Molecular Mechanism Analysis Of Low Phosphate Stress Response Mediated By Calcium Signal In Arabidopsis

Phosphorus (P), one of the essential macronutrient elements, plays important roles in plant growth and development. Low phosphate stress leads to rhizosphere acidification, by which insoluble phosphates are turned into usful phosphates. Rhizosphere acidification increases the utilization efficiency of phosphorus. For plant, however, how rhizosphere acidification is induced by phosphate starvation, what kinds of factor can mediate rhizosphere acidification process, and how to achieve efficient absorption of phosphate by acidification, these problems are still unclear.In this study, we abtained a rhizosphere acidification-deficiency mutant prad-1, which was screened from T-DNA insertion mutant library. Compared with wild type plants, prad-1significantly decreased its rhizosphere acidification capacity under low phosphate condition. Meanwhile, the mutant accumulated more anthocyanin than wild-type plants. PRAD gene was cloned by Tail-PCR and its TAIR access number is Atlg73805. Semi-quantitative RT-PCR showed that prad-1is a loss of function mutant. We generated transgenic complementation lines in prad-1background, and found rhizosphere acidification-deficiency phenotype in prad-1can be restored by PRAD under low phosphate. Also, an allele mutant prad-2showed the same phenotype as prad-1. These results provided evidence that disruption of PRAD results in significantly reduced capacity of rhizosphere acidification which should be induced by low phosphate. RT-PCR and GUS staining showed that PRAD gene is expressed in various tissues including roots, and that transcription level of PRAD is induced mildly, as well as quickly under low phosphate stress.It has been known that PRAD encodes a calmodulin binding protein which functions as a transcription factor. Through yeast two-hybrid, BiFC and in vitro calmodulin binding assays, PRAD was found to have potential interaction with plant calmodulin protein CAM4, CAM5, and CAM9. We constructed transient expression system in which PRAD-binding cis-element drives the reporter gene. Transient expression activation assay showed that CAM5can significantly increase PRAD transcription activity. The double mutant prad-1Icam5displayed rhizosphere acidification-deficiency phenotype and slso accumulated more anthocyanin than wild type under low phosphate treatment. All these results indicated that PRAD, together with CAM5, modulated low phosphate-induced rhizosphere acidification process.The study revealed that upon low phosphate condition, the specific inhibitor of membrane H+-ATPase suppressed the rhizosphere acidification, inferring that the membrane H+-ATPase functions in precess of rhizosphere acidification. We tested the membrane H+-ATPase activity of root cell under low phosphate stress, and found that H+-ATPase activity in prad-1is weaker than that in WT. Quantitative RT-PCR demenstrateded that upon the low phosphate stress, the transcritption level of AHA1in prad-1mutant is lower than that in wild type plants, suggesting that PRAD may regulates the transcription of AHA1to mediate the rhizosphere acidification in low phosphate.The transgenic plants carrying calcium ion probe YC3.6were used to detect the alteration of intracellular calcium concentration before and after low phosphate treatment. We found that low phosphate caused the decrease of calcium ion level in root cytoplasm; RT-PCR results also showed that exogenous calcium supressed the expression of PRAD and the rhizosphere acidification capacity under low phosphate condition. Meanwhile, it was also found that excess calcium can relief the stress resulted from low phosphate. These results suggested that calcium may be involved in the response to low phosphate stress in plant. We hypothesized that the decreased level of calcium in cytoplasm activate phosphate starvation response. However, further experiments are required to explain this mechanism.