mutants. This confirms the importance of PHT1;1 in Pi
acquisition under both Pi-sufficient and Pi-deficient con-
ditions. However, although pht1;4 mutants also display
a 2343% reduction in Pi uptake under low Pi conditions,
the Pi content in pht1;4 mutants is similar to that in wild-
type plants (Misson et al., 2004; Shin et al., 2004). The
authors concluded that PHT1;4-dependent Pi uptake may
not be a limiting factor for growth. Nevertheless, the pht1;1/
pht1;4 double mutant has a significant decrease in Pi uptake
and accumulation of shoot Pi under both Pi-sufficient and
Pi-deficient conditions, which indicates that the effect of
PHT1;1 and PHT1;4 knockout is additive (Shin et al.,
2004). Because of inadequate internal Pi concentrations, Pi
starvation symptoms are accelerated in the pht1;1/pht1;4
plants when Pi was limited. In parallel with the studies in
Arabidopsis, in tomato, Pi uptake and biomass are reduced
in a transposon-insertion line defective in a Pi transporter,
LePT4 (Xu et al., 2007).
PHT2;1 is the only member of the PHT2 family in
Arabidopsis (Daram et al., 1999). PHT2;1 is highly
expressed in leaves but barely detected in roots, and its
expression is not regulated by Pi status (Daram et al., 1999).
PHT2;1 encodes a low-affinity Pi transporter and is localized
in the chloroplast envelope (Versaw and Harrison, 2002).
Although pht2;1 and wild-type plants do not differ in total P
content of whole plants, the allocation of Pi between shoots
and roots is altered in pht2;1 (Versaw and Harrison, 2002).
Under Pi-limited conditions, the pht2;1 mutant exhibits
reduced root Pi concentration (5270% of the wild-type
level), but increased shoot Pi concentration (129156% of
wild-type level). Moreover, the redistribution of Pi between
old and young leaves is impaired in pht2;1 (Versaw and
Harrison, 2002). This suggests that PHT2;1 affects whole-
plant Pi allocation, especially under Pi-limited conditions.
By contrast, overexpression or suppression of the PHT2;1
orthologues in potato, SOLtu;Pht2;1, does not change the
leaf Pi content and Pi allocation (Rausch et al., 2004).
Whether PHT2;1 plays different roles in different plant
species requires further study.
TPSI1/MT4/At4 is a group of Pi starvation-induced
transcripts. They were initially isolated by differential or
subtractive hybridization because of their high induction by
Pi deficiency. They are classified as non-coding RNAs and
have no sequence homology among them except for a 22
24 nt conserved sequence (Burleigh and Harrison, 1999;
Martn et al., 2000). They are found in many plant species,
including TPSI1 from tomato (Liu et al., 1997), Mt4 from
Medicago truncatula (Burleigh and Harrison, 1997, 1998),
IPS1, At4, At4.1, and At4.2 from Arabidopsis (Burleigh
and Harrison, 1999; Martn et al., 2000; Shin et al., 2006),
and OSPI1 from rice (Wasaki et al., 2003). The Pi-starved
at4 mutant shows a 17% higher shoot Pi content than wild-
type plants but a normal root Pi content, for a 36% higher
ratio of shoot to root Pi (Shin et al., 2006). This effect is
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Molecular regulators of Pi homeostasis | 1433
more severe in the at4/pht1;1/pht1;4 triple mutant. Under
Pi-sufficient conditions, the triple mutant has 49% higher
shoot Pi content and 48% greater ratio of shoot to root Pi
than the pht1;1/pht1;4 double mutant (Shin et al., 2006).
The fresh weight of the triple mutant is also higher than
that of the double mutant. These observations suggest that
At4 regulates Pi distribution between the roots and the
shoots and ultimately affects plant growth. Of note, the
2224 nt conserved sequence in the TPSI1/MT4/At4 non-
coding RNA family has partial complementarity to
miR399s, with mismatches at the middle of miR399
sequences (Chiou, 2007; Franco-Zorrilla et al., 2007). Like
TPSI1/MT4/At4 non-coding RNAs, miR399s are also up-
regulated by Pi deficiency. Overexpression of miR399
decreases the PHO2 mRNA level and increases shoot Pi
content, but overexpression of IPS1 or At4 alone or
together with miR399 increases the level of PHO2 mRNA,
leading to reduced shoot Pi content (Franco-Zorrilla et al.,
2007). The reduced shoot Pi content in IPS1- or At4-
overexpressing plants is consistent with the moderate but
significant increase in shoot Pi content in the at4 mutant
(Shin et al., 2006). These findings suggest that At4/IPS1
non-coding RNAs may function as riboregulators to
inhibit the cleavage of miR399s to PHO2 mRNA through
sequestration of miR399s by mimicking the target sequen-
ces of PHO2 (Franco-Zorrilla et al., 2007). The relation-
ship between PHO2, miR399s, and At4/IPS1 was
discussed in a recent review (Doerner, 2008).
