Analysis Of Signal Pathway Components Of Arabidopsis P455 Regulating Low Phosphorus Induced Rhizosphere Acidification

Phosphorus is one of the essential nutrients for plant growth and development.Phosphorus in soil mainly exists in the form of insoluble phosphorus,but plants can only directly absorb soluble inorganic phosphorus,so plants often face low phosphorus stress.Rhizosphere acidification is the plant's root secretion of protons and organic acids to the soil and promote the dissolution of undissolved phosphorus in the soil,thus improving the utilization of phosphorus in the soil.Therefore,rhizosphere acidification is a way to adapt to low phosphorus stress.However,the molecular mechanism by which plants perceive low-phosphorus signals and regulate the acidification reaction in the rhizosphere remains unclearIn the previous study,we monitored the changes of acidification in Arabidopsis thaliana by bromocresol purple color indicator method and screened a mutant p455 with low phosphorous-induced root acidification.Under low-phosphorus stress conditions,the rhizosphere acidification of p455 mutants increased and the accumulation of anthocyanin in the leaves decreased,and the tolerance to insoluble phosphorus increased.Sequence analysis showed that P455 gene encodes a calmodulin binding protein and belongs to the IQD protein family.Further experimental results showed that P455 was located on the cytoplasmic microtubule skeleton and negatively regulate the low-phosphorus induced rhizosphere acidification.However,its specific molecular mechanism is not clearTo analyze the role of P455 protein in the low-phosphorus-induced rhizosphere acidification of Arabidopsis thaliana,In this experiment,we first observed the GFP fluorescence of p455::GFP fusion transgenic Arabidopsis thaliana plants using TIRFM,and analyzed the cell localization characteristics of P455 protein under low phosphorus conditions.The results showed that p455::GFP was localized on cytoplasmic microtubules,but compared with the control,treatment with low-phosphorus stress could affect the distribution of microtubule localized p455::GFP fluorescence,and low phosphorous stress for 3 days resulted in a decrease in the number of filament-like fluorescence and shortened length;however,the RFP fluorescence observation of TUA5::RFP fusion transgenic plants showed that low phosphorus stress did not affect the structure of the microtubules themselves.Protein immunoassay showed that low-phosphorus treatment resulted in ubiquitination and degradation of P455 protein.Therefore,it is speculated that low phosphorus stress can affect the microtubule localization of P455 protein and lead to its degradation by ubiquitination.The yeast two-hybrid and BiFC showed that the microtubule-localized P455 protein does not interact directly with the tubulins TUA3 and TUA6,but interacts directly with the kinesin protein KLCR1/KLCR2 that binds to the tubulin TUA6.So we presumably,P455 may be localized on microtubules through KLCR1/KLCR2 mediation.Rhizosphere acidification experiments showed that the kinesin mutant klcrl and double mutant p455 klcr1 had stronger acidification ability than WT and p455 under low-phosphorus stress conditions,suggesting that KLCR1 may be the main component of downstream signaling pathway of P455 protein.The results of co-immunoprecipitation and mass spectrometry analysis showed that membrane proton pump protein AHA1 and membrane transporter related proteins PATL1 and PCAP1 may interact with P455.Yeast two-hybrid and BiFC did not detect the direct interaction of the above proteins on P455,but found that these proteins interact directly with the P455-binding protein KLCR1/KLCR2.Presumably,P455 may interact with AHA1,PATL1,and PCAP1 through KLCR1/KLCR2.Under low-phosphorus stress conditions,the rhizosphere acidification ability of the plasma membrane proton pump mutant ahal is weaker than that of WT,and the rhizosphere acidification ability of p455 ahal is weaker than p455.These results indicate that AHA1 may be a component of downstream signaling pathways of the P455 and KLCR1 proteins,mediating low-phosphorus signaling and regulating H+-ATPase.However,how AHA1 affects the rhizosphere acidification of p455 remains to be further studied.In addition,it has been reported that Ser/Thr protein kinase PKS5 can phosphorylate Ser-931 of plasma membrane protein AHA2 and negatively regulate PMH+-ATPase.Therefore,we suspect that PKS5 may interact with P455.Yeast two-hybrid and two-molecule fluorescence complementation experiments confirmed the direct interaction between PKS5 and KLCR1/KLCR2.The above results suggest that P455 may interact with other proteins indirectly through KLCR1/KLCR2.