Molecular Mechanism Of GhADA2b And Histone Acetylation Mediated By Vesicular Trafficking In Plant Trichomes Development Regulation

Cotton fiber is an important material for energy and textile industry.Improving yield and quality of fiber are the key issue in cotton breeding work.The development of cotton fibers is a complex process that is regulated by kinds of genetic factors,transcription regulation,and environmental signals.With the continuous development of science and technology,more and more genes have been discovered and investigated to be related to fiber initiation and elongation development.However,the epigenetic regulatory mechanism in controling fiber development is not yet well understood.Histone acetylation is one of the most important epigenetic modification.Previous studies have shown that ADA2 b in the subunits of SAGA comlex can regulate the trichomes development by interacting with different factors.However,it is unclear whether GhADA2 b has a regulatory effect on fiber development.Therefore,we analyzed and cloned the GhADA2 b gene and transformed it into Arabidopsis and cotton of ZM24,to explore its function and molecular mechanism in regulating the development of Arabidopsis trichomes and cotton fibers.Meanwhile,we performed RNA-Seq with a novel fiberless mutant ZM24 fl to discover novel lncRNA transcripts related to fiber development,which may improve the epigenetic regulation mechanism of non-coding RNA on cotton fiber development.The main results are as follows:1)GhADA2 b regulates cotton fiber development by promoting histone acetylation.We created transgenic cotton that over-expressed GhADA2 b in Arabidopsis and ZM24 through genetic transformation.Analysis of phenotypes between OE-GhADA2 b Arabidopsis lines and WT showed that GhADA2 b promoted the trichomes initiation including root hairs and leaf epidermal hairs in Arabidopsis.Moreover,the density of root hair promoted by OE-GhADA2 b responded to the treatment of histone(de)acetylation inhibitor MB-3 and TSA.In addition,GhADA2 b can also promote the ratio of four-branched trichomes in the first pair of leave of Arabidopsis.Western blot showed that the overexpresion of GhADA2 b increased the levels of H3K9 ac and H3K14 ac.In cotton,the over-expression of GhADA2 b also promotes an increase in H3K9 ac levels,but a decrease in H3K14 ac levels.Unlike Arabidopsis trichomes,the over-expression of GhADA2 b inhibited fiber initiation and elongation,which is consistent with the results that MB-3 and TSA showed positive and negative effect on fiber intiation from ovule in vitro,respectively,suggesting that appropriate inhibition of histone acetylation can promote fiber initiation and elongation.These results indicate that GhADA2 b,as the core subunit of the SAGA complex,can promote the levele of histone acetylation to regulate the development of Arabidopsis trichomes and cotton fiber,with different functions and different mechanism.2)Analysis of GhADA2 b interaction protein in G.hirsutumTo elucidate the mechanism of GhADA2 b function,the domain analysis divided the GhADA2 b as several truncated peptides,which were then used to explore interaction relationship with GhGCN5 using Y2 H.The results indicate that the domain of myb＿binding in GhADA2 b contains 18 amino acids(aa,376-393)that can interact with GhGCN5 in yeast.In addtion,the N-terminal ZZ＿Zn F to SNAT domains(aa,44-153),or SNAT-L1(aa,104-244)of GhADA2 b could also interact with GhGCN5,suggesting the complexity and diversity of histone acetylation produced by GhADA2b-GhGCN5,which also provide novel clues in analysis of three-dimensional interaction model.To explore the other key members of the GhADA2b-GhGCN5 complex during fiber development,GhADA2 b was used as bait,30 candidate interacting proteins were screened from a fiber tissue c DNA library using yeast library mating.Combining analysis of public transcriptome,prediction of bioinformatics,Y2 H and BiFC experiments,9proteins,which were ubiquitin 60 S ribosome protein L40,pectin lyase(PEL48＿Dt),1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase 2,AGAMOUS,high chlorophyll fluorescence 107,HD-2 histone deacetylase,and two xyloglucan enzymes,and a clathrin light chain,were confirmed to interact with GhADA2 b.Moreover,pectin lyase(PEL48＿Dt)has been reported to be involved in fiber elongation development,and xyloglucans are also involved in cell wall development.These results indicate that GhADA2 b may interact with different factors and participate in multiple pathways,thus have many functions in regulating plant growth and development.3)GhCLC2 affects histone acetylation and trichomes development by promoting the trafficking of GhADA2bOne of the interacting protein,GhCLC2,is a clathrin light chain.It has documented that the double mutant of clc2 clc3 showed poor trichomes and lateral root primordia,which guided us to explore relationship between GhCLC and GhADA2 b.Further BiFC and Pull-down experiment demonstrate stable interaction between GhCLC2 and GhADA2 b.Moreover,GhADA2 b and GhCLC2 showed co-localization signal in nucleus.Furthermore,there is also an interaction between At CLC2 and At ADA2 b in Arabidopsis by Y2 H.Therefore,we cloned the GhCLC2 gene and transformed it into Arabidopsis.Phenotypic analysis showed that overexpression of GhCLC2 in Arabidopsis resulted in shortened hypocotyl and decreased density of root hair.Western blot experiments showed a decrease in histone acetylation H3K9 ac levels in the clc2 clc3 double mutants,while OE-GhCLC2 promoted histone acetylation.These results indicating that GhCLC2 may promote the protein abundance of GhADA2 in the nucleus by affecting the trafficking of GhADA2 b,thereby affecting histone acetylation and regulating trichomes develpmet.4)Identification and epigenetic transcription regulation analysis of lncRNAs related to fiber initiation and development in the fiberless mutant ZM24flWe identified 3288 lncRNAs using ZM24 fl and the wild-type at-2,0 and 5 DPA using RNA-Seq.Analysis of differential expression transcripts showed that 87,27,and201 DELs were identified at the three stages,respectively.Similarly,452,389 and 3986 genes were differentially expressed at-2,0,and 5 DPA stages.GO enrichment analysis of these DEGs shows that they are mainly enriched in jasmonic acid,chitin and fatty acid biosynthesis.etc.pathways.By comparing the common DELs and DEGs between "ZM24＿0 vs fl＿0" and "ZM24＿0 vs ZM24＿-2" using Venn plots,these transcripts may be potential factors for fiber initiation.As a result,a lncRNA MSTRG.2723.1 and 273 DEGs were identified.Further analysis showed that MSTRG.2723.1 targeted 36 DEGs.KEGG analysis showed that these DEGs were enriched in pathways such as fatty acid elongation,starch and sucrose metabolism,and phenylpropanoid biosynthesis.qPCR experiments showed that MSTRG.2723.1,fatty acid elongation pathway genes such as KCS9,KCS19,and some documented genes related to fiber development(eg,Gh MYB25-like)were down-regulated in ZM24 fl.The combined analysis of lncRNA-mRNA showed that MSTRG.2723.1,MSTRG.3390.1,MSTRG.48719.1,and MSTRG.31176.1 also have potential regulatory roles in fiber development.In summary,the accumulation and function of GhADA2 b in the SAGA complex might be affected by clathrin-mediated visicular trafficking thus affecting histone acetylation and regulating trichomes development in plant.Meanwhile,the RNA-Seq analysis of the fiberless mutant ZM24 fl provide novel regulation mechanism in lncRNA regulating fiber initiation development.Our results provide novel clues for exploring epigenetic modification in regulating cotton fiber development.