Phosphate starvation induced OsPHR4 mediates Pi-signaling and homeostasis in rice

Key message

OsPHR4 mediates the regulation of Pi-starvation signaling and Pi-homeostasis in a PHR1-subfamily dependent manner in rice.

Abstract

Phosphate (Pi) starvation response is a sophisticated process for plant in the natural environment. In this process, PHOSPHATE STARVATION RESPONSE 1 (PHR1) subfamily genes play a central role in regulating Pi-starvation signaling and Pi-homeostasis. Besides the three PHR1 orthologs in Oryza sativa L. (Os) [(Os) PHR1, (Os) PHR2, and (Os) PHR3], which were reported to regulated Pi-starvation signaling and Pi-homeostasis redundantly, a close related PHR1 ortholog [designated as (Os) PHR4] is presented in rice genome with unknown function. In this study, we found that OsPHR4 is a Pi-starvation induced gene and mainly expresses in vascular tissues through all growth and development periods. The expression of OsPHR4 is positively regulated by OsPHR1, OsPHR2 and OsPHR3. The nuclear located OsPHR4 can respectively interact with other three PHR1 subfamily members to regulate downstream Pi-starvation induced genes. Consistent with the positive role of PHR4 in regulating Pi-starvation signaling, the OsPHR4 overexpressors display higher Pi accumulation in the shoot and elevated expression of Pi-starvation induced genes under Pi-sufficient condition. Besides, moderate growth retardation and repression of the Pi-starvation signaling in the OsPHR4 RNA interfering (RNAi) transgenic lines can be observed under Pi-deficient condition. Together, we propose that OsPHR4 mediates the regulation of Pi-starvation signaling and Pi-homeostasis in a PHR1-subfamily dependent manner in rice.

     

SPX proteins regulate Pi homeostasis and signaling in different subcellular level

To cope with low phosphate (Pi) availability, plants have to adjust its gene expression profile to facilitate Pi acquisition and remobilization. Sensing the levels of Pi is essential for reprogramming the gene expression profile to adapt to the fluctuating Pi environment. AtPHR1 in Arabidopsis and OsPHR2 in rice are central regulators of Pi signaling, which regulates the expression of phosphate starvation-induced (PSI) genes by binding to the P1BS elements in the promoter of PSI genes. However, how the Pi level affects the central regulator to regulate the PSI genes have puzzled us for a decade. Recent progress in SPX proteins indicated that the SPX proteins play important role in regulating the activity of central regulator AtPHR1/OsPHR2 in a Pi dependent manner at different subcellular levels.     

OsSPX1 suppresses the function of OsPHR2 in the regulation of expression of OsPT2 and phosphate homeostasis in shoots of rice

Phosphate (Pi) homeostasis in plants is required for plant growth and development, and is achieved by the coordination of Pi acquisition, translocation from roots to shoots, and remobilization within plants. Previous reports have demonstrated that over‐expression of OsPHR2 (the homolog of AtPHR1) and knockdown of OsSPX1 result in accumulation of excessive shoot Pi in rice. Here we report that OsPHR2 positively regulates the low‐affinity Pi transporter gene OsPT2 by physical interaction and upstream regulation of OsPHO2 in roots. OsPT2 is responsible for most of the OsPHR2‐mediated accumulation of excess shoot Pi. OsSPX1 suppresses the regulation on expression of OsPT2 by OsPHR2 and the accumulation of excess shoot Pi, but it does not suppress induction of OsPT2 or the accumulation of excessive shoot Pi in the Ospho2 mutant. Our data also show that OsSPX1 is a negative regulator of OsPHR2 and is involved in the feedback of Pi‐signaling network in roots that is defined by OsPHR2 and OsPHO2. This finding provides new insight into the regulatory mechanism of Pi uptake, translocation, allocation and homeostasis in plants.     

A constitutive expressed phosphate transporter, OsPht1;1, modulates phosphate uptake and translocation in phosphate-replete rice

A number of phosphate (Pi) starvation- or mycorrhiza-regulated Pi transporters belonging to the Pht1 family have been functionally characterized in several plant species, whereas functions of the Pi transporters that are not regulated by changes in Pi supply are lacking. In this study, we show that rice (Oryza sativa) Pht1;1 (OsPT1), one of the 13 Pht1 Pi transporters in rice, was expressed abundantly and constitutively in various cell types of both roots and shoots. OsPT1 was able to complement the proton-coupled Pi transporter activities in a yeast mutant defective in Pi uptake. Transgenic plants of OsPT1 overexpression lines and RNA interference knockdown lines contained significantly higher and lower phosphorus concentrations, respectively, compared with the wild-type control in Pi-sufficient shoots. These responses of the transgenic plants to Pi supply were further confirmed by the changes in depolarization of root cell membrane potential, root hair occurrence, (33)P uptake rate and transportation, as well as phosphorus accumulation in young leaves at Pi-sufficient levels. Furthermore, OsPT1 expression was strongly enhanced by the mutation of Phosphate Overaccumulator2 (OsPHO2) but not by Phosphate Starvation Response2, indicating that OsPT1 is involved in the OsPHO2-regulated Pi pathway. These results indicate that OsPT1 is a key member of the Pht1 family involved in Pi uptake and translocation in rice under Pi-replete conditions.     

