Studies On The Functions Of Two BHLH/HLH Transcription Factors And