Function Prediction And Validation For Arabidopsis PHO1;H10 Gene Based On Multi-dimensional Omics Data Mining

The Arabidopsis PH01 gene family consists of 11 members,named PH01 and PHO1;H1-H10,respectively.All the protein sequences of this family contain SPX and EXS domains.It was reported that the expression of PHO1;H10 gene can be induced by various biotic and abiotic stresses in Arabidopsis,including wounding,drought,cold,salt,pathogen-infection,etc.,as well as abscisic acid(ABA)and 12-oxy-phytodienoic acid(OPDA,the precursor of jasmonic acid).Through analysis of many transcriptome data in Arabidopsis,the PHO1;H10 gene was identified to be involved in stress responses,plant hormone metabolism and signal transduction pathways,such as water stress(i.e.osmotic stress,drought stress,and salt stress),UV-B treatment,ABA and salicylic acid(SA)signal transduction,etc.However,the studies about the molecular regulation mechanism of PHO1;H10 gene expression and the experimental function verification are very limited.Thus in this study,I conducted bioinformatics approaches for multi-dimensional omics data mining to predict gene function of PHO1;H10 and possible molecular mechanism.Furthermore,molecular biology and genetics methods were applied to verify the predicted functions of PHO1;H10.Firstly,through motif analysis of the promoter region of PHO1;H10 gene in the Plant cis-acting regulatory DNA elements database,many cis-elements were predicted,including ABA response related cis-element ABRE and drought response related cis-elements ACGTATERD1,etc.Then we used our in-house developed plant chromatin state database(PCSD)to explore the chromatin state and transcription factor(TF)binding cites around PHO1;H10.Several TFs were shown with significant binding signals in the promoter region of PHO1;H10,such as ABI5,HY5,and SPCH,etc.ABI5 is a key transcription factor in the ABA signaling pathway,with binding to the ABRE cis-element on PHO1;H10 promoter.HY5 plays essential roles in light signal transduction pathway.HY5 is involved in the regulation of ABI5 gene expression,suggesting that PHO1;H10 gene may be regulated by both light and ABA signals.SPCH(SPEECHLESS)is a key transcription factor regulating stomatal development.It is well known that gene expression is closely related to the regulation of transcription factors.By using the Arabidopsis Electronic Fluorescent Pictograph Browser(Arabidopsis eFP Browser),we found the gene expression of PHO1;H10 was induced under ABA treatment,especially in guard cells.In addition,the expression of PHO1;H10 gene in senescent leaves and sepals of flowers was significantly higher than that in other tissues.In the meanwhile,co-expression network of PHO1;H 10 gene was constructed.Through gene ontology analysis for the genes co-regulated together with PHO1;H10 in the network,some ABA response and drought response related GO terms were significantly enriched,which indicated that PHOl;H10 gene is involved in ABA and drought signal transduction pathways.Secondly,to validate the predicted functions of PHO1;H10 gene,the mutants of PHO1;H 10 gene were screened,several complementary and overexpression transgenic lines were constructed.During seed germination,it was found that the mutant was more sensitive to the inhibition of ABA,which suggested that PHO1;H10 gene might play a negative role in the inhibition of seed germination by ABA.With drought treatment in the soil,it was found that,under drought condition,the survival rates of the over-expression and complementary materials as well as wild type plants were higher than those of mutant plants,which suggested that PHO1;H10 gene might be related to drought tolerance.Through investigation of epidermis cells in cotyledon,the results showed that,the number of stomata in the abaxial leaf surface for the overexpression and complementary lines was significantly decreased compared with that for wild type and mutants,but the change in the adaxial leaf surface was not obvious,which indicated that PHO1;H10 may inhibit stomatal formation.Further infrared thermal imaging system was used to detect surface temperature of plants under one week drought treatment.The results showed that the leaf surface temperature of mutant was significantly lower than those of wild type and over-expressed line.To study the regulation pathway of PHO1;H10 gene involved and the Arabidopsis ATH1 microarray was conducted to study the differential gene expression between wild type and mutant under polyethylene glycol(PEG)treatment and normal condition.Under normal condition,178 down-regulated genes and 324 up-regulated genes were found in mutant plants.Under PEG treatment for 6 hours,104 genes were down-regulated and only 24 genes were up-regulated in mutant plants.GO enrichment analysis of these down-regulated genes in the mutant showed that these genes were significantly enriched in response to stress and in response to hormone signals,which may explain the defense response ability of the mutant was decreased compared with the wild type.In summary,by using multi-dimensional omics data analysis platform,we did data-mining and predicted the functions of Arabidopsis PHO1;H10 gene,and explored the transcriptional regulation mechanism of PHO1;H10.Through analyzing the phenotypes and gene expression profiles,PHO1;H10 gene was discovered to be involved in the inhibition of seed germination by ABA,drought stress resistance and stomatal development,etc.In addition,the research approaches of systems biology developed in this study might provide a reference for other gene functional analysis.