The Screening And Identification Of Rhizosphere-Acidification-Deficiency Mutants In Arabidopsis Thaliana Under Low Phosphorus

Phosphorus is one of the macronutrients essential for plant growth and development, phosphorus also participates in many cellular processes. Though phosphorus is abundant in soil, it often forms insoluble salts, which decrease the utilization efficiency of phosphorus. Therefore, low phosphorus is a kind of stress for plants.Under low phosphorus, plants can secrete organic acids, which facilitate the breakdown of some insoluble matters to release Pi for plants to absorb. However, how plants perceive low phosphorus signal and transduct the signal is not clear. Rhizosphere acidification is a complex physiological process, which is regulated by a series of genes. The rapid development of molecular biology is beneficial to understand rhizosphere acidification mechanism under low phosphorus, and thus provide the foundation for breeding high utilization efficiency of phosphorus of crops.Bromocresol purple is a kind of pH indicators; it will gradually change from yellow to purple between pH5.2 6.8. Under low phosphorus, the plant could secrete organic acids leading to pH around roots as low as 5.5 for phosphorus being absorbed and utilized effectively, this process of biochemical and physiological change can be indicated by bromocresol purple sensitively. By observing the color change of the medium containing bromocresol purple, 200 mutants deficient in rhizosphere acidification were screened from the mutant library, which contains about 20,000 seeds induced by T-DNA. These mutants provides us materials for the researching the molecular mechanism of rhizosphere acidification mechanisms induced by low phosphorus.In this study, the mutant p122 deficient in rhizosphere acidification was examined, under low phosphorus stress, the mutant p122 behaved shorter of main roots, more lateral roots and root hairs and accumulated more anthocyanin than WT did. The content of Pi in mutant p122 is lower than in WT by 25% to 40%, and this Pi-deficiency symptoms can be inherited. TAIL-PCR analysis indicated that two T-DNA were inserted into the gene P122, and the mutant p122M of the gene P122 inserted by T-DNA from SALK institute showed the same Pi-deficiency symptoms as P122. Acorrding to bioinformatics analysis, the gene P122 encoding a calmodulin-binding protein is located on chromosome 1 of Arabidopsis, and can be induced by low phosphorus treatment. P122::GFP fusion protein showed that P122 protein was localized in the nucleus. Gene P122 was expressed in leaf, flowers, and roots and so on by GUS staining. The mutation of the gene P122 partially inhibited the expression of calmodulin CAM9, calcium anion exchanger CAX3 and transcription factor WRKY75. Increasing of concentrations can enhance the resistance to low phosphorus stress in p122 plants. The yeast two-hybrid experiment showed that P122 can bind calmodulin, this may imply that P122 regulate rhizosphere acidification induced by low phosphorus through Ca²⁺ signaling. P122 over-expression vector was also constructed to further study the gene P122.