Genes involved in transcriptional regulation
In addition to PHR1, several other transcription factors
involved in the regulation of Pi starvation responses were
recently characterized. Using subtractive hybridization,
OsPTF1 (Pi starvation-induced transcription factor 1) encod-
ing a basic helix-loop-helix (bHLH) transcription factor
similar to the yeast PHO4 gene was isolated from rice (Yi
et al., 2005). OsPTF1 is up-regulated by Pi deficiency in
roots, but it is expressed constitutively in shoots. Over-
expression of OsPTF1 in rice enhances tolerance to Pi
deficiency (Yi et al., 2005). Compared with wild-type plants,
transgenic rice plants show an increased Pi content, 30%
higher biomass, and 40% higher tiller number under Pi-
deficient conditions. The improved performance of
OsPTF1-overexpressing rice is probably the result of in-
creased total root length and root surface area and greater
Pi uptake activity (Yi et al., 2005). Results from microarray
and northern blot analyses of 35S::OsPTF1 transgenic
plants revealed that several genes up-regulated by OsPTF1
were involved in Pi uptake and Pi homeostasis. Further
identification of these components downstream of OsPTF1
will broaden our understanding of the OsPTF1-mediated
regulatory network in rice.
In Arabidopsis, three transcription factors, WRKY75,
ZAT6, and BHLH32, identified from microarray analysis
and showing transcriptional induction by Pi deficiency were
characterized (Misson et al., 2005; Chen et al., 2007;
Devaiah et al., 2007a, b). RNAi suppression of WRKY75
 1434 | Lin et al.
results in the early accumulation of anthocyanin, reduced
expression of Pi responsive genes, and Pi uptake (Devaiah
et al., 2007a). However, total P concentration in older
RNAi plants is increased, which may be associated with the
increase in lateral root length and the number of lateral
roots and root hairs at the later growth stage. WRKY75
may function as a positive regulator of Pi starvation
responses while down-regulating lateral root and root hair
growth regardless of Pi status (Devaiah et al., 2007a).
ZAT6 (zinc finger of Arabidopsis 6) encodes a cysteine-2/
histidine-2 (C2H2) zinc finger transcription factor (Devaiah
et al., 2007b). ZOe (ZAT6 Overexpression) Arabidopsis
seedlings exhibit increased anthocyanin accumulation and
extracellular acid phosphatase activity, reduced Pi uptake
and total P content, short primary roots, and retarded
growth (Devaiah et al., 2007b). By contrast, the root
architecture of older ZOe plants is altered, with decreased
primary root length and lateral root number but increased
lateral root length, which leads to an increased root to
shoot ratio, total P content, and Pi uptake (Devaiah et al.,
2007b). Moreover, expression of several Pi starvation-
induced genes is reduced in ZOe plants. These results
suggest that ZAT6 controls root architecture, Pi uptake,
and accumulation. The different performance between
young seedlings and mature plants of WRKY75 RNAi or
ZOe plants in terms of Pi accumulation is interesting, but
the differential regulation of WRKY75 and ZAT6 at
different growth and developmental stages remains to be
elucidated.
The bhlh32 mutant shows increased root hair number
with sufficient Pi and displays higher anthocyanin and Pi
content under both Pi-sufficient or Pi-deficient conditions.
BHLH32 thus serves as a negative regulator of a variety of
biochemical and morphological responses induced by Pi
starvation in Arabidopsis (Chen et al., 2007). Genes encod-
ing PPCK (phosphoenolpyruvate carboxylase kinase),
which is involved in metabolic adjustment under
Pi deficiency, are negatively regulated by BHLH32, perhaps
as a result of BHLH32 interference with TTG1 (TRANS-
PARENT TESTA GLABRA1)-containing complexes
(Chen et al., 2007).
Genes involved in altering membrane lipid composition
A great proportion of P reserve is phospholipids in cell
membranes. To adjust internal Pi homeostasis during Pi
deprivation, phospholipids are hydrolysed to release Pi and
diacylglycerol (DAG), which is subsequently converted into
galactolipids or sulpholipids to compensate for reduced
phospholipids (Hartel et al., 2000; Yu et al., 2002). SQD1
and SQD2 are two enzymes required for sulpholipid
biosynthesis in Arabidopsis (Yu et al., 2002). The sqd2 T-
DNA insertion mutant showed reduced growth under Pi-
limited conditions, but there were no changes in morphol-
ogy or growth for the sqd2 mutant grown in Pi-replete
medium, which suggests that sulpholipids are an important
substitute for phospholipids when Pi is deprived (Yu et al.,
2002).
