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 Lin
1,2,3,
*, Shu-I Lin
1,4,
* and Tzyy-Jen Chiou
1,2,3,4,
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, signicant 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 identication
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-afnity 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 identied 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
The Author [2009]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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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
At4g28610
(unchanged)
d a MYB transcription factor
d activates a subset of Pi starvation-induced genes by the binding to P1BS element
d phr1 shows reduced Pi concentration
Rubio et al., 2001
Nilsson et al., 2007
Zhou et al., 2008
SIZ1
At5g60410
(up)
d SUMO E3 ligase
d facilitates the sumoylation of PHR1
d regulates the expression of several Pi starvation-responsive genes
d siz1 shows higher shoot Pi concentration
Miura et al., 2005
PHF1
At3g52190
(up)
d a ER-located SEC12-related protein
d facilitates the trafcking of PHT1 proteins to plasma membrane
d regulated by PHR1
d phf1 shows reduced Pi content
Gonzalez et al., 2005
PHO1
At3g23430
(up)
d a protein with SPX and EXS domains
d involved in Pi loading into the xylem
d pho1 displays Pi starvation symptoms with reduced shoot Pi
Poirier et al., 1991
Hamburger et al., 2002
Stefanovic et al., 2007
PHO2 (UBC24)
At2g33770
(down)
d a ubiquitin E2 conjugase
d regulates Pi uptake, allocation and remobilization
d a target gene of miR399s
d pho2 displays Pi toxicity with excessive shoot Pi
Delhaize and Randall, 1995
Dong et al., 1998
Aung et al., 2006
Bari et al., 2006
MiR399a,b,c,d,e,f
At1g29265, At1g63005,
At5g62162, At2g34202,
At2g34204, At2g34208
(up)
d a microRNA
d negatively regulates PHO2
d serves as a shoot-derived long-distance signal
d regulated by PHR1
d overexpression of miR399 mimics the Pi toxic phenotype of pho2
Fujii et al., 2005
Chiou et al., 2006
Bari et al., 2006
Lin et al., 2008
Pant et al., 2008;
PHO3 (SUC2)
At1g22710
d a sucrose/H
+
symporter
d regulates Pi starvation responses
d pho3 shows reduced total P concentration
Zakhleniuk et al., 2001
Lloyd and Zakhleniuk, 2004
pup1
pup3
d pup mutants show aberrant APase activity and less accumulation of
Pi in organic phosphate-containing medium
Trull and Deikman, 1998
Tomscha et al., 2004
pdr2 d pdr2 cannot utilize organic phosphate, leading to less accumulation of P
d pdr2 mutant is oversensitive to Pi starvation and has higher
Pi content under P decient conditions
d likely to be involved in Pi signalling or sensing
Ticconi et al., 2004
PHT1;1(AtPT1)
At5g43350
(up)
d high afnity Pi transporters involved in Pi acquisition
d pht1;1 mutant shows reduced Pi uptake and shoot Pi content
d increased Pi uptake by overexpression of PHT1;1 in tobacco cells
d pht1;1/pht1;4 double mutant shows signicant decrease
in Pi uptake, shoot Pi content and fresh weight
Mitsukawa et al., 1997
Misson et al., 2004
Shin et al., 2004
PHT1;4, (AtPT2)
At2g38940
(up)
PHT2;1
At3g26570
(unchanged)
d a low-afnity Pi transporter in chloroplast
d involved in Pi allocation between roots and shoots and
Pi remobilization between old and young leaves.
Daram et al., 1999
Versaw and Harrison, 2002
1428 | Lin et al.

