Study Of Novel Functions Of Arabidopsis AtSPX1through Multi-dimension Omics Data-mining, And The Role Of OsSPX1Involved In Oxidative Stresses In Rice Seedlings

The SPX (SYG1/PHO81/XPR1) domain with180amino acid residues is located at the N-terminal of the proteins. Many proteins possessing the SPX domain have been suggested to be involved in the phosphate-related signal transduction pathways and regulatory pathways. At present, the studies of AtSPX1gene mostly focus onthe regulation of phosphorus (Pi) signal. Pi can induce expression of AtSPX1gene. AtSPX1is involved in Pi homeostasis in Arabidopsis by inhibiting the activity of AtPHR1in a Pi-dependent manner. OsSPX1is the highest sequence similarity homologous gene of AtSPX1in Rice, OsSPX1cooperating with OsSPX2was reported to interact with OsPHR2to suppress Pi starvation. The SPX1/PHR1module links Pi sensing and signaling. With rapid development of bioinformatics, especially throughdata-mining of high-throughput omics data, we couldpredict the old genes with novel functions, which could be validated by bench studies.In the first part of this study, we mainly conducted multidimensional omics data-mining methodto study the possible novel functions of AtSPX1gene in Arabidopsis. Our work confirmed the function of AtSPX1gene under low Pi treatment. The AtSPX1protein-protein interaction network was constructed through data-mining of the public proteomics data. Based on analysis of protein TGA1which interacts with AtSPX1, we predicted that AtSPX1may involve in SA-mediated immune signaling pathways. We successfully verified our hypothesis through SA treatment experiment. The atspx1mutant was not sensitive to SA treatment. In addition, we found that IAA11was interacted with AtSPX1, thus we predicted that AtSPX1may involve in the auxin-regulated lateral root development. In phenotype tests of various plant materials under low Pi and auxin or auxin analogue treatment, atspxl mutant was less sensitive to WT, and the lateral roots numbers of atspxl mutant was more than those in WT under low Pi condition, but the phenotype of atspxl mutant was not significant under auxin or auxin analogue treatment, indicating that AtSPX1might involve in auxin-regulated lateral root development in a Pi-dependent manner. Furthermore, ATH1GeneChip analysis was conducted to study the differential gene expression profiles in atspx1mutant and wild-type plants. According to GO annotation analysis of the differentially expressed genes, lots of genes were involved in plant defense responses including biotic stress, system immune resistance, andmetabolic pathway, etc. The genes involved in biotic stress signal transduction pathways and related metabolism pathways, including WRKY transcription factor genes, PR proteins,signal transducer,and proteins related to cell wall and secondary metabolites. The GeneChip results were further validated by real-time RT-PCR.In the second part of this study, my research mainly focused on the possible function of AtSPX1involved in circadian. Through data-mining and bench validation, the expression of AtSPX1gene was regulated by circadian rhythmic fluctuations and the total Pi content measurement of the over ground parts in Arabidopsis was also showed the rhythmic fluctuations. To elucidate the possible mechanism about the AtSPX1gene on circadian rhythm, we applied RNA-seq to do transcriptomics analysis for the wild-type plants and atspxl mutant materials through timeseries experiments. AtSPX1gene was higher expression at daytime and lower expression at night. Lots of genes involved in immune-related signaling pathways differentially expressed in atspxl mutant vs. wild-type plants. We also conducted ChIP-seq experimentwith anti-H3K27ac antibody to further study the molecular mechanism of AtSPX1on the epigenetic level. Our results showed that H3K27ac modified levels of many genes were significantly changed over times and some genes with changed H3K27ac modified level in atspxl mutant were also involved in SA-mediated immune signaling pathways mediated.Through combining omics data-mining analysis, we predicted that AtSPX1gene might be involved in SA-mediated immune signaling pathway and auxin-regulated lateral root development, and further verified these hypotheses with bench work.Moreover, ourstudies also suggested that AtSPX1gene function might depend on circadian rhythm. The AtSPX1genewith properly rhythm might be necessary for its functions related to immune defense responses and Pi homeostasis, etc.The last of my study is that OsSPXl affecting anti-oxidative ability in Rice. Our previous work showed that constitutive over-expression of OsSPX1in tobacco and Arabidopsis plants improved cold tolerance. OsSPX1-antisense transgenic lines were sensitivity to cold. Rice whole-genome GeneChip analysis showed that some oxidative-stress marker genes were significantly down-regulated in the antisense of OsSPX1compared to wild-type and OsSPX1-sense transgenic lines. Our study indicated that OsSPX1-antisense transgenic lines displayed a reduced ability to oxidative stress. The hypothesis was verified with the fact that OsSPX1-antisense transgenic lines were sensitive to MV treatment. Compared to wild-type, peroxidase activity was higher in OsSPX1-antisense transgenic lines, which may be was a way to make up its reduced ability to eliminate of ROS. As for cold treatment and oxidative stress, when GSH solution was added to the MV, the sensitive phenotype of OsSPX1-antisense transgenic plants under MV treatment was better, and the above result also verified that OsSPX1-antisense transgenic plants sensitivity to cold and oxidative stress was due to reducing ability to eliminate of ROS. This part of my study provided theoretical support to how to deal with the relationship between the fertilizer and pesticide scientifically in agricultural production.In summary, we conducted multi-dimension omics approaches, including proteomics, transcriptomics, and epigenomics, together with network and GO analysis, to study the possible novel functions of SPX1in Arabidopsis and rice. Through bench validation, we successfully identified that AtSPX1is possibly involved in SA-mediated signaling transduction pathway and auxin-regulated lateral root development. We also found that the function of AtSPX1gene may depend on circadian. In addition, we also found that down-regulation of OsSPXl causes high sensitivity to oxidative stresses in rice seedlings. Our system biology study showed in this study offered a referential stradegy for the genes function data mining.