Oxygen deficit alleviates phosphate overaccumulation toxicity in OsPHR2 overexpression plants

Overexpression of OsPHR2 increases phosphate (Pi) uptake and causes overaccumulation of Pi in rice plants, which is toxic to rice plants when they are grown in media with a sufficient Pi supply. The toxicity that results from OsPHR2 overexpression can be significantly relieved by growing the plants in a waterlogged paddy field. A comparison of the Pi uptake and growth status of OsPHR2-overexpression plants (PHR2-Oe plants) grown in paddy fields or in a laboratory setting in aerated or stagnant hydroponic conditions indicated that the oxygen limitation that is present in paddy fields and in stagnant rice culture solutions inhibits the Pi overaccumulation toxicity of PHR2-Oe plants by reducing their Pi uptake. Quantitative RT-PCR demonstrated that the expression of Pi-starvation-induced (PSI) genes was induced by oxygen limitation in both wild-type and PHR2-Oe plants. The induction of PSI genes is the consequence of reducing the Pi concentration in stagnant plants. Thus, when evaluating the efficiency of Pi use in rice germplasm or transgenic materials under hydroponic conditions, the impact of the low oxygen condition that exists in waterlogged paddies should be considered.     

Cytokinin represses phosphate-starvation response through increasing of intracellular phosphate level

The involvement of cytokinins (CTKs) in the repression of phosphate (Pi)-starvation signalling has been widely documented. However, the full physiological and molecular relevance of this role remains unclear. To gain further insights into the regulation system of CTK repression of Pi-starvation signalling, a global analysis of gene expression events in rice seedlings under Pi starvation, and the exogenous CTK treatment under Pi-sufficient (+P) and Pi-deficient (-P) conditions, was conducted using oligonucleotide array analysis. Physiological and biochemical adaptation was observed after 10 d Pi starvation in rice seedlings. A global reduction of the Pi-starvation signalling was detected after 3 d treatment of exogenous CTK. Expression profiling data indicate that, together with a significant increase of intracellular Pi content, many expression changes responsive to Pi starvation were reversed by exogenous CTK treatment while CTK-responsive genes behaved normally under -P condition. These results suggest that the interplay of CTK signal and Pi-starvation response can be partially explained by the rise of Pi concentration after exogenous CTK treatment. Microarray data also revealed that a small number of genes have different CTK response patterns under different Pi levels, suggesting a subtle interaction between CTK and Pi-starvation signalling pathway.     

Does OsPHR2, central Pi-signaling regulator,regulate some unknown factors crucial for plant growth?

OsPHR2, the homolog of AtPHR1, is a central Pi-signaling regulator. The Pi-signaling pathway downstream of AtPHR1, similarly of OsPHR2,^1,2 involves a noncoding RNA which targets mimicry of miR399. miRNA399 mediates cleavage of PHO2.^3,4 The regulating pathway downstream of OsPHR2 is negatively regulated by the Pi-signaling responsive gene OsSPX1.^5,6 Overexpression of AtPHR1 and OsPHR2 leads to an increased concentration of Pi in the shoot tissues with leaf toxic symptom and growth retardation similar as the phenotype of pho2 mutant, especially under Pi abundant conditions.^2,6,7 It has been known that the low affinity Pi transporter OsPT2 mainly contributes to the shoot Pi accumulation mediated by OsPHR2, and overexpression of OsPT2 results in shoot Pi accumulation and leaf toxic symptom and growth retardation under Pi abundant conditions.⁶ Two curious questions are emerging from the reported results: How Os SPX1 functions on the negative regulation of the pathway and what mechanism of the growth retardation mediated by OsPHR2. For the second question, our favored hypothesis is that the growth inhibition mediated by overexpression of OsPHR2 is caused by toxic physiological effects due to excessive Pi accumulation in shoots (Pi toxicity). In fact, the toxic symptoms become diminished with decreased Pi levels in growth medium. However, the plant growth retardation mediated by overexpression of OsPHR2 may be caused by some unknown genetic factor(s) regulated by OsPHR2.Key words: Oryza Sativa L, OsPHR2, OsSPX1, pi-signaling, plant growth     

Membrane lipid alteration during phosphate starvation is regulated by phosphate signaling and auxin/cytokinin cross-talk

