Functional Characterization Of The Rice SPX-MFS Family

Phosphorus (P) is an essential and important element for growth and development in plants. Although it is abundant in the Earth’s crust, few can be available to plants. Thus, P deficiency is a common nutrient stress limiting crop yield.. To solve the problem, excessive phosphorus fertilizer has been used for the crop production. While improving crop production, over-fertilization increase the economic cost as well as environmental pollution. Genetic improvement of crop P use efficiency is of important.Plant can adapt to the low P condition by expansion of the root system to accelerate soil exploration, increasing Pi uptake capacity of Pi high-affinity transporters, the adjustment of metabolism to maintain intracellular Pi homeostasis, as well as, the induction and secretion of phosphatases and organic acids to mobilize Pi from organic matters. To date, molecular and physiological mechanisms regarding plant phosphate starvation signaling have been studied extensively, which is greatly contributed to the efforts of improvement of P use efficiency in crops.. In this study, we cloned and functionally analyzed the rice OsSPX-MFS genes. Our result showed that OsSPX-MFS1gene acts as a P transporter and play a key role in maintaining the Pi homeostasis in rice.Plant SPX domain-containing proteins can be divided into four classes. While three of which, SPX, SPX-EXS and SPX-RING subfamilies had been reported to be involved in inorganic phosphate transport or signaling, the role of SPX-MFS family is still unclear. According to gene structure analysis of OsSPX-MFS family, a osa-miR827binding site has been found in both sequences from OsSPX-MFS1and OsSPX-MFS2, suggesting these two genes may be the targets of the Pi starvation induced osa-miR827and under the control of osa-miR827. Protein blast analysis shows the high identity and similarity between OsSPX-MFS1and OsSPX-MFS2, which means the function of these two genes could probably be the same.The transmembrane domain assay shows the SPX domain locate in the extracellular, while the MFS domain containing10transmembrane domains locate in the membrane, suggesting the importance of MFS domain in protein location.Analysis of OsSPX-MFS transcripts revealed that the responses to Pi deficiency are different. The expression of OsSPX-MFS1and OsSPX-MFS3are suppressed under Pi deficiency condition, while that of the OsSPX-MFS2was induced. Time-course experiment showed that responses of OsSPX-MFS genes to Pi starvation are gradual.Transcript abundance of OsSPX-MFS family genes in OsPHR2overexpression lines was significantly lower than that in the wildtype plant, suggesting osa-miR827-OsSPX-MFS1/2pathway is under the control of the transcription factor OsPHR2.In addition, using the osa-miR827overexpression and OsSPX-MFSl mutant plants, we demonstrated that the induction extent in a series of Pi starvation induced genes, such as OsSPXl, OsPAP10α, OsIPSl, OsACP and parts of OsPT genes, are attenuated. As the MFS-domain containing proteins had always been found could transport a certain of substrate, our hypothesis was that some of the SPX-MFS family members could be involved in mediating Pi transport. To test this hypothesis, we characterized the Pi concentration in osa-miR827overexpression and OsSPX-MFS1mutant plants, results show us the Pi concentration in the shoots appears to be increased significant. After testing the Pi concentration in different leaves, respectively, we confirm this increase comes from accumulation in old leaves, whereas, the difference in young leaves is not apparent.Furthermore, we confirm OsSPX-MFS1could function as Pi relative ions transporter with yeast complementation test by using two yeast mutant lines, respectively, one is PAM2, which loss the function of yeast high affinity phosphate transporters, while another one is EY917, which loss the function of all the phosphate transporters in yeast. The results prove the Pi transport ability of OsSPX-MFS1. In conclusion, we found that OsSPX-MFS1is involved in controlling Pi tranportation in rice.