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Degradation of phospholipids is carried out by phospho-
lipases C and D. The expression of phospholipases PLDf1
and PLDf2 is induced by low Pi, and they are involved in
lipid turnover during limited Pi (Cruz-Ramirez et al., 2006;
Li et al., 2006a, b). The accumulation of digalactosyldiacyl-
glycerol (DGDG), a galactolipid, is reduced in the roots of
Pi-starved pldf2 single and pldf1/pldf2 double mutants. In
addition, PLDf1 and PLDf2 regulate changes in root
architecture in response to Pi limitation. Phosphatidic acid
(PA) generated by PLDf was suggested to be dephosphory-
lated by a phosphatase to release Pi and DAG or to serve as
a signal to activate a protein kinase cascade that regulates
root growth. In contrast to PLDf functioning in roots, non-
specific phospholipase C5 (NPC5) is responsible for phos-
pholipid degradation and DGDG accumulation in the
leaves during Pi starvation, as was found recently (Gaude
et al., 2008). Synthesis of galactolipids during Pi starvation
is independent of the PHR1 transcription factor (Gaude
et al., 2008). Despite no description of changes in Pi
concentration in these phospholipase mutants, the roles of
PLDf and NPC5 in maintaining cellular Pi homeostasis in
terms of changes in membrane lipid composition are
indisputable.
Genes involved in sensing and signalling
Calcium ion (Ca2+) and inositol polyphosphate (InsP) are
well-documented secondary messengers in response to many
aspects of cellular stimuli (Irvine and Schell, 2001; Sanders
et al., 2002; Hetherington and Brownlee, 2004; Krinke
et al., 2007). Oscillation of cytosolic Ca2+ concentration
triggers the cascade of signal transduction, achieved by
modulating Ca2+ flux across the plasma membranes or
endomembranes, including the ER, Golgi apparatus, and
tonoplast. When a signal is perceived, inositol 1,4,5-tri-
sphosphate [Ins(1,4,5)P3, InsP3] is generated by the activa-
tion of phospholipase C. Increased InsP3 can facilitate Ca2+
release from the ER and tonoplast through InsP3-gated
Ca2+ channels (Allen et al., 1995; Sanders et al., 2002;
Krinke et al., 2007). The cross-talk between these two
intracellular signals results in defined and delicate networks
to ensure precise transduction of signals. Besides InsP3,
many higher-order inositol polyphosphates, such as InsP4,
InsP5, and InsP6 regulate a range of biological processes
(Irvine and Schell, 2001).
The association between InsP signals and Pi homeostasis
was first revealed through the characterization of an ipk1
(inositol polyphosphate kinase 1) mutant. The ipk1-1 mutant
is defective in an InsP4 and InsP5 2-kinase that is involved
in the biosynthesis of phytate (inositol hexakisphosphate,
InsP6) (Stevenson-Paulik et al., 2005). Impaired IPK1 not
only reduces the level of InsP6 but also causes excessive
accumulation of Pi in the leaves and leads to Pi toxicity
under Pi-sufficient conditions. Moreover, the ipk1-1 mutant
shows longer root hairs under Pi starvation conditions and
is less sensitive to high external Pi concentrations with
decreased root hair length (Stevenson-Paulik et al., 2005).
These findings suggest the involvement of InsP signals in the

integration of Pi sensing and starvation responses. Down-
stream of InsP3 signalling, altered InsP4, InsP5 or InsP6 in
ipk1-1 mutants could be possible candidates for a messenger
for Pi signalling. Notably, recent studies in yeast cells
showed that intracellular concentrations of inositol heptaki-
sphosphate (InsP7) increased upon Pi starvation, and InsP7
may function as a signalling molecule to control the yeast
PHO signalling pathway (Lee et al., 2007). Whether InsP7 is
a general signal for Pi limitation in other organisms remains
to be determined.
As mentioned above, cytosolic Ca2+ concentrations can
be adjusted by sequestration of Ca2+ into endomembranes.
One way to sequester cytosolic Ca2+ into vacuoles
is mediated by a tonoplast Ca2+/H+ antiporter, CAX
(CALCIUM EXCHANGER) (Hirschi et al., 1996). Impair-
ment of such activity can result in elevated Ca2+ concen-
trations in the cytosol. CAX1 was identified from
Arabidopsis by complementation of a yeast mutant defective
in Ca2+ transport across vacuolar membranes (Hirschi
et al., 1996). CAX1 and CAX3, a close homologue of
CAX1, are localized in the tonoplast (Cheng et al., 2003,
2005). The cax1 and cax1/cax3 double mutants show
reduced tonoplast Ca2+/H+ antiporter activity (Cheng
et al., 2003, 2005). The cax1/cax3 double mutant displays
a severe reduction in growth and perturbation of hormone
sensitivity and ionome, which indicates that CAX1 and
CAX3 are required for growth, hormone responses and ion
homeostasis (Catala et al., 2003; Cheng et al., 2003, 2005).
Significantly, the cax1/cax3 double mutant but not the cax1
or cax3 single mutant shows 66% more shoot Pi than wild-
type plants (Cheng et al., 2005), which provides the first hint
of the involvement of Ca2+ in Pi homeostasis.
