Phosphate Homeostasis In Rice Research Articles

Transcriptome analysis with different leaf blades identifies the phloem-specific phosphate transporter OsPHO1;3 required for phosphate homeostasis in rice.

Phosphate (Pi) is essential for plant growth and development. One strategy to improve Pi use efficiency is to enhance Pi remobilization among leaves. Using transcriptome analysis with first (top) and fourth (down) leaf blades from rice (Oryza sativa) in Pi-sufficient and deficient conditions, we identified 1384 genes differentially expressed among these leaf blades. These genes were involved in physiological processes, metabolism, transport, and photosynthesis. Moreover, we identified the Pi efflux transporter gene, OsPHO1;3, responding to Pi-supplied conditions among these leaf blades. OsPHO1;3 is highly expressed in companion cells of phloem, but not xylem, in leaf blades and induced by Pi starvation. Mutation of OsPHO1;3 led to Pi accumulation in second to fourth leaves under Pi-sufficient conditions, but enhanced Pi levels in first leaves under Pi-deficient conditions. These Pi accumulations in leaves of Ospho1;3 mutants resulted from induction of OsPHT1;2 and OsPHT1;8 in root and reduction of Pi remobilization in leaf blades, revealed by the decreased Pi in phloem of leaves. Importantly, lack of OsPHO1;3 caused growth defects under a range of Pi-supplied conditions. These results demonstrate that Pi remobilization is essential for Pi homeostasis and plant growth irrespective of Pi-supplied conditions, and OsPHO1;3 plays an essential role in Pi remobilization for normal plant growth.

Protein phosphatase 5 mediates plant growth and phosphate homeostasis in rice

Protein phosphorylation is a reversible post-translational modification, and protein phosphatase is the most important regulator for protein dephosphorylation. Protein phosphatase 5 (PP5), which functions in many cellular processes essential for cell survival and growth in animals, has not been functionally reported in rice. In this study, we functionally identified OsPP5 in rice. OsPP5 is localized in both the nucleus and the cytoplasm which functions as a manganese (Mn2+)-dependent phosphatase. OsPP5 is expressed in most tissues of rice and is positively regulated by Pi supply at transcript and protein levels. Compared with the wild-type plants, OsPP5 overexpression lines showed increased Pi concentration under both Pi sufficient and Pi deficient conditions, while OsPP5 RNA interfering plants showed the opposite phenotype. Expression of phosphate transporters OsPT2, OsPT9, and OsPT10 were increased in OsPP5 overexpression plants. Our findings show that OsPP5 plays an important role in rice Pi homeostasis which could be a potential target for improving Pi absorption under low Pi environment in rice.

Phosphate-dependent regulation of vacuolar trafficking of OsSPX-MFSs is critical for maintaining intracellular phosphate homeostasis in rice

Vacuolar storage of inorganic phosphate (Pi) is essential for Pi homeostasis in plants. The SPX-MFS family proteins have been demonstrated to be vacuolar Pi transporters in many plant species. Transcriptional regulation of the predominant transporter among rice SPX-MFSs, OsSPX-MFS3, was only moderately suppressed by Pi starvation. Thus, post-transcriptional mechanisms were hypothesized to regulate the activity of OsSPX-MFS3. In this study, we found that the tonoplast localization of OsSPX-MFSs is inhibited under Pi-depleted conditions, resulting in their retention in the pre-vacuolar compartments (PVCs). A yeast two-hybrid screen identified that two SNARE proteins, OsSYP21 and OsSYP22, interact with the MFS domain of OsSPX-MFS3. Further genetic and cytological analyses indicate that OsSYP21 and OsSYP22 facilitate trafficking of OsSPX-MFS3 from PVCs to the tonoplast. Although a homozygous frameshift mutation in OsSYP22 appeared to be lethal, tonoplast localization of OsSPX-MFS3 was significantly inhibited in transgenic plants expressing a negative-dominant form of OsSYP22 (OsSYP22-ND), resulting in reduced vacuolar Pi concentrations in OsSYP22-ND plants. Under Pi-depleted conditions, the interaction between OsSYP22 and OsSPX-MFS3 was disrupted, and this process depended on the presence of the SPX domain. Deleting the SPX domains of OsSPX-MFSs resulted in their tonoplast localization under both Pi-depleted and Pi-replete conditions. Complementation of the osspx-mfs1/2/3 triple mutants with the MFS domain or the SPX domain of OsSPX-MFS3 confirmed that the MFS and SPX domains are responsive to Pi transport activity and Pi-dependent regulation, respectively. These data indicated that the SPX domains of OsSPX-MFSs sense cellular Pi (InsP) levels and, under Pi-depleted conditions, inhibit the interaction between OsSPX-MFSs and OsSYP21/22 and subsequent trafficking of OsSPX-MFSs from PVCs to the tonoplast.

