Functional Analysis Of MKK9-MPK3/MPK6-mediated MAPK Cascade In Regulating Phosphate Acquisition In Arabidopsis Thaliana

Phosphorus (P) is one of the essential macronutrients required for plant growth and development. Despite abundant of phosphorus in soil, very little is available as phosphate (Pi) for plants. For always experiencing low Pi (LP) stress, plants have developed diverse responsive mechanisms to resist Pi deficiency, while Pi sensing and signaling transduction are two indispensable steps to achieve and regulate theses responses. Mitogen-activated protein kinase (MAPK) cascades, consisting of a MAPKKK-MAPKK-MAPK module, transmits and amplifies the stimulus by cascaded phosphorylating, and plays important roles in plant growth, development and stress responses. Genome-wide transcriptional profiling by Microarray has revealed that multiple MAPK kinases are induced by LP stress, which suggests that MAPK cascades may involve in Pi signaling. This dissertation focuses on exploring the relationship between MAPK cascades and LP stress, and its functions in the regulation of Pi starvation responses.To find out whether MAPKs are involved in the response of different Pi conditions in Arabidopsis, the Pi-starving wild-type plant was used for measuring the activities of MAPKs, the results showed that the activities of MPK3and MPK6were increased under LP stress. Measurements of Pi contents and Pi uptake shown that mpk3and mpk6seedlings absorbed and accumulated less Pi than wild-type seedlings. Activation of MAPKs requires the phosphorylation of activated upstream MAPKKs, and many studies have shown that MKK9could activate both MPK3and MPK6in Arabidopsis. Therefore, Vector (empty vector), MKK9KR (an inactive allele of MKK9) and MKK9DD (an active allele of MKK9) transgenic plants were used to further explore the functions of MPK3and MPK6in Phosphate Starvation Responses (PSR). MKK9DD seedlings accumulated less anthocyanin than Vector and MKF9KR seedlings under LP conditions, and Pi content in MKK9DD seedlings was significantly higher than MKK9KR seedlings under either LP or Pi-sufficient (MS) conditions. The results of Pi uptake assay showed that Pi uptake in MKK9DD seedlings was significantly higher than that in MKK9KR seedlings. While LP-induced anthocyanin accumulation, Pi content and Pi uptake rate in MKK9DD/mpk3and MKKSDDlmpk6seedlings recovered to the levels of that in MKK9KR seedlings. Microarray and Q-PCR results showed that the transcriptions of some Pi acquisition related genes were up-regulated in MKK9DD seedlings. These results suggest that MKK9-MPK3/MPK6cascade suppresses LP-induced anthocyanin accumulation and positively regulates Pi uptake in Arabidopsis.WRKY75is a WRKY family transcription factor, which positively regulates Pi uptake. The transcription of WRKY75is up-regulated by MKK9DD induction. Comparing MKK9DD/wrky75with MKKSDD seedlings, loss of WRKY75function in MKK9DD/wrky75seedlings reduced MKK9DD-enhanced Pi accumulation, increased anthocyanin production and reduced the transcription level of Pi acquisition related genes. These data suggest that regulation of PSR by MKK9-MPK3/MPK6requires WRKY75.In conclusion, activation of MKK9-MPK3/MPK6cascade enhances Pi acquisition in Arabidopsis thaliana, and WRKY75is required in this process. It is a great significance for comprehensive understanding of Pi signaling network.