Introduction
The molecular mechanisms involved in regulating Pi-signaling in plants remain to be elucidated, although several transcription factors have identified that each influence only a part of the profile of changes induced by Pi starvation in
Arabidopsis. These transcription factors include PHR1 (PHOSPHATE STARVATION RESPONSE 1) (
Rubio et al., 2001), WRKY75 (
Devaiah et al., 2007a), ZAT6 (
Devaiah et al., 2007b), MYB62 (
Devaiah et al., 2009), BHLH32 (
Chen et al., 2007), and WRKY96 (
Chen et al., 2009). In rice (
Oryza sativa L.), a bHLH transcription factor, OsPTF1 (Pi starvation-induced transcription factor 1) has been reported to be involved in Pi-signaling and Pi uptake (
Yi et al., 2005). AtPHR1 plays a key role in the phosphorus (P) signaling system in
Arabidopsis (
Arabidopsis thaliana). AtPHR1 is a transcription factor with a MYB domain and a predicted coiled-coil (CC) domain, and is defined as a member of the MYB-CC family.
AtPHR1 and
CrPSR1 (PHOSPHORUS STARVATION RESPONSE 1) belong to a large gene family (including another 14 proteins in
Arabidopsis) with a conserved MYB DNA binding domain (BD) and a predicted CC domain in each family member (
Rubio et al., 2001). Based on the protein sequence similarity of AtPHR1, two homologous single copy genes were isolated in rice (
Oryza sativa L.) and designated
OsPHR1 and
OsPHR2 (
Zhou et al., 2008). Although both are involved in Pi-signal transduction, only the ectopic overexpression of
OsPHR2 results in excessive shoot Pi accumulation and plant growth inhibition, especially under Pi abundant conditions (
Zhou et al., 2008). This raises the intriguing question of whether the increase in Pi uptake and translocation in plants would upset the nutrient homeostasis in plant cells required for normal metabolism in plant growth. We prefer the hypothesis that the excessive Pi in shoots results in Pi-toxicity. In support of this, ectopic overexpression of a low affinity Pi transporter, OsPT2, creates excessive shoot Pi accumulation and inhibition of plant growth (
Ai et al., 2009;
Liu et al. 2010). However, at shoot Pi concentration levels similar to wild type plants, the OsPHR2-overexpressed plants still showed inhibition of plant growth. This raises the possibility that OsPHR2 regulates some unknown factors crucial for plant growth or phosphate physiologic utilization (
Wu and Xu, 2010). In this review, we focus on the molecular mechanisms involved in regulating Pi-signaling and Pi homeostasis under OsPHR2, as well as on the recent progress in discovering new signaling players involved in the Pi-starvation responses in rice.
OsPHR2 regulates low affinity Pi-transporter through both physiologic and reciprocal regulation of OsPHO2
Downstream of AtPHR1,
miRNA399, as a target of PHR1, is specifically induced by Pi starvation.
miRNA399 reciprocally regulates the gene
PHO2 at the post-transcriptional level (
Fujii et al., 2005;
Bari et al., 2006;
Chiou et al., 2006). PHO2 functions as a ubiquitin-conjugating E2 enzyme (UBC24;
Aung et al., 2006;
Bari et al., 2006), and the loss of function of PHO2/UBC24 leads to excessive accumulation of Pi in the shoot tissue (
Fujii et al., 2005;
Chiou et al., 2006). In addition, repression of OsPT2, the low affinity of Pi transporter (
Ai et al., 2009), in a background of
OsPHR2 overexpression, remarkably reduces the excessive shoot Pi accumulation, but this is not observed in a
pho2 mutant (
Liu et al. 2010). These results provide evidence that OsPT2 makes a major contribution to excessive Pi accumulation in shoots driven by OsPHR2 and makes us speculate that OsPHR2 may physically regulate OsPT2, in addition to the reciprocal regulation of PHO2 at the transcriptional level (
Liu et al., 2010).
The cis-element of AtPHR1 (designated as P1BS) is conserved in promoters of many Pi-signaling responsive genes (
Rubio et al., 2001). In the promoter of
OsPT2, one P1SB element is found between -346 and -338 bp upstream of the ATG of
OsPT2. The expression and response of
OsPT2 to Pi-starvation signaling was greatly reduced in a T-DNA insertion mutant at -569 bp of
OsPT2 promoter. This result suggests that the T-DNA insertion interferes with binding of OsPHR2 to the cis-element or that the cis-element alone is not sufficient for
OsPT2 to respond to Pi-starvation signaling (
Liu et al., 2010). In fact, the P1BS element exists in many promoters of genes both responsive and non-responsive to Pi-starvation stress. It will be interesting to clarify what elements coordinate Pi-signal transduction in plants.
OsSPX1 is a suppressor on function of OsPHR2
The hydrophilic and poorly conserved SPX domain (SYG1/Pho81/XPR1) is found at the N-termini of various proteins, particularly signal transduction proteins (
Barabote et al., 2006). Most identified plant SPX gene products are involved in responses to environmental cues or internal regulation of nutrition homeostasis. Barley IDS4 (iron-deficiency specific clone 4) contains part of the SPX domain and is preferentially expressed in Fe-deficient roots (
Nakanishi et al., 1993).