OsUEV1B, an Ubc enzyme variant protein, is required for phosphate homeostasis in rice.

Phosphorus is a crucial macronutrient for plant growth and development. The mechanisms for maintaining inorganic phosphate (Pi) homeostasis in rice are not well understood. The ubiquitin-conjugating enzyme variant protein OsUEV1B was previously found to interact with OsUbc13 and mediate lysine63-linked polyubiquitination. In the present study, we found OsUEV1B was specifically inhibited by Pi deficiency, and was localized in the nucleus and cytoplasm. Both osuev1b mutant and OsUEV1B-RNA interference (RNAi) lines displayed serious symptoms of toxicity due to Pi overaccumulation. Some Pi starvation inducible and phosphate transporter genes were upregulated in osuev1b mutant and OsUEV1B-RNAi plants in association with enhanced Pi acquisition, and representative Pi starvation responses, including stimulation of acid phosphatase activity and root hair growth, were also activated in the presence of sufficient Pi. A yeast two-hybrid screen revealed an interaction between OsUEV1B and OsVDAC1, which was confirmed by bimolecular fluorescence complementation and firefly split-luciferase complementation assays. OsVDAC1 encoded a voltage-dependent anion channel protein localized in the mitochondria, and OsUbc13 was shown to interact with OsVDAC1 via yeast two-hybrid and bimolecular fluorescence complementation assays. Under sufficient Pi conditions, similar to osuev1b, a mutation in OsVDAC1 resulted in significantly greater Pi concentrations in the roots and second leaves, improved acid phosphatase activity, and enhanced expression of the Pi starvation inducible and phosphate transporter genes compared with wild-type DongJin, whereas overexpression of OsVDAC1 had the opposite effects. OsUEV1B or OsVDAC1 knockout reduced the mitochondrial membrane potential and adenosine triphosphate levels. Moreover, overexpression of OsVDAC1 in osuev1b partially restored its high Pi concentration to a level between those of osuev1b and DongJin. Our results indicate that OsUEV1B is required for rice phosphate homeostasis.

Knockdown of OsSAE1a affects the growth and development and phosphate homeostasis in rice

SUMOylation is a post-translational modification process that comprises a tandem enzymatic cascade, i.e., maturation, activation, conjugation, and ligation of a small ubiquitin-like modifier, which triggers the modulated activities and transport of the cellular proteins to other areas of the cell. In Oryza sativa (rice), OsSIZ1/2 encoding E3 SUMO ligase exerts regulatory influences on Pi homeostasis and developmental responses. However, the role of OsSAE1a, SUMO E1 activating enzyme, in regulating phosphate (Pi) utilization and/or growth and development is not known in rice and was thus investigated in this study. The qRT-PCR assay revealed a constitutive and variable spatiotemporal expression pattern of OsSAE1a in the vegetative and reproductive tissues and was comparable in the root and shoot grown under different Pi regimes. RNAi-mediated suppression of OsSAE1a exerted variable effects on the concentrations of Pi and total P in different tissues, uptake and distribution of 32Pi, and relative expression levels of several genes that play pivotal roles in the maintenance of Pi homeostasis. The effects of the mutation in OsSAE1a were also evident in the vegetative and reproductive traits of rice during growth in a hydroponic system and pot soil, respectively. Overall, these results suggest a broad-spectrum role of OsSAE1a in the maintenance of Pi homeostasis and regulating growth and development.