The SPX domain is found in many proteins of the major
eukaryotes, and plays a role in Pi sensing and G-protein-
mediated signal pathways (Lenburg and OShea, 1996;
Wykoff and OShea, 2001). The transcript levels of several
SPX domain-containing genes of Arabidopsis (AtSPX1-
SPX4) are regulated by Pi starvation, in part through the
regulation of PHR1 and SIZ1 (Duan et al., 2008). Over-
expression of AtSPX1 promotes the expression of several
Pi-starvation responsive genes. Inhibition of AtSPX3 by
RNA interference leads to increased shoot P concentra-
tions, probably from the enhancement of Pi starvation-
induced genes involved in Pi uptake, allocation, and
remobilization (Duan et al., 2008). A potential negative
feedback regulation of Pi signalling by AtSPX3 was pro-
posed. Nevertheless, how SPX family proteins participate in
the Pi signalling network in plants has not yet been
delineated.
Conclusions and perspectives
Maintenance of Pi homeostasis is achieved by the co-
ordination of multiple physiological processes, from the
initial Pi acquisition from the rhizospheres to subsequent Pi
loading in the roots, followed by Pi allocation and
remobilization within plants. Such coordination requires
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Molecular regulators of Pi homeostasis | 1435
proper communications among different tissues, organs,
and cells to synchronize plant growth and development with
changes in environmental Pi supply. Recent findings by
forward or reverse genetics approaches have seen great
advances in our understanding of the molecular mechanism
regulating Pi homeostasis. Forward genetic approaches are
usually time-consuming and labour-intensive, but yield
direct information of gene function. On the other hand,
while many genes can be the targets for reverse genetic
studies, they do not always disclose the genes function
because of gene redundancy or the existence of alternative
pathways. Recent development of the technique of leaf
ionomics (Baxter et al., 2008; Salt et al., 2008), high-
throughput profiling of elemental composition, will facili-
tate the identification of mutants directly linked with
changes in P concentration. In future, it will be interesting
to explore the possible networks among different molecular
regulators, such as among PHR1, WRKY75, ZAT6, and
BHLH32 transcriptional factors. Examining the interaction
between genes whose alteration results in a similar pheno-
type, such as pho2 and ipk1 mutants, will also be
worthwhile. In addition, further investigations of the cross-
talk between Ca2+ homeostasis and Pi homeostasis will be
an exciting topic to pursue. Identification of miR399 as
a systemic shoot-derived signal to control Pi homeostasis
has added another new dimension to our knowledge of the
communication of Pi status between the shoots and the
roots (Lin et al., 2008; Pant et al., 2008). The future
challenge will be to dissect the specific signals of local/
external and long-distance/internal sensing, that maintain
the homeostasis of cellular Pi for essential physiological and
biochemical processes.
Acknowledgements
The authors thank Tzu-Yin Liu and Yee-yung Charng for
critically reading this article. Research in Chious labora-
tory is supported by the grants from Academia Sinica (AS-
97-FP-L20-1) and the National Science Council of the
Republic of China (97-2321-B-001-018).
References
Allen GJ, Muir SR, Sanders D. 1995. Release of Ca2+ from
individual plant vacuoles by both InsP3 and cyclic ADP-ribose.
Science 268, 735737.
Aung K, Lin S-I, Wu C-C, Huang Y-T, Su C-l, Chiou T-J. 2006.
pho2, a phosphate overaccumulator, is caused by a nonsense
mutation in a microRNA399 target gene. Plant Physiology 141,
10001011.
Bari R, Datt Pant B, Stitt M, Scheible W-R. 2006. PHO2,
microRNA399, and PHR1 define a phosphate-signaling pathway in
plants. Plant Physiology 141, 988999.
Baxter IR, Vitek O, Lahner B, Muthukumar B, Borghi M,
Morrissey J, Guerinot ML, Salt DE. 2008. The leaf ionome as
 1436 | Lin et al.
a multivariable system to detect a plants physiological status.
Proceedings of the National Academy of Sciences, USA 105, 12081
12086.
Burleigh SH, Harrison MJ. 1997. A novel gene whose expression in
Medicago truncatula roots is suppressed in response to colonization
by vesiculararbuscular mycorrhizal (VAM) fungi and to phosphate
nutrition. Plant Molecular Biology 34, 199208.
Burleigh SM, Harrison MJ. 1998. Characterization of the Mt4 gene
from Medicago truncatula. Gene 216, 4753.
Burleigh SH, Harrison MJ. 1999. The down-regulation of Mt4-like
genes by phosphate fertilization occurs systemically and involves
phosphate translocation to the shoots. Plant Physiology 119,
241248.
