PDRAD1 Is Involved In Phosphorus Deficiency Induced Rhizosphere Acidification By Regulating The Activities Of H~+-ATPase And Exudation Of Organic Acids

Phosphorus is one of the macronutrient elements which are required by plants for normal growth.Total phosphorus in the soil is abundant, but most exist in the form of organophosphorus and insoluble phosphorus compounds, which cannot be directly used by plants, so plants are often faced with low phosphorus stress. Under the condition of phosphorus deficiency, plants can promote the dissolution of sparingly soluble phosphorus in soil and increase the concentration of available phosphorus around the root through rhizosphere acidification, to increase the absorption and utilization of phosphorus. But how plants perceive signals of low phosphorus and regulate the molecular mechanisms of rhizosphere acidification is still unclear.The subject in previous work using bromocresol purple as the pH indicator in situ colorimetric method,screening a strain of rhizosphere acidification deletion mutant, named pdrad1-1. Under the condition of phosphorus deficiency, the mutant ability of rhizosphere acidification weaker than WT, lateral root density and anthocyanin accumulation less than WT, showing the characteristics of insensitive to low phosphorus stress. In addition, the phosphorus content in mutant's leaf is higher than WT, and the ratio of phosphorus content between aboveground and underground is higher than WT, which shows the capability of phosphorus transfer is better than WT. Map-based cloning results show that, PDRAD1 positioned at the upper end of chromosome 5. The mutant in the coding region missed 25 bp bases, frame shift mutations resulted in premature termination of the protein translation, loss of function. Allelic T-DNA insertion mutant pdrad1-2 collected from Salk has the same phenotype as pdrad1-1.This experiment is using mutant pdrad1-1, pdrad1-2, transgenic complementation lines and overexpression lines, further study the role of PDRAD1 in phosphorus-deficience induced rhizosphere acidification and nutrient utilization of phosphorus. Using the method of pH chromogenic in situ,determination of plant rhizosphere acidification ability, the results showed that under low phosphorus stress,the mutant pdrad1-1, pdrad1-2 rhizosphere acidification ability weaker than WT, transgenic complementation lines rhizosphere acidification ability consistent with WT, and overexpression lines rhizosphere acidification ability significantly stronger than the WT. In addition, under the stress of lowphosphorus, transgenic complementation lines' anthocyanin accumulation and lateral density consistent with WT, and the capability of phosphorus transfer from underground to aboveground also recover consistent with WT. In conclusion, PDRAD1 participate in the regulation of phosphorus-deficience induced rhizosphere acidification and the transport of inorganic phosphorus in plants.The vector of PDRAD1::PCAMBIA1381-GUS was constructed in this experiment, and analyzed its tissue localization. The results showed that the gene was expressed in both the leaves and roots, and under the condition of phosphorus deficiency, staining deepened and GUS enzyme activity increased. The above results showed that PDRAD1 was induced by low phosphorus, and the expression level increased under the condition of phosphorus deficiency. After addition the H⁺-ATPase specific inhibitor Na₃VO₄ to the medium,the rhizosphere acidification reaction was obviously inhibited, in addition, this experiment determined the activity of H⁺-ATPase on the root plasma membrane, found that under low phosphorus stress, the activity of H⁺-ATPase in mutant pdrad1-1 was significantly lower than WT, while the activity of H⁺-ATPase in overexpression lines were higher than WT, in addition the experiment using liquid chromatography and mass spectrometry determined the content of organic acids in root exudates of plants, showed that under low phosphorus stress, the content of organic acid in mutant's root exudates is generally lower than that of WT, and the content of succinate with the most obvious difference. The above results indicate that the mutant gene through regulation the activity of H⁺-ATPase on the root plasma membrane and organic acid secretion to influence the rhizosphere acidification ability of mutant. In addition, the density of lateral roots and root hairs with the mutant and overexpression lines were analyzed, it was found that the density of lateral roots and root hairs with pdrad1-1 were less than WT, and overexpression lines' were greater than WT. From the above results speculate that PDRAD1 influences the formation and development of lateral roots and root hairs.In addition, we measured the physiological and biochemical indexes and the content of phosphorus under different concentrations of N sources, results showed that the mutant under the condition of ammonium-deficiency, the ability of phosphorus transport to further improve, in addition, under the condition of low phosphorus, the ammonium in plant tissue will get a lot of accumulation, these results indicate that the gene has a regulatory effect on nitrogen and phosphorus in nutrient elements. Besides that,PDRAD1::pCAMBIA1300-GFP vector was also constructed, prepared materials for the further analysis ofthe relationship between the subcellular localization of PDRAD1 and phosphorus.