Are we ready to improve phosphorus homeostasis in rice?

Phosphorus (P) is an essential macronutrient which often limits plant growth, but the phosphate rock used for fertilizer production is a finite resource. On the other hand, large amounts of P compounds are entering surface waters, leading to eutrophication. Therefore, improvement of phosphate use efficiency of crop plants is a major task for plant science. Rice as a staple crop has recently been a focus of such efforts with several major discoveries. New transporters controlling phosphate homeostasis in rice have been discovered. Manipulation of expression of the corresponding genes improves different components of phosphate use efficiency, such as delivery of phosphate to the developing seeds and synthesis of phytic acid. Here these new findings are discussed in the context of general P nutrition and with the aim of finding out how far we can optimize P homeostasis in rice.

OsSIZ2 exerts regulatory influences on the developmental responses and phosphate homeostasis in rice

OsSIZ1, a small ubiquitin-related modifier (SUMO) E3 ligase, exerts regulatory influences on the developmental responses and phosphate (Pi) homeostasis in rice (Oryza sativa). Whether paralogs OsSIZ1 and OsSIZ2 are functionally redundant or the latter regulates these traits independent of the former is not known. To determine this, in this study, OsSIZ2 was functionally characterized by employing reverse genetic approaches. Although the relative expression of OsSIZ2 was spatiotemporally regulated, it showed constitutive expression in root and leaf blade irrespective of Pi regime. Analysis of T-DNA insertion knockout (ossiz2) and RNAi-mediated knockdown (Ri1-3) mutants revealed positive influences on growth and developmental responses including yield-related traits. On the contrary, these mutants exhibited negative effects on the concentrations of Pi and total P in different tissues. The relative expression levels of some of the genes that are involved in Pi sensing and signaling cascades were differentially modulated in the mutants. Further, attenuation in the expression levels of OsSIZ2 in the roots of ossiz1 and relatively similar trend of the effects of the mutation in OsSIZ1 and OsSIZ2 on growth and development and total P concentration in different tissues suggested a prevalence of partial functional redundancy between these paralogs.

OsNLA1, a RING-type ubiquitin ligase, maintains phosphate homeostasis in Oryza sativa via degradation of phosphate transporters.

Inorganic phosphate (Pi) transporters (PTs) play vital roles in Pi uptake and translocation in plants. Under Pi sufficient conditions, PTs are degraded to prevent excess Pi accumulation. The mechanisms targeting PTs for degradation are not fully elucidated. In this study, we found that the Oryza sativa (rice) ortholog of Arabidopsis thaliana nitrogen limitation adaptation (NLA), OsNLA1 protein, a RING-type E3 ubiquitin-ligase, was predominantly localized in the plasma membrane, and could interact with rice phosphate transporters OsPT2 and OsPT8. Mutation of the 265th cysteine residue in OsNLA1 that was required for ubiquitination prevented breakdown of OsPT2/PT8, suggesting OsNLA1 targeted OsPT2/PT8 for degradation. Mutation in OsNLA1 (osnla1) led to a significant increase of Pi concentration in leaves in a nitrate-independent manner. Overexpression of OsNLA1 or repression of OsPT2/PT8 restored the high leaf Pi concentration in osnla1 mutants to a level similar to that of wild-type plants. In contrast to what has been observed in Arabidopsis, the transcript abundance of OsNLA1 did not decrease under Pi limited conditions or in OsmiR827 (microRNA827)- or OsPHR2 (PHOSPHATE STARVATION RESPONSE 2)-overexpressing transgenic lines. Moreover, there was no interaction of OsNLA1 and OsPHO2, an E2 ubiquitin-conjugase, suggesting that OsPHO2 was not the partner of OsNLA1 involved in ubiquitin-mediated PT degradation. Our results show that OsNLA1 is involved in maintaining phosphate homeostasis in rice by mediating the degradation of OsPT2 and OsPT8, and OsNLA1 differs from the ortholog in Arabidopsis in several aspects.