Calderon-Vazquez C, Ibarra-Laclette E, Caballero-Perez J, Her-
rera-Estrella L. 2008. Transcript profiling of Zea mays roots reveals
gene responses to phosphate deficiency at the plant- and species-
specific levels. Journal of Experimental Botany 59, 24792497.
Catala R, Santos E, Alonso JM, Ecker JR, Martnez-Zapater JM,
Salinas J. 2003. Mutations in the Ca2+/H+ transporter CAX1 increase
CBF/DREB1 expression and the cold-acclimation response in Arabidop-
sis. The Plant Cell 15, 29402951.
Catala R, Ouyang J, Abreu IA, Hu Y, Seo H, Zhang X, Chua N-H.
2007. The Arabidopsis E3 SUMO ligase SIZ1 regulates plant growth
and drought responses. The Plant Cell 19, 29522966.
Chardin P, Callebaut I. 2002. The yeast Sar exchange factor Sec12,
and its higher organism orthologs, fold as b-propellers. FEBS Letters
525, 171173.
Chen DL, Delatorre CA, Bakker A, Abel S. 2000. Conditional
identification of phosphate-starvation-response mutants in Arabidopsis
thaliana. Planta 211, 1322.
Chen Z-H, Nimmo GA, Jenkins GI, Nimmo HG. 2007. BHLH32
modulates several biochemical and morphological processes that
respond to Pi starvation in Arabidopsis. Biochemical Journal 405,
191198.
Cheng N-H, Pittman JK, Barkla BJ, Shigaki T, Hirschi KD. 2003.
The Arabidopsis cax1 mutant exhibits impaired ion homeostasis,
development, and hormonal responses and reveals interplay among
vacuolar transporters. The Plant Cell 15, 347364.
Cheng N-H, Pittman JK, Shigaki T, Lachmansingh J, LeClere S,
Lahner B, Salt DE, Hirschi KD. 2005. Functional association of
Arabidopsis CAX1 and CAX3 is required for normal growth and ion
homeostasis. Plant Physiology 138, 20482060.
Chiou T-J. 2007. The role of microRNAs in sensing nutrient stress.
Plant, Cell and Environment 30, 323332.
Chiou T-J, Aung K, Lin S-I, Wu C-C, Chiang S-F, Su C-l. 2006.
Regulation of phosphate homeostasis by microRNA in Arabidopsis.
The Plant Cell 18, 412421.
Ciereszko I, Johansson H, Hurry V, Kleczkowski LA. 2001.
Phosphate status affects the gene expression, protein content and
enzymatic activity of UDP-glucose pyrophosphorylase in wild-type and
pho mutants of Arabidopsis. Planta 212, 598605.
Cruz-Ramirez A, Oropeza-Aburto A, Razo-Hernandez F,
Ramirez-Chavez E, Herrera-Estrella L. 2006. Phospholipase DZ2
plays an important role in extraplastidic galactolipid biosynthesis and
Downloaded from https://academic.oup.com/jxb/article-abstract/60/5/1427/480099/Molecular-regulators-of-phosphate-homeostasis-in
by Agricultural University of Athens user
on 06 September 2017
phosphate recycling in Arabidopsis roots. Proceedings of the National
Academy of Sciences, USA 103, 67656770.
Daram P, Brunner S, Rausch C, Steiner C, Amrhein N,
Bucher M. 1999. Pht2;1 encodes a low-affinity phosphate transporter
from Arabidopsis. The Plant Cell 11, 21532166.
Delhaize E, Randall PJ. 1995. Characterization of a phosphate-
accumulator mutant of Arabidopsis thaliana. Plant Physiology 107,
207213.
Devaiah BN, Karthikeyan AS, Raghothama KG. 2007a. WRKY75
transcription factor is a modulator of phosphate acquisition and root
development in Arabidopsis. Plant Physiology 143, 17891801.
Devaiah BN, Nagarajan VK, Raghothama KG. 2007b. Phosphate
homeostasis and root development in Arabidopsis are synchronized
by the zinc finger transcription factor ZAT6. Plant Physiology 145,
147159.
Doerner P. 2008. Phosphate starvation signaling: a threesome
controls systemic Pi homeostasis. Current Opinion in Plant Biology 11,
536540.
Dong B, Rengel Z, Delhaize E. 1998. Uptake and translocation of
phosphate by pho2 mutant and wild-type seedlings of Arabidopsis
thaliana. Planta 205, 251256.
Duan K, Yi K, Dang L, Huang H, Wu W, Wu P. 2008.
Characterization of a sub-family of Arabidopsis genes with the SPX
domain reveals their diverse functions in plant tolerance to phosphorus
starvation. The Plant Journal 54, 965975.
Franco-Zorrilla JM, Martn AC, Leyva A, Paz-Ares J. 2005.
Interaction between phosphate-starvation, sugar, and cytokinin signal-
ing in Arabidopsis and the roles of cytokinin receptors CRE1/AHK4 and
AHK3. Plant Physiology 138, 847857.