Arabidopsis PHO1, harboring both SPX and EXS domains, plays a role in loading root Pi into the xylem vessels, and loss of PHO1 function in
pho1 mutants results in Pi deficiency in above-ground tissues (
Poirier et al., 1991;
Hamburger et al., 2002). A PHO1 homolog in
Arabidopsis may have a similar role in Pi loading and signaling (
Wang et al., 2004). The product of the tomato IDS4-like gene interacts with the leucine zipper domain of a hypoxia-induced transcription factor involved in the low-oxygen response (
Sell and Hehl, 2005). Homologous to yeast SYG1, the
Arabidopsis SHORT HYPOCOTYL UNDER BLUE 1 (SHB1) protein acts in cryptochrome signaling and seed development (
Kang and Ni, 2006;
Zhou and Ni, 2009;
Zhou et al., 2009;
Zhou and Ni, 2010).
Four genes with unique SPX domains in
Arabidopsis were identified to be involved in Pi-signaling pathways controlled by PHR1 and SIZ1 (
Duan et al., 2008). The rice (
Oryza sativa L.) genome contains at least six genes exclusively with the SPX (SYG1/PHO81/XPR1) domain at the N-terminal, designated as OsSPX1-6. The diverse expression patterns of the
OsSPX genes in different tissues and their responses to Pi-starvation have been reported (Wang et al., 2009b). Among them, five genes,
OsSPX1,
2,
3,
5 and
6 are responsive to Pi-starvation in shoots and/or in roots. Subcellular localization analysis indicates that OsSPX1 and OsSPX2 is exclusively located in nucleus, OsSPX3 is in cytoplasm and/or in nucleus, and OsSPX4 is a membrane-localized protein. OsSPX1 regulates
OsSPX2, 3 and
5 at the transcriptional level and is positively involved in the responses of the genes to Pi-starvation. Overexpression of
OsSPX3 regulates
OsSPX5 in shoots under Pi sufficiency, and OsSPX3 negatively regulates PSI (Pi-starvation induced) genes (
Wang et al., 2009b). The effect of OsSPX1 on Pi-signaling and its negative regulation on shoot Pi excessive accumulation has also been reported (
Wang et al., 2009a). Because of the reciprocal effect of OsPHR2 and OsSPX1 on shoot Pi accumulation, it could be reasoned that OsSPX1 may be a genetic repressor of function of OsPHR2 on Pi uptake and translocation.
Evidence from the physiological analysis of transgenic plants with double overexpression of
OsPHR2 and
OsSPX1 supports the above hypothesis (
Liu et al., 2010) and reveals that OsSPX1 has a counteracting effect on the upregulation of
OsPT2 in roots driven by
OsPHR2 overexpression and the accumulation of excess shoot Pi under abundant Pi. In contrast, OsSPX1 did not, however, show the counteracting effect on the negative regulation of OsPHO2 on
OsPT2. The results suggest that OsSPX1 may be a repressor involved in the physical regulation of OsPHR2 on OsPT2, but not in the reciprocal regulation of OsPHO2 on OsPT2, although a feedback Pi-signaling network is defined by OsPHR2, OsSPX1 and OsPHO2 in roots under abundant Pi (Fig. 1).
OsPHR2 may control some unknown factors crucial for physiologic utilization of cell Pi
The growth inhibition of transgenic plants with shoot Pi excessive accumulation caused by ectopic overexpression of
OsPHR2 raises an intriguing question: whether the excessive Pi in shoots exerts a physiologic toxicity? Our favored hypothesis is that the growth inhibition mediated by overexpression of
OsPHR2 is caused by toxic physiologic effects due to excessive Pi accumulation in shoots (Pi toxicity). In fact, toxic symptoms become diminished with decreased Pi levels in growth medium (
Zhou et al., 2008). However, under low Pi supply conditions, the shoot Pi concentration in plants with
OsPHR2-overexpression is similar to that in wild type plants under higher Pi supply conditions, but the growth of the transgenic plant is still retarded (
Wu and Xu, 2010). Therefore, it could be reasoned that the inhibition of plant growth caused by overexpression of
OsPHR2 may be due to the repression of physiologic utilization of cell Pi or to some inhibitor(s) under regulation of OsPHR2. This hypothesis is supported by the fact that some mutants isolated from an EMS-generated seed stock under background of overexpression of
OsPHR2 showed diminished growth inhibition (Fig. 2). Although experimental evidence is still required to confirm this hypothesis, a complex network of Pi uptake, translocation and utilization under the control of OsPHR2 has emerged (Fig. 2).
Prospects
It is increasingly clear that the response of plants to Pi-signaling and the maintenance of Pi homeostasis in cells are defined by a complex feedback loop. Currently, the regulatory systems and their molecular basis are still poorly understood, in spite of their importance for biotechnological and agronomic strategies aimed at improving crops with more efficient utilization of Pi nutrition as well as other nutrients. It is obvious that many pieces of this puzzle representing the Pi regulatory network are missing, and future exciting research will aim to resolve the mystery of how plants sense Pi, transmit signals both locally and at long distance, and maintain the nutrient homeostasis to efficiently utilize Pi coordinated with other nutrients. Mutants isolated from EMS-generated seed stock show rescue of growth inhibition caused by overexpression of OsPHR2 (Fig. 2), giving us new insight into the understanding of molecular network regulating Pi uptake, translocation and utilization in plants. Loss-of-function of some repressor(s) or gain-of-function of some gene(s) may be involved in the rescue of plant growth inhibition due to Pi excessive accumulation driven by OsPHR2, which is worthy of further study. Finally, a curious question is how SPX1 negatively regulates the function of OsPHR2 on Pi transporters.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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