Identification and expression analysis of OsLPR family revealed the potential roles of OsLPR3 and 5 in maintaining phosphate homeostasis in rice.

BackgroundPhosphorus (P), an essential macronutrient, is often limiting in soils and affects plant growth and development. In Arabidopsis thaliana, Low Phosphate Root1 (LPR1) and its close paralog LPR2 encode multicopper oxidases (MCOs). They regulate meristem responses of root system to phosphate (Pi) deficiency. However, the roles of LPR gene family in rice (Oryza sativa) in maintaining Pi homeostasis have not been elucidated as yet.ResultsHere, the identification and expression analysis for the homologs of LPR1/2 in rice were carried out. Five homologs, hereafter referred to as OsLPR1-5, were identified in rice, which are distributed on chromosome1 over a range of 65 kb. Phylogenetic analysis grouped OsLPR1/3/4/5 and OsLPR2 into two distinct sub-clades with OsLPR3 and 5 showing close proximity. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed higher expression levels of OsLPR3-5 and OsLPR2 in root and shoot, respectively. Deficiencies of different nutrients ie, P, nitrogen (N), potassium (K), magnesium (Mg) and iron (Fe) exerted differential and partially overlapping effects on the relative expression levels of the members of OsLPR family. Pi deficiency (−P) triggered significant increases in the relative expression levels of OsLPR3 and 5. Strong induction in the relative expression levels of OsLPR3 and 5 in osphr2 suggested their negative transcriptional regulation by OsPHR2. Further, the expression levels of OsLPR3 and 5 were either attenuated in ossiz1 and ospho2 or augmented in rice overexpressing OsSPX1.ConclusionsThe results from this study provided insights into the evolutionary expansion and a likely functional divergence of OsLPR family with potential roles of OsLPR3 and 5 in the maintenance of Pi homeostasis in rice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0853-x) contains supplementary material, which is available to authorized users.

The Phosphate Transporter Gene OsPht1;4 Is Involved in Phosphate Homeostasis in Rice.

A total of 13 phosphate transporters in rice (Oryza sative) have been identified as belonging to the Pht1 family, which mediates inorganic phosphate (Pi) uptake and transport. We report the biological property and physiological role of OsPht1;4 (OsPT4). Overexpressing OsPT4 resulted in significant higher Pi accumulation in roots, straw and brown rice, and suppression of OsPT4 caused decreased Pi concentration in straw and brown rice. Expression of the β-glucuronidase reporter gene driven by the OsPT4 promoter showed that OsPT4 is expressed in roots, leaves, ligules, stamens, and caryopses under sufficient Pi conditions, consistent with the expression profile showing that OsPT4 has high expression in roots and flag leaves. The transcript level of OsPT4 increased significantly both in shoots and roots with a long time Pi starvation. OsPT4 encoded a plasma membrane—localized protein and was able to complement the function of the Pi transporter gene PHO84 in yeast. We concluded that OsPT4 is a functional Pi-influx transporter involved in Pi absorption in rice that might play a role in Pi translocation. This study will enrich our understanding about the physiological function of rice Pht1 family genes.