Franco-Zorrilla JM, Valli A, Todesco M, Mateos I, Puga MI,
Rubio-Somoza I, Leyva A, Weigel D, Garca JA, Paz-Ares J.
2007. Target mimicry provides a new mechanism for regulation of
microRNA activity. Nature Genetics 39, 10331037.
Fujii H, Chiou T-J, Lin S-I, Aung K, Zhu J-K. 2005. A miRNA
involved in phosphate-starvation response in Arabidopsis. Current
Biology 15, 20382043.
Gaude N, Nakamura Y, Scheible W, Ohta H, Dormann P. 2008.
Phospholipase C5 (NPC5) is involved in galactolipid accumulation
during phosphate limitation in leaves of Arabidopsis. The Plant Journal
56, 2839.
Goldstein AH, Baertlein DA, McDaniel RG. 1988. Phosphate
starvation inducible metabolism in Lycopersicon esculentum. I. excre-
tion of acid phosphatase by tomato plants and suspension-cultured
cells. Plant Physiology 87, 711715.
Gonzalez E, Solano R, Rubio V, Leyva A, Paz-Ares J. 2005.
PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 is a plant-
specific SEC12-related protein that enables the endoplasmic reticulum
exit of a high-affinity phosphate transporter in Arabidopsis. The Plant
Cell 17, 35003512.
Guo B, Jin Y, Wussler C, Blancaflor EB, Motes CM, Versaw WK.
2008. Functional analysis of the Arabidopsis PHT4 family of intracellu-
lar phosphate transporters. New Phytologist 177, 889898.
Hamburger D, Rezzonico E, MacDonald-Comber Petetot J,
Somerville C, Poirier Y. 2002. Identification and characterization of

the Arabidopsis PHO1 gene involved in phosphate loading to the
xylem. The Plant Cell 14, 889902.
Hammond JP, Bennett MJ, Bowen HC, Broadley MR,
Eastwood DC, May ST, Rahn C, Swarup R, Woolaway KE,
White PJ. 2003. Changes in gene expression in Arabidopsis shoots
during phosphate starvation and the potential for developing smart
plants. Plant Physiology 132, 578596.
Hammond JP, White PJ. 2008. Sucrose transport in the phloem:
integrating root responses to phosphorus starvation. Journal of
Experimental Botany 59, 93109.
Hartel H, Dormann P, Benning C. 2000. DGD1-independent
biosynthesis of extraplastidic galactolipids after phosphate deprivation
in Arabidopsis. Proceedings of the National Academy of Sciences,
USA 97, 1064910654.
Hetherington AM, Brownlee C. 2004. The generation of
Ca2+ signals in plants. Annual Review of Plant Biology 55,
401427.
Hirschi KD, Zhen R-G, Cunningham KW, Rea PA, Fink GR. 1996.
CAX1, an H+/Ca2+ antiporter from Arabidopsis. Proceedings of the
National Academy of Sciences, USA 93, 87828786.
Irvine RF, Schell MJ. 2001. Back in the water: the return of the
inositol phosphates. Nature Reviews Molecular Cell Biology 2, 327
338.
Krinke O, Novotna Z, Valentova O, Martinec J. 2007. Inositol
trisphosphate receptor in higher plants: is it real? Journal of
Experimental Botany 58, 361376.
Lai F, Thacker J, Li Y, Doerner P. 2007. Cell division activity
determines the magnitude of phosphate starvation responses in
Arabidopsis. The Plant Journal 50, 545556.
Lee Y-S, Mulugu S, York JD, OShea EK. 2007. Regulation of
a cyclin-CDK-CDK inhibitor complex by inositol pyrophosphates.
Science 316, 109112.
Lenburg M, OShea E. 1996. Signaling phosphate starvation. Trends
in Biochemical Sciences 21, 383387.
Li M, Qin C, Welti R, Wang X. 2006a. Double knockouts of
phospholipases Df1 and Df2 in Arabidopsis affect root elongation
during phosphate-limited growth but do not affect root hair patterning.
Plant Physiology 140, 761770.
Li M, Welti R, Wang X. 2006b. Quantitative profiling of Arabidopsis
polar glycerolipids in response to phosphorus starvation. roles of
phospholipases Df1 and Df2 in phosphatidylcholine hydrolysis and
digalactosyldiacylglycerol accumulation in phosphorus-starved plants.
Plant Physiology 142, 750761.
Lin S-I, Chiang S-F, Lin W-Y, Chen J-W, Tseng C-Y, Wu P-C,
Chiou T-J. 2008. Regulatory network of microRNA399 and PHO2 by
systemic signaling. Plant Physiology 147, 732746.
Liu C, Muchhal US, Raghothama KG. 1997. Differential expression
of TPS11, a phosphate starvation-induced gene in tomato. Plant
Molecular Biology 33, 867874.
