P67 Involves In Phosphorus Deficiency-induced Rhizosphere Acidification In Arabidopsis

Phosphorus(P), one of the essential macronutrients in plants, plays an important role in plant growth and development. P is not only an indispensable component of nucleic acids, nucleotides, ATP,phospholipids, kinase and phosphorylase in plants, but also widely involved in biochemical reactions such as signal transduction, material metabolism and energy metabolism in plants. However, most of soil phosphorus exists in the form of organophosphorus and insoluble phosphorus compounds, which cannot be directly used by plants. The available phosphorus which plant can directly use is far less than the demand of the plant, so that plant is often under low phosphorus stress. Under low phosphorus environment, plants have evolved a variety of mechanisms to improve the utilization rate of soil phosphorus. Rhizosphere acidification is one of the important mechanisms of plant response to low phosphorus stress. It is widely noticed that plant roots can secrete organic acid to promote the insoluble phosphorus compounds in soil convert into soluble phosphorus around rhizosphere, improving the efficiency of phosphorus nutrition under phosphorus derivation condition. However, the specific molecular mechanism of plant rhizosphere acidification induced low phosphorus stress by is still not clear.Preliminary work has screened a mutant line named p67 from T-DNA insertion mutant library based on pH chromogenic method. Mutant p67 showed weak rhizosphere acidification ability under low phosphorus condition and accumulation more anthocyanin than wild type. P67 gene has been cloned through TAIL-PCR technology and a T-DNA was inserted in the P67 gene’s upstream 1000 bp. P67 gene has a weak expression in p67 mutant. T-DNA insertion mutant p67 M collected from Salk has the same phenotype as p67. All of above give us an assumption that P67 gene has effect on rhizosphere acidification.In this experiment, we use mutant p67, p67 M and the P67 overexpressing lines to study the role of P67 in phosphorus deficiency-induced rhizosphere acidification in Arabidopsis. Through rhizosphere in suit p H chromogenic method, we found, under low P condition, the rhizosphere acidification ability in p67 and p67M was decreased, while it was increased in overexpressing lines OE-4, OE-11 and OE-14. It is confirmed that P67 plays the role of phosphorus deficiency-induced rhizosphere acidification. In the media containing insoluble P, the overexpressing lines grow well which shows the overexpressing lines have better adaptability in insoluble phosphorus environment. Under low phosphorus stress, the accumulation ofanthocyanin content of the mutant was enhanced, while the accumulation of anthocyanin content of the overexpressing lines was on the opposite. The difference of inorganic phosphorus content in the mutant,wild type and the overexpressing lines is not distinct. To sum up, P67 gene plays a role in phosphorus deficiency-induced accumulation of anthocyanin, but may be not involved in the absorption and transport of phosphorus. To identify P67’s distribution, we also construct the vector P67::supper1300-cGFP and P67::pEGAD-nGFP, and have already got the positive transgene plants. Subcellular localization in perpetual P67-cGFP T1 generation plants indicates P67 protein locates in cell membrane and cell wall and distributes unevenly in cell membrane, which prepares materials for further study of the function of P67 and its relation with P.Na3VO4 is an inhibitor for cell membrane H+-ATPase. When we add Na3VO4 into p H chromogenic medium, rhizosphere acidification is strongly suppressed under P deprivation condition. This result builds a connection between H+-ATPase activity and rhizosphere acidification. P67 gene may influence the activity of plasma membrane H+-ATPase. To study the signaling pathways that P67 involves in regulating the rhizosphere acidification reaction induced by low phosphorus, we conducted yeast-two hybrid assay using P67 protein and 9 14-3-3 proteins, we expressed fusion protein of bait protein and whole P67 protein, two motif fragments DUF1 and DUF2. We find that DUF1 fusion protein has interaction with GRF8 and GRF10 by the yeast two-hybrid. BIFC results confirmed those interactions. 14-3-3 proteins can play a widely regulatory role in plant, for example material transportation, 14-3-3 protein can combine with H+-ATPase and regulate its activity to control material transmembrane transportation. P67 may change the activity of H+-ATpase through combing with GRF8, GRF10 and regulate rhizosphere acidification under low P stress.We further screened Arabidopsis cDNA library through yeast two hybrid system to find possible interaction proteins with P67, DUF1 and DUF2, after rescreening and rotary validation, we concluded that DUF1 protein has the intertion with PEPCK protein and At1g49570 encoding protein. All those results help us to further define P67’s function.