A rice cis-natural antisense RNA acts as a translational enhancer for its cognate mRNA and contributes to phosphate homeostasis and plant fitness.

cis-natural antisense transcripts (cis-NATs) are widespread in plants and are often associated with downregulation of their associated sense genes. We found that a cis-NAT positively regulates the level of a protein critical for phosphate homeostasis in rice (Oryza sativa). PHOSPHATE1;2 (PHO1;2), a gene involved in phosphate loading into the xylem in rice, and its associated cis-NATPHO1;2 are both controlled by promoters active in the vascular cylinder of roots and leaves. While the PHO1;2 promoter is unresponsive to the plant phosphate status, the cis-NATPHO1;2 promoter is strongly upregulated under phosphate deficiency. Expression of both cis-NATPHO1;2 and the PHO1;2 protein increased in phosphate-deficient plants, while the PHO1;2 mRNA level remained stable. Downregulation of cis-NATPHO1;2 expression by RNA interference resulted in a decrease in PHO1;2 protein, impaired the transfer of phosphate from root to shoot, and decreased seed yield. Constitutive overexpression of NATPHO1;2 in trans led to a strong increase of PHO1;2, even under phosphate-sufficient conditions. Under all conditions, no changes occurred in the level of expression, sequence, or nuclear export of PHO1;2 mRNA. However, expression of cis-NATPHO1;2 was associated with a shift of both PHO1;2 and cis-NATPHO1;2 toward the polysomes. These findings reveal an unexpected role for cis-NATPHO1;2 in promoting PHO1;2 translation and affecting phosphate homeostasis and plant fitness.

Auxin response factor (OsARF12), a novel regulator for phosphate homeostasis in rice (Oryza sativa)

Phosphorus (P) is crucial nutrient element for crop growth and development. However, the network pathway regulating homeostasis of phosphate (Pi) in crops has many molecular breeding unknowns. Here, we report that an auxin response factor, OsARF12, functions in Pi homeostasis. Measurement of element content, quantitative reverse transcription polymerase chain reaction analysis and acid phosphatases (APases) activity assay showed that the osarf12 mutant and osarf12/25 double mutant with P-intoxicated phenotypes had higher P concentrations, up-regulation of the Pi transporter encoding genes and increased APase activity under Pi-sufficient/-deficient (+Pi/-Pi, 0.32/0 mM NaH2 PO4) conditions. Transcript analysis revealed that Pi-responsive genes--Phosphate starvation (OsIPS)1 and OsIPS2, SYG1/Pho81/XPR1(OsSPX1), Sulfoquinovosyldiacylglycerol 2 (OsSQD2), R2R3 MYB transcription factor (OsMYB2P-1) and Transport Inhibitor Response1 (OsTIR1)--were more abundant in the osarf12 and osarf12/25 mutants under +Pi/-Pi conditions. Knockout of OsARF12 also influenced the transcript abundances of the OsPHR2 gene and its downstream components, such as OsMiR399j, OsPHO2, OsMiR827, OsSPX-MFS1 and OsSPX-MFS2. Results from -Pi/1-naphthylphthalamic acid (NPA) treatments, and auxin reporter DR5::GUS staining suggest that root system alteration and Pi-induced auxin response were at least partially controlled by OsARF12. These findings enrich our understanding of the biological functions of OsARF12, which also acts in regulating Pi homeostasis.

The Phosphate Transporter Gene OsPht1;8 Is Involved in Phosphate Homeostasis in Rice

Plant phosphate transporters (PTs) are active in the uptake of inorganic phosphate (Pi) from the soil and its translocation within the plant. Here, we report on the biological properties and physiological roles of OsPht1;8 (OsPT8), one of the PTs belonging to the Pht1 family in rice (Oryza sativa). Expression of a β-glucuronidase and green fluorescent protein reporter gene driven by the OsPT8 promoter showed that OsPT8 is expressed in various tissue organs from roots to seeds independent of Pi supply. OsPT8 was able to complement a yeast Pi-uptake mutant and increase Pi accumulation of Xenopus laevis oocytes when supplied with micromolar (33)Pi concentrations at their external solution, indicating that it has a high affinity for Pi transport. Overexpression of OsPT8 resulted in excessive Pi in both roots and shoots and Pi toxic symptoms under the high-Pi supply condition. In contrast, knockdown of OsPT8 by RNA interference decreased Pi uptake and plant growth under both high- and low-Pi conditions. Moreover, OsPT8 suppression resulted in an increase of phosphorus content in the panicle axis and in a decrease of phosphorus content in unfilled grain hulls, accompanied by lower seed-setting rate. Altogether, our data suggest that OsPT8 is involved in Pi homeostasis in rice and is critical for plant growth and development.