Lloyd JC, Zakhleniuk OV. 2004. Responses of primary and
secondary metabolism to sugar accumulation revealed by microarray
expression analysis of the Arabidopsis mutant, pho3. Journal of
Experimental Botany 55, 12211230.
Downloaded from https://academic.oup.com/jxb/article-abstract/60/5/1427/480099/Molecular-regulators-of-phosphate-homeostasis-in
by Agricultural University of Athens user
on 06 September 2017
Molecular regulators of Pi homeostasis | 1437
Marschner H. 1995. Mineral nutrition of higher plants. London:
Academic Press.
Martn AC, del Pozo JC, Iglesias J, Rubio V, Solano R, de la
Pena A, Leyva A, Paz-Ares J. 2000. Influence of cytokinins on the
expression of phosphate starvation responsive genes in Arabidopsis.
The Plant Journal 24, 559567.
Misson J, Thibaud M-C, Bechtold N, Raghothama K,
Nussaume L. 2004. Transcriptional regulation and functional proper-
ties of Arabidopsis Pht1;4, a high affinity transporter contributing
greatly to phosphate uptake in phosphate deprived plants. Plant
Molecular Biology 55, 727741.
Misson J, Raghothama KG, Jain A, et al. 2005. A genome-wide
transcriptional analysis using Arabidopsis thaliana Affymetrix gene
chips determined plant responses to phosphate deprivation. Proceed-
ings of the National Academy of Sciences, USA 102, 1193411939.
Mitsukawa N, Okumura S, Shirano Y, Sato S, Kato T,
Harashima S, Shibata D. 1997. Overexpression of an Arabidopsis
thaliana high-affinity phosphate transporter gene in tobacco cultured
cells enhances cell growth under phosphate-limited conditions.
Proceedings of the National Academy of Sciences, USA 94, 7098
7102.
Miura K, Jin JB, Lee J, Yoo CY, Stirm V, Miura T, Ashworth EN,
Bressan RA, Yun D-J, Hasegawa PM. 2007. SIZ1-mediated
sumoylation of ICE1 controls CBF3/DREB1A expression and freezing
tolerance in Arabidopsis. The Plant Cell 19, 14031414.
Miura K, Rus A, Sharkhuu A, et al. 2005. The Arabidopsis SUMO
E3 ligase SIZ1 controls phosphate deficiency responses. Proceedings
of the National Academy of Sciences, USA 102, 77607765.
Morcuende R, Bari R, Gibon Y, et al. 2007. Genome-wide
reprogramming of metabolism and regulatory networks of Arabidopsis
in response to phosphorus. Plant, Cell and Environment 30, 85112.
Mudge SR, Rae AL, Diatloff E, Smith FW. 2002. Expression
analysis suggests novel roles for members of the Pht1 family of
phosphate transporters in Arabidopsis. The Plant Journal 31,
341353.
Nilsson L, Muller R, Nielsen TH. 2007. Increased expression of the
MYB-related transcription factor, PHR1, leads to enhanced phosphate
uptake in Arabidopsis thaliana. Plant, Cell and Environment 30,
14991512.
Pant BD, Buhtz A, Kehr J, Scheible W-R. 2008. MicroRNA399 is
a long-distance signal for the regulation of plant phosphate homeo-
stasis. The Plant Journal 53, 731738.
Poirier Y, Bucher M. 2002. Phosphate transport and homeostasis in
Arabidopsis. In: Somerville CR, Meyerowitz EM, eds. The Arabidopsis
book. Rockville, MD: The American Society of Plant Biologists, 135.
Poirier Y, Thoma S, Somerville C, Schiefelbein J. 1991. Mutant of
Arabidopsis deficient in xylem loading of phosphate. Plant Physiology
97, 10871093.
Raghothama KG. 1999. Phosphate acquisition. Annual Review of
Plant Physiology and Plant Molecular Biology 50, 665693.
Rausch C, Zimmermann P, Amrhein N, Bucher M. 2004.
Expression analysis suggests novel roles for the plastidic phosphate
transporter Pht2;1 in auto- and heterotrophic tissues in potato and
Arabidopsis. The Plant Journal 39, 1328.
 1438 | Lin et al.
Rubio V, Linhares F, Solano R, Martn AC, Iglesias J, Leyva A,
Paz-Ares J. 2001. A conserved MYB transcription factor involved in
phosphate starvation signaling both in vascular plants and in
unicellular algae. Genes and Development 15, 21222133.
Salt DE, Baxter I, Lahner B. 2008. Ionomics and the study of the
plant ionome. Annual Review of Plant Biology 59, 709733.
Sanders D, Pelloux J, Brownlee C, Harper JF. 2002. Calcium at
the crossroads of signaling. The Plant Cell 14, S401S417.
Shin H, Shin H-S, Dewbre GR, Harrison MJ. 2004. Phosphate
transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in
phosphate acquisition from both low- and high-phosphate environ-
ments. The Plant Journal 39, 629642.