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Table 1. Continued
Gene/mutant Description of function Reference
Locus no.
(transcriptional
regulation by low Pi)
At4
At5g03545
(up)
d non-coding RNAs
d regulates Pi allocation between roots and shoots
d inhibits the cleavage of miR399 to PHO2 mRNA by mimicking
the target sequence of miR399
Shin et al., 2006
Franco-Zorrilla et al., 2007
IPS1
At3g09922
(up)
OsPTF1
Os06g09370
(up)
d a bHLH transcription factor
d regulates the expression of several Pi starvation responsive genes
d overexpression of OsPTF1 increases Pi content, root growth and tiller number
Yi et al., 2005
WRKY 75
At5g13080
(up)
d a WRKY transcription factor
d positively regulates Pi starvation responses but negatively
regulates lateral root and root hair growth
Devaiah et al., 2007a
ZAT6
At5g04340
(up)
d a C
2
H
2
zinc nger transcription factor
d regulates several Pi starvation-responsive genes
d controls root architecture, Pi uptake and Pi accumulation
Devaiah et al., 2007b
BHLH32
At3g25710
(up)
d a bHLH transcription factor
d a negative regulator of Pi starvation responses.
d bhlh32 has hypersensitive Pi starvation responses and
increased Pi concentration
Chen et al., 2007
SQD2
At5g01220
(up)
d involved in sulfolipid biosynthesis
d sqd2 mutant shows reduced growth under Pi decient conditions
Yu et al., 2002
PLDz1
At3g16785
(up) d involved in phospholipid degradation and synthesis of galactolipid in roots
d regulates root architecture in response to Pi starvation
Cruz-Ramirez et al., 2006
Li et al., 2006a
Li et al., 2006b
PLDz2
At3g05630 (up)
NPC5
At3g03540
(up)
d involved in phospholipid degradation and synthesis of galactolipid in leaves Gaude et al., 2008
CAX1
At1g08960
d tonoplast Ca
2+
/H
+
antiporters
d cax1/cax3 double mutant has increased shoot Pi content
Cheng et al., 2003
Cheng et al., 2005
CAX3
At3g51860
IPK1
At5g42810
d an inositol polyphosphate kinase
d reduced phytate but increased Pi concentration in ipk1 mutant seeds
d ipk1 mutant has high leaf Pi concentration
Stevenson-Paulik et al., 2005
SPX1
At5g20150
(up) d SPX domain-containing proteins
d regulates the expression of several Pi starvation-responsive
genes involved in Pi uptake, allocation and remobilization
Duan et al., 2008
SPX3
At2g45130
(up)
Molecular regulators of Pi homeostasis | 1429

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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 specically responsive to Pi starvation.
Characterization of these mutants and subsequent
identication 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-sufcient conditions but
highly induced by low Pi (Mart n 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 rst identied 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
specic binding sequence) cis-element (GNATATNC)
(Rubio et al., 2001). In agreement with this nding,
genome-wide expression proling 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-sufcient and Pi-decient 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
identied to be involved in regulating several Pi starvation-
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 modication 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-sufcient 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
identied a phf1 (phosphate transporter trafc facilitator1)
mutant with constitutive expression of GUS regardless of
whether the plant grew under Pi-sufcient or -decient
conditions, because of low internal Pi concentrations
(Gonzalez et al., 2005). In Pi-sufcient 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-specic protein that functions as an endoplasmic
reticulum (ER)-located facilitator to assist the trafcking 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 deciency, which is mediated, in part, by the
transcriptional activation of PHR1.
1430 | Lin et al.

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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-decient 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 deciency; however, PHO1;H1
but not PHO1 is regulated by PHR1 (Stefanovic et al.,
2007). Interestingly, PHO1 homologues have also been
identied 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 deciency. P. patens and higher plants may share some
common features to adapt to Pi deciency (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 identied 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-
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-
sufcient 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 deciency 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 rst 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 identied. 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 nding
supports the notion that secreted phosphatase is important
for the utilization of organic phosphates in soil. Arabidopsis
contains ve 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
Molecular regulators of Pi homeostasis | 1431

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no change in the transcription level of AtPAP12. PUP3 may
be involved in the post-transcriptional modication of
APase. pup1 and pup3 are mapped to chromosomes 2 and 5,
respectively, and the genes responsible for these mutations
await identication.
A pho3 mutant shows aberrant APase induction in re-
sponse to Pi deciency (Zakhleniuk et al., 2001). Under Pi-
sufcient 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
signicantly 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 deciency 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 specic for
Pi and is not induced by other nutrient deciency. In-
terestingly, a recent nding suggests that the meristem
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-
afnity 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
1432 | Lin et al.