Increased expression of OsSPX1 enhances cold/subfreezing tolerance in tobacco and Arabidopsis thaliana

Low temperature is a major environmental stress for plants. Many important cultivated crops have limited capacity to survive below freezing/subfreezing temperatures. Low inorganic phosphate (Pi) is reportedly important in triggering cold acclimatization. SPX (SYG1/Pho81/XPR1: SYG1, suppressor of yeast gpal; Pho81, CDK inhibitor in yeast PHO pathway; XPR1, xenotropic and polytropic retrovirus receptor) domain proteins have been shown to be involved in the phosphate-related signal transduction and regulation pathways. Recently, Arabidopsis AtSPX family genes have been found to possess diverse functions in plant tolerance to phosphorus starvation, and OsSPX1 is involved in phosphate homeostasis in rice and optimizes growth under phosphate-limited conditions through a negative feedback loop. In this study, our phylogenetic and gene expression profiling approaches identified six rice OsSPX genes up-regulated during cold stress. Transgenic tobacco plants with constitutive expression of OsSPX1 were more tolerant to cold stress than were wild-type plants, and showed better seedling survival and reduced cellular electrolyte leakage. In addition, there was decreased total leaf Pi content and accumulation of free proline and sucrose in transgenic tobacco plants during cold stress. To further establish a cause-and-effect relationship between intracellular Pi level and cold acclimatization in transgenic plants, we generated transgenic Arabidopsis plants with constitutive expression of OsSPX1. Cold stress resulted in reduced leaf Pi levels in Arabidopsis transgenic relative to wild-type plants. From real-time reverse transcriptase-polymerase chain reaction analysis, several Pi starvation-related genes, such as AtSPX1 (orthologue of OsSPX1), PHO2, PLDZ2 and ATSIZ1, showed differential expression between wild-type and transgenic plants during cold stress. Our results indicate that OsSPX1 may play an important role in linking cold stress and Pi starvation signal transduction pathways.

Involvement of OsSPX1 in phosphate homeostasis in rice

Arabidopsis thaliana SPX (SYG/PHO81/XPR1) domain genes have recently been shown to be involved in the phosphate (Pi) signaling pathway. We show here that a rice (Oryza sativa) SPX gene, OsSPX1, is specifically induced by Pi starvation in roots. Suppression of OsSPX1 by RNA interference resulted in severe signs of toxicity caused by the over-accumulation of Pi, similar to that found in OsPHR2 (phosphate starvation response transcription factor 2) overexpressors and pho2 (phosphate-responsive mutant 2). Quantitative RT-PCR showed that expression of OsSPX1 was strongly induced in OsPHR2 overexpression and pho2 mutant plants, indicating that OsSPX1 occurs downstream of OsPHR2 and PHO2. The expression of 10 genes associated with the phosphate-starvation signal pathways was analyzed. Expression of OsPT2 (phosphate transporter 2) and OsPT8 was significantly induced in OsSPX1-RNAi (OsSPX1-Ri) plants, suggesting that over-accumulation of Pi in OsSPX1-Ri plants results from an increase in Pi transport. In contrast, overexpression of OsSPX1 suppressed the induction of expression by Pi starvation of all 10 phosphate starvation-induced genes tested: IPS1 (induced by phosphate starvation 1), IPS2, OsPAP10 (purple acid phosphatase 10), OsSQD2 (sulfoquinovosyldiacylglycerol 2), miR399d and miR399j (microRNA 399), OsPT2, OsPT3, OsPT6 and OsPT8. This suggests that OsSPX1 acts via a negative feedback loop to optimize growth under phosphate-limited conditions.