During phosphate (Pi) starvation in plants, membrane phospholipid content decreases concomitantly with an increase in non-phosphorus glycolipids. Although several studies have indicated the involvement of phytohormones in various physiological changes upon Pi starvation, the regulation of Pi-starvation induced membrane lipid alteration remains unknown. Previously, we reported the response of type B monogalactosyl diacylglycerol synthase genes (atMGD2 and atMGD3) to Pi starvation, and suggested a role for these genes in galactolipid accumulation during Pi starvation. We now report our investigation of the regulatory mechanism for the response of atMGD2/3 and changes in membrane lipid composition to Pi starvation. Exogenous auxin activated atMGD2/3 expression during Pi starvation, whereas their expression was repressed by cytokinin treatment in the root. Moreover, auxin inhibitors and the axr4 aux1 double mutation in auxin signaling impaired the increase of atMGD2/3 expression during Pi starvation, showing that auxin is required for atMGD2/3 activation. The fact that hormonal effects during Pi starvation were also observed with regard to changes in membrane lipid composition demonstrates that both auxin and cytokinin are indeed involved in the dynamic changes in membrane lipids during Pi starvation. Phosphite is not metabolically available in plants; however, when we supplied phosphite to Pi-starved plants, the Pi-starvation response disappeared with respect to both atMGD2/3 expression and changes in membrane lipids. These results indicate that the observed global change in plant membranes during Pi starvation is not caused by Pi-starvation induced damage in plant cells but rather is strictly regulated by Pi signaling and auxin/cytokinin cross-talk.     

Molecular mechanisms regulating Pi-signaling and Pi homeostasis under OsPHR2, a central Pi-signaling regulator, in rice

Phosphorus (P) is one of the most important major mineral elements for plant growth and metabolism. Plants have evolved adaptive regulatory mechanisms to maintain phosphate (Pi) homeostasis by improving phosphorus uptake, translocation, remobilization and efficiency of use. Here we review recent advances in our understanding of the OsPHR2-mediated phosphate-signaling pathway in rice. OsPHR2 positively regulates the low-affinity Pi transporter OsPT2 through physical interaction and reciprocal regulation of OsPHO2 in roots. OsPT2 is responsible for most of the OsPHR2-mediated accumulation of excess Pi in shoots. OsSPX1 acts as a repressor in the OsPHR2-mediated phosphate-signaling pathway. Some mutants screened from ethyl methanesulfonate (EMS)-mutagenized M2 population of OsPHR2 overexpression transgenic line removed the growth inhibition, indicating that some unknown factors are crucial for Pi utilization or plant growth under the regulation of OsPHR2.     

OsARF16, a transcription factor,is required for auxin and phosphate starvation response in rice (Oryza sativa L.)

Plant responses to auxin and phosphate (Pi) starvation are closely linked. However, the underlying mechanisms connecting auxin to phosphate starvation (?Pi) responses are largely unclear. Here, we show that OsARF16, an auxin response factor, functions in both auxin and ?Pi responses in rice (Oryza sativa L.). The knockout of OsARF16 led to primary roots (PR), lateral roots (LR) and root hair losing sensitivity to auxin and ?Pi response. OsARF16 expression and OsARF16::GUS staining in PR and LR of rice Nipponbare (NIP) were induced by indole acetic acid and ?Pi treatments. In ?Pi conditions, the shoot biomass of osarf16 was slightly reduced, and neither root growth nor iron content was induced, indicating that the knockout of OsARF16 led to loss of response to Pi deficiency in rice. Six phosphate starvation‐induced genes (PSIs) were less induced by ?Pi in osarf16 and these trends were similar to a knockdown mutant of OsPHR2 or AtPHR1, which was a key regulator under ?Pi. These data first reveal the biological function of OsARF16, provide novel evidence of a linkage between auxin and ?Pi responses and facilitate the development of new strategies for the efficient utilization of Pi in rice.     

LEAF TIP NECROSIS1 plays a pivotal role in the regulation of multiple phosphate starvation responses in rice

Although phosphate (Pi) starvation signaling is well studied in Arabidopsis (Arabidopsis thaliana), it is still largely unknown in rice (Oryza sativa). In this work, a rice leaf tip necrosis1 (ltn1) mutant was identified and characterized. Map-based cloning identified LTN1 as LOC_Os05g48390, the putative ortholog of Arabidopsis PHO2, which plays important roles in Pi starvation signaling. Analysis of transgenic plants harboring a LTN1 promoter::β-glucuronidase construct revealed that LTN1 was preferentially expressed in vascular tissues. The ltn1 mutant exhibited increased Pi uptake and translocation, which led to Pi overaccumulation in shoots. In association with enhanced Pi uptake and transport, some Pi transporters were up-regulated in the ltn1 mutant in the presence of sufficient Pi. Furthermore, the elongation of primary and adventitious roots was enhanced in the ltn1 mutant under Pi starvation, suggesting that LTN1 is involved in Pi-dependent root architecture alteration. Under Pi-sufficient conditions, typical Pi starvation responses such as stimulation of phosphatase and RNase activities, lipid composition alteration, nitrogen assimilation repression, and increased metal uptake were also activated in ltn1. Moreover, analysis of OsmiR399-overexpressing plants showed that LTN1 was down-regulated by OsmiR399. Our results strongly indicate that LTN1 is a crucial Pi starvation signaling component downstream of miR399 involved in the regulation of multiple Pi starvation responses in rice.