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reduced to ;80% of the wild-type level under low Pi
conditions and ;60% of the wild-type level under Pi-
sufcient conditions (Shin et al., 2004). The decrease in Pi
uptake results in lower Pi content in the shoots of pht1;1
mutants. This conrms the importance of PHT1;1 in Pi
acquisition under both Pi-sufcient and Pi-decient 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 signicant decrease in Pi uptake
and accumulation of shoot Pi under both Pi-sufcient and
Pi-decient 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-afnity 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 deciency. They are classied as non-coding RNAs and
have no sequence homology among them except for a 22
24 nt conserved sequence (Burleigh and Harrison, 1999;
Mart n 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; Mart n 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
more severe in the at4/pht1;1/pht1;4 triple mutant. Under
Pi-sufcient 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 deciency. 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
signicant increase in shoot Pi content in the at4 mutant
(Shin et al., 2006). These ndings 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 deciency in
roots, but it is expressed constitutively in shoots. Over-
expression of OsPTF1 in rice enhances tolerance to Pi
deciency (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-
decient 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
identication 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, identied from microarray analysis
and showing transcriptional induction by Pi deciency were
characterized (Misson et al., 2005; Chen et al., 2007;
Devaiah et al., 2007a, b). RNAi suppression of WRKY75
Molecular regulators of Pi homeostasis | 1433

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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 nger of Arabidopsis 6) encodes a cysteine-2/
histidine-2 (C2H2) zinc nger 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 sufcient Pi and displays higher anthocyanin and Pi
content under both Pi-sufcient or Pi-decient 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 deciency, 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).
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-
specic 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 (Ca
2+
) 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 Ca
2+
concentration
triggers the cascade of signal transduction, achieved by
modulating Ca
2+
ux 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)P
3
, InsP
3
] is generated by the activa-
tion of phospholipase C. Increased InsP
3
can facilitate Ca
2+
release from the ER and tonoplast through InsP
3
-gated
Ca
2+
channels (Allen et al., 1995; Sanders et al., 2002;
Krinke et al., 2007). The cross-talk between these two
intracellular signals results in dened and delicate networks
to ensure precise transduction of signals. Besides InsP
3
,
many higher-order inositol polyphosphates, such as InsP
4
,
InsP
5
, and InsP
6
regulate a range of biological processes
(Irvine and Schell, 2001).
The association between InsP signals and Pi homeostasis
was rst revealed through the characterization of an ipk1
(inositol polyphosphate kinase 1) mutant. The ipk1-1 mutant
is defective in an InsP
4
and InsP
5
2-kinase that is involved
in the biosynthesis of phytate (inositol hexakisphosphate,
InsP
6
) (Stevenson-Paulik et al., 2005). Impaired IPK1 not
only reduces the level of InsP
6
but also causes excessive
accumulation of Pi in the leaves and leads to Pi toxicity
under Pi-sufcient 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 ndings suggest the involvement of InsP signals in the
1434 | Lin et al.

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integration of Pi sensing and starvation responses. Down-
stream of InsP
3
signalling, altered InsP
4
, InsP
5
or InsP
6
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 (InsP
7
) increased upon Pi starvation, and InsP
7
may function as a signalling molecule to control the yeast
PHO signalling pathway (Lee et al., 2007). Whether InsP
7
is
a general signal for Pi limitation in other organisms remains
to be determined.
As mentioned above, cytosolic Ca
2+
concentrations can
be adjusted by sequestration of Ca
2+
into endomembranes.
One way to sequester cytosolic Ca
2+
into vacuoles
is mediated by a tonoplast Ca
2+
/H
+
antiporter, CAX
(CALCIUM EXCHANGER) (Hirschi et al., 1996). Impair-
ment of such activity can result in elevated Ca
2+
concen-
trations in the cytosol. CAX1 was identied from
Arabidopsis by complementation of a yeast mutant defective
in Ca
2+
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 Ca
2+
/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).
Signicantly, 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 rst hint
of the involvement of Ca
2+
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
proper communications among different tissues, organs,
and cells to synchronize plant growth and development with
changes in environmental Pi supply. Recent ndings 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 proling of elemental composition, will facili-
tate the identication 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 Ca
2+
homeostasis and Pi homeostasis will be
an exciting topic to pursue. Identication 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 specic 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).
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