Functional Analysis Of Rice B-bZIP Subfamily Members OsbZIP81 And OsbZIP84

Rice(Oryza sativa)is one of the most important cereals for human and regarded as one of the model plants for monocotyledonous and cereal crop research.The application of Agrobacterium-mediated rice transformation has been greatly developed,which provides a strong technical foundation for rice research and genetic improvement.However,the efficiency for some rice varieties or large fragments transformation is greatly limited.In addition,only a few members of the B subgroup of the bZIP TFs family were reported to be involved in plant growth and development,disease and cold resistance,while the function of the other members were rarely reported.In this study,we cloned and studied a new gene OsbZIP81 which is a member of the B-bZIP subgroup,and further studied the OsbZIP84 gene which is another B-bZIP subgroup member and was studied by a former member of our group.Finally,we found that OsbZIP81.1 may positively regulate JA levels by directly targetting the genes in JA signaling and metabolism pathway,and OsbZIP81.2 can interact with Agrobacterium virulence protein VirE2.Furthermore,we found a new cis-element,designated as OVRE for OsVIP1 response element,which can be bound by both OsbZIP81 and OsbZIP84.In addition,we investigated a potential mechanism that OsbZIP84 regulates rice height.The main results are summarized as follows:We found OsbZIP81 is an alternative splicing gene,and successfully cloned its three transcripts by RT-PCR.The three alternative transcripts were designated as OsbZIP81.1,OsbZIP81.2 and OsbZIP81.3.OsbZIP81.1 is a typical bZIP transcription factor,while OsbZIP81.2 is not.Both OsbZIP81.1 and OsbZIP81.2 could be induced by Agrobacterium tumefaciens(EHA105),MeJA and PEG6000.Through yeast two-hybrid(Y2H)assay between three types of VirE2 and all B-bZIP members,we found only OsbZIP81.2 could interact with three types of VirE2.By using OsbZIP81.1 overexpression rice plants and chromatin immunoprecipitation and high throughput sequencing(ChIP-Seq),we found 14,245 peaks with 8,169 potential target genes and a new bZIP TFs binding DNA element,OVRE,which was verified by EMSA assay.At the same time,using RNA-Seq assay,we found 5,143 and 5,002 significantly differentially expressed genes(DEGs)for OsbZIP81.1ox and OsbZIP81.2ox compared with wild type ZH11,respectively.In addition,combining the ChIP-Seq data and OsbZIP81.1ox DEGs,we found 1,332 target genes of OsbZIP81.1.With KEGG analysis of these target genes,the enriched pathways are mainly in starch and sucrose metabolism,pyruvate metabolism,nicotinic acid and nicotinamide metabolism,flavonoid biosynthesis and ?-linolenic acid metabolism,and so on.In OsbZIP81.1 overexpression transgenic rice plant,JA and SA were up-regulated,while ABA was down-regulated.By yeast two-hybrid library screening and immunoprecipitation-mass spectrometry(IP-MS)assays,we identified four OsbZIP81 interacting proteins,including two PR proteins,RSOsPR10 and PBZ1/OsPR10,one MADS TF,OsMADS1,and a putative 14-3-3 protein,designated as Os11g34450.Furthermore,we successfully constructed and obtained OsbZIP81.1 and OsbZIP81.2 overexpression transgenic tobacco plants,which laid a material foundation for the future study of Agrobacterium-mediated transformation.OsbZIP84 is a typical bZIP transcription factor.In the plants with down-regulated expression of OsbZIP84,the plant height became shorter that caused by the shortened cell length.Additionally,OsbZIP84 was regulated by exogenous bioactive gibberellin and gibberellin inhibitor.We believe that the growth of rice could be regulated by regulating the synthesis of OsGA20ox2 in gibberellin metabolism pathway and the content of intracellular gibberellin.Based on RDSA assay,we found a cis-element which is exactly the same as OsbZIP81.1 response cis-element OVRE.In addition,we obtained homozygous transgenic knockout plants of OsbZIP84 by CRISPR/Cas9 gene knockout technology,which provide us the materials for the further study of OsbZIP84.In conclusion,we found that OsbZIP81.1 may positively regulate JA levels by directly targetting the genes in JA signaling and metabolism pathway in rice,and only OsbZIP81.2 can interact with Agrobacterium virulence protein VirE2.Furthermore,we identified an OsbZIP81.1 and OsbZIP84 response cis-element,OVRE,which is a novel bZIP TFs response element.At last,we found that OsbZIP84 may regulate the height of rice through directly regulating the expression of OsGA20ox2.