Shin H, Shin H-S, Chen R, Harrison MJ. 2006. Loss of At4 function
impacts phosphate distribution between the roots and the shoots during
phosphate starvation. The Plant Journal 45, 712726.
Shin R, Berg RH, Schachtman DP. 2005. Reactive oxygen species
and root hairs in Arabidopsis root response to nitrogen, phosphorus
and potassium deficiency. Plant and Cell Physiology 46, 13501357.
Shin R, Schachtman DP. 2004. Hydrogen peroxide mediates plant
root cell response to nutrient deprivation. Proceedings of the National
Academy of Sciences, USA 101, 88278832.
Stefanovic A, Ribot C, Rouached H, Wang Y, Chong J,
Belbahri L, Delessert S, Poirier Y. 2007. Members of the PHO1
gene family show limited functional redundancy in phosphate transfer
to the shoot, and are regulated by phosphate deficiency via distinct
pathways. The Plant Journal 50, 982994.
Stevenson-Paulik J, Bastidas RJ, Chiou S-T, Frye RA, York JD.
2005. Generation of phytate-free seeds in Arabidopsis through
disruption of inositol polyphosphate kinases. Proceedings of the
National Academy of Sciences, USA 102, 1261212617.
Ticconi CA, Delatorre CA, Lahner B, Salt DE, Abel S. 2004.
Arabidopsis pdr2 reveals a phosphate-sensitive checkpoint in root
development. The Plant Journal 37, 801814.
Tomscha JL, Trull MC, Deikman J, Lynch JP, Guiltinan MJ.
2004. Phosphatase under-producer mutants have altered phosphorus
relations. Plant Physiology 135, 334345.
Trull MC, Deikman J. 1998. An Arabidopsis mutant missing one acid
phosphatase isoform. Planta 206, 544550.
Versaw WK, Harrison MJ. 2002. A chloroplast phosphate trans-
porter, PHT2;1, influences allocation of phosphate within the plant and
phosphate-starvation responses. The Plant Cell 14, 17511766.
Downloaded from https://academic.oup.com/jxb/article-abstract/60/5/1427/480099/Molecular-regulators-of-phosphate-homeostasis-in
by Agricultural University of Athens user
on 06 September 2017
Wang Y, Ribot C, Rezzonico E, Poirier Y. 2004. Structure and
expression profile of the Arabidopsis PHO1 gene family indicates
a broad role in inorganic phosphate homeostasis. Plant Physiology
135, 400411.
Wang Y, Secco D, Poirier Y. 2008. Characterization of the PHO1
gene family and the responses to phosphate deficiency of Physcomi-
trella patens. Plant Physiology 146, 646656.
Wasaki J, Yonetani R, Shinano T, Kai M, Osaki M. 2003.
Expression of the OsPI1 gene, cloned from rice roots using cDNA
microarray, rapidly responds to phosphorus status. New Phytologist
158, 239248.
Wu P, Ma L, Hou X, Wang M, Wu Y, Liu F, Deng XW. 2003.
Phosphate starvation triggers distinct alterations of genome expres-
sion in Arabidopsis roots and leaves. Plant Physiology 132, 1260
1271.
Wykoff DD, Grossman AR, Weeks DP, Usuda H,
Shimogawara K. 1999. Psr1, a nuclear localized protein that
regulates phosphorus metabolism in Chlamydomonas. Proceedings
of the National Academy of Sciences, USA 96, 1533615341.
Wykoff DD, OShea EK. 2001. Phosphate transport and sensing in
Saccharomyces cerevisiae. Genetics 159, 14911499.
Xu G-h, Chague V, Melamed-Bessudo C, Kapulnik Y, Jain A,
Raghothama KG, Levy AA, Silber A. 2007. Functional characteriza-
tion of LePT4: a phosphate transporter in tomato with mycorrhiza-
enhanced expression. Journal of Experimental Botany 58, 24912501.
Yi K, Wu Z, Zhou J, Du L, Guo L, Wu Y, Wu P. 2005. OsPTF1,
a novel transcription factor involved in tolerance to phosphate
starvation in rice. Plant Physiology 138, 20872096.
Yu B, Xu C, Benning C. 2002. Arabidopsis disrupted in SQD2
encoding sulfolipid synthase is impaired in phosphate-limited growth.
Proceedings of the National Academy of Sciences, USA 99, 5732
5737.
Zakhleniuk OV, Raines CA, Lloyd JC. 2001. pho3: a phosphorus-
deficient mutant of Arabidopsis thaliana (L.) Heynh. Planta 212, 529
534.
Zhou J, Jiao F, Wu Z, Li Y, Wang X, He X, Zhong W, Wu P. 2008.
OsPHR2 is involved in phosphate-starvation signaling and excessive
phosphate accumulation in shoots of plants. Plant Physiology 146,
16731686.