A Novel RR-TZF Gene,GhTZF2,Regulates Cotton Fiber Cell Development

Cotton,one of the most popular economic crops around the world,provides a desirable resource for the global textile industry.Upland cotton(Gossypium hirsutum L.)is the most widely cultivated cotton cultivar as well as the most important subject of considerable studies in both cotton molecular biology and cotton genetics.Exploring key regulators will contribute to understanding of the molecular mechanisms during cotton fiber development.In eukaryotes,gene expression is regulated at multiple levels,including the transcriptional,pre-and post-transcriptional,translational as well as epigenetic levels.Histone modifications,as a key part of epigenetic regulation,have been reported to play an important role during plant developments.Hence,analyzing wholegenome histone modifications of cotton fibers will help revealing the changes of gene expression patterns during fiber development.In this study,global distribution patterns of H3K4me3,H3K27 ac,H3K4me1,H3K36me3 and H3K27me3 were investigated in Upland cotton Xu142(wild-type and fuzzless-lintless mutant,WT and fl)fiber cells at 0 DPA(days post anthesis)via Ch IPseq(Chromatin Immunoprecipitation followed by sequencing)analysis.Through a comprehensive analysis,we obtained the following major discoveries:1.Ch IP-seq analysis of various histone modifications in allotetraploid cotton(AADD)revealed significant sub-genomic bias.About 7% to 37% of homologous gene pairs showed obvious At-or Dt-biased marker pattern on histone modification levels.Also,this epigenomic bias was found positively correlated with transcriptional bias.2.Comparative analysis of H3K4me3 contents found a total of 119 genes with WT specific modification pattern,while 80 genes displayed fl specific pattern.H3K4me3 modification analysis,genome sequencing and PCR amplification experiments pointed out that,a ～23 kb DNA fragment deletion on Chromosome A13 of the fl mutant was responsible for the significant difference on H3K4me3 level between WT and fl.3.Systematic genome sequence analysis uncovered that,as a result of the ～23 kb deletion,two genes were lost in the fl mutant.Ghi＿A13G12816 which is responsible for protein sorting and transportation,while Ghi＿A13G12821,which encodes a RR-TZF protein containing an Arg-rich domain(R-rich domain,RR)with two tandem CCCH zinc finger domains(TZF).Ch IP-seq and transcriptome analysis indicated that Ghi＿A13G12821 expressed at a much higher level than Ghi＿A13G12816,so that we decided to take Ghi＿A13G12821 as the candidate gene for further research.It was named GhTZF2 because of its homology with the reported cotton gene GhTZF1.It is highly expressed during the early cotton fiber developmental stages(from –2 DPA to 10 DPA)as confirmed by q RT-PCR and western blot assays.4.GhMORF8(Multiple Organellar RNA Editing Factor 8)was identified to interact with GhTZF2 through yeast two-hybrid(Y2H)screening.LCI(Luciferase Complementation Imaging)and Co-IP(Co-Immunoprecipitation)assays further confirmed direct interactions between them,suggesting that GhTZF2 might be involved in RNA editing in organelles,including mitochondria and plastid,by forming protein complexes with GhMORF8.5.A total of 27 mutant lines of GhTZF2(GhTZF2＿CR)were obtained by using the CRISPR-Cas9 system.Two homozygotic GhTZF2-CR lines were obtained by screening T3 generation individual plants.Compared with WT,the thickness of mature fiber cell wall in GhTZF2-CR lines was reduced by more than 50%.In both these lines,the expression level of 424 transcripts was significantly altered,with about 40% of these genes contained an ARE motif in their 3’ UTR region.Sub-cellular localization studies showed that GhTZF2 was found mainly in cell of granule shaped organelles,which further indicated that GhTZF2 might regulate the m RNA turnover in cytoplasmic foci(Processing Bodies and Stress Granules,PBs and SGs).Taken together,whole-genome distributions and the sub-genome-biased pattern of histone modifications,including H3K4me3,H3K27 ac,H3K4me1,H3K36me3 and H3K27me3 were investigated in Upland cotton.GhTZF2 was first identified by comparative analysis between WT and fl on H3K4me3 level and was proposed to regulate cotton fiber cell development through interacting with GhMORF8 or regulating cytoplasmic foci-mediated m RNA turnover.This study laid the foundation for exploring the role of RR-TZF zinc finger proteins during cotton fiber development.It not only helps to advance our understanding of the molecular mechanisms during cotton fiber developing processes,but also provides valuable information and gene resources for improving cotton breeding.