Molecular Mechanism Of Wheat Transcription Factor TaARF9 Involved In Saline-alkali And Oxidative Stress

Saline-alkali stress and oxidative stress are common abiotic stress factors that have a great impact on crop yield.Excavation of new genes that enhance abiotic stress resistance in plants and exploring their mechanisms are one of the important tasks of current crop breeding.ARFs are mainly responsible for regulating the expression of auxin response genes.Current reports on the function of ARFs have focused on their regulation of synthesis and metabolism,transportation,and signal transduction in plants.In recent years,reports have gradually confirmed that ARFs also play an important role in response to stress.Its value in crop breeding is also reflected.Shanrong No.4(SR4)was selected from the wheat introgression lines through asymmetric somatic "hybridization between wheat and tall wheatgrass,which showed significantly stronger salt and alkali tolerance than its parents.And TaARF9,a gene whose expression was significantly up-regulated in SR4 under alkali treatment,was found through transcriptome analysis.We overexpressed TaARF9 in the model plant Arabidopsis for preliminary functional analysis and found that TaARF9 can significantly enhance the ability of resistance to saline-alkali and oxidative stress.Therefore,we speculated that TaARF9 played an important role in the response to abiotic stress.On this basis,we further studied the function and molecular mechanism of TaARF9 in responding to abiotic stress.The main research contents include:1.Functional analysis of TaARF9Multiple sequence alignment results show that TaARF9 is very similar to ARF9 sequences from Brachypodium distachyon,Aegilops tauschii,Sorghum bicolor,Oryza sativa,Setaria italica,and Zea mays,especially with up to 99.85%homology to that of Aegilops tauschii.This shows that TaARF9 is highly conserved in evolution.Conserved domain analysis showed that the protein contains the typical structure of the ARFs family,including the conserved domain of the ARF and AUX/IAA family at the C-terminus,the B3-DNA binding domain near the N-terminus,and the middle domain(MD).Subcellular localization results show that TaARF9 is mainly located in the nucleus,which is consistent with the characteristics of transcription factors.The results of promoter analysis showed that a large number of light-responsive elements and stress-responsive elements existed in the promoter region of the TaARF9,indicating that it may participate in the response process of wheat to abiotic stress.Phenotypic experiments of TaARF9 transgenic Arabidopsis show that the overexpression of TaARF9 can significantly increase the resistance of Arabidopsis to alkali and oxidative stress.In addition,the genes related to ROS production in the overexpression lines including AtRbohC,AtRbohE,and AtRbohG had a raised expression,and further research found that TaARF9 can directly increase the expression of AtRbohC,AtRbohE and other genes to promote ROS synthesis in plants.Then whether the ROS accumulation increases is related to the enhancement of plant resistance needs further investigation.2.Stress resistance analysis of TaARF9Previous studies have reported that TaARF9 has transcriptional activation activity in vitro.In order to find the interaction protein of TaARF9,we screened the self-activation domain of TaARF9 by manual truncation,and found that the transcriptional activation region is located in the two regions of FN(1-123aa)and F2(330-434aa).After the deletion of two regions,the remaining fragment Δ2N was used as bait to screen the yeast twohybrid library.After screening the yeast two-hybrid library and verifying by the BiFC experiment,we determined that TaIAA34 was the interaction protein of TaARF9.The results of homologous multiple sequence alignment showed that the sequence of TaIAA34 is very similar to the sequence of IAA25 of Aegilops tauschii and Hordeum vulgare,with a 97.62%homology,which showed that TaIAA34 is highly conserved in evolution.How TaIAA34 and TaARF9 interact and regulate the function of TaARF9 remains to be further studied.3.Genetic analysis of TaARF9 in wheatIn order to analyze the function of TaARF9 in wheat,we constructed the overexpression vector for wheat genetic transformation and obtained some wheat transgenic lines for alkali tolerance analysis.Contrary to the results of transgenic Arabidopsis,wheat transgenic lines showed obvious sensitive phenotypes after alkali stress treatments.But the specific reason is unclear.We searched for several marker genes of ROS pathway in wheat.After GUS reporter gene experiments,we determined TaRbohF as the target genes regulated by TaARF9 in wheat.TaARF9 could activate its expression by binding to its promoter.But how exactly TaARF9,TaIAA34 and TaRbohF interact with each other requires further exploration.