Functions Of The Florigen GhFT And Receptor 14-3-3 Proteins In Flowering Regulation In Cotton

Flowering transformation is an important process of plant reproduction,which is affected by the external environment and internal development mechanism.FLOWERING LOCUS T(FT)protein,as a "florigen",can form a florigen activation complex(FAC)with b ZIP transcription factor FD and 14-3-3 receptor protein in the nucleus,which affects the expression of downstream target genes to regulate plant flowering time and establishment of plant type.In cotton,the research on function of FT gene is not deep was not deep.However,previous studies on 14-3-3genes were mainly focused on fiber cell initiation and elongation,salt and drought stress signaling,and Verticillium dahlia resistance.Whether the GhFT and 14-3-3 proteins are also part of cotton FAC,the molecular network on regulating flowering in cotton is still unclear.In this study,the functions of GhFT and 14-3-3(GRF)genes in cotton were studied to clarify the relationship between Gh GRF proteins and GhFT,and to analyze part of molecular regulatory networks in the regulation of cotton flowering and plant type,so as to provide direction for regulating flowering time and the vegetative cycle of cotton,and then provide theoretical basis for improving cotton yield and other breeding work.The main contents and results of this study are as follows.1.Studies on the function of GhFT geneRegulatory sequence analysis found that there are five insertion deletion sites in 5.9-kb FT promoters of Gossypium arboreum,G.hirsutum and G.raimondii.Because of repeat sequence(RS)existed in the 1.0-kb promoter,the 1.8-kb FT promoter was truncated to 1.0-kb.Complementation analyses revealed that these constructs could partially rescue the late-flowering phenotype of ft-10 plants.However,the 1.0-kb FT promoters were more effective than that of1.8-kb in inrescuing the late-flowering mutant ft-10.The virus induced gene silencing(VIGS)exprements showed that the flowering time in GhFT-silenced plants were later than that of control plants and the height of plants was significantly higher than that of control.The CRISPR/Cas9 gene editing vectors in the first and second exon of GhFT gene were futher constructed and transgenic lines were selected for sequencing,and the editing types were diverse.The editing plants showed indeterminate growth,apical cluster and serration of tip leaf edge.The expression levels of floral meristem-identity genes,AP1 homologues Gh AP1 and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1(SOC1)homologues Gh SOC1 were significantly decreased,while the expression of antiflorigen TERMINAL FLOWER1(TFL1)homologous Gh TFL1 was increased.Using GhFT as bait protein to screen the interaction proteins by yeast two-hybrid(Y2H).A total of 39 interacting proteins were identified,which were involved in oxidation-reduction,RNA synthesis,transcriptional regulation,protein folding and electron transport,auxin response and various metabolic processes and so on.In addition,the full-length CDS sequence annotated as 14-3-3 protein 6 was cloned and named Gh GRF.Forthermore,the interaction between Gh GRF protein and GhFT in cytoplasm and nucleus was verified by Y2 H and bimolecular fluorescence complementation(Bi FC).2.The function research of 14-3-3(GRF)gene family in cotton17,17,31,and 17 GRF genes were identified form Gossypium herbaceum,G.arboreum,G.hirsutum,and G.raimondii,respectively,by genome-wide analyses.The Gh GRF protein sinteracting with GhFT screened from c DNA library was identified as Gh GRF15.Gene structural,motif composition,synteny,and duplicated gene analyses of the identified GRF genes were comprehensively and systematically investigated,which provided insights into the evolution of this family in cotton.Quantitative real-time PCR confirmed that Gh GRF gene family members exhibited diverse expression patterns in different tissues and stress responses,suggesting the diversity function of Gh GRF gene family.RT-PCR was used to clone the Gh GRF3/6/9/14/15 genes.Y2H and Bi FC experiments showed that the Gh GRF3/6/9/14/15 interacted with GhFT in the cytoplasm and nucleus,while Gh GRF3/6/9/14/15 interacted with Gh FD only in the nucleus.The interaction between Gh GRF3/6/9/14/15,GhFT,and Gh FD protein formed different FACs.In addition,Y2 H indicated that other Gh GRF proteins could also interact with GhFT.VIGS exprements showed that the flowering time in Gh GRF3,–6,–9,and –15-silenced plants were earlier than that of control plants and the expression levels of floral meristem-identity genes,Gh AP1 and Gh SOC1,were up-regulated.Howere,silenceing Gh GRF14,the cotton flowering later and the expression of Gh AP1 and Gh SOC1 were downregulated.Ectopic overexpressions of Gh GRF3,–6,–9,and –15 in Arabidopsis inhibited the expression of At AP1 and At SOC1 to repress flowering,while ectopic overexpressions of Gh GRF14 activated the expression of At AP1 and At SOC1 to promote flowering.In conclusion,GhFT from upland cotton not only affected flowering,but also regulated cotton plant type.The Gh GRF3/6/9/14/15 protein were the adaptor protein of GhFT3/6/9/14/15 and they combined with Gh FD to form GhFT–Gh GRF3/6/9/14/15–Gh FD complex.The function of FAC was affected by its component component Gh GRF,which promoted or inhibited cotton flowering by regulating the expression of AP1 and SOC1.The molecular network of cotton flowering regulation was preliminarily elucidated,which provided reference for changing flowering time of cotton to change the growth cycle of cotton,and also provided theoretical basis and guidance for the cultivation of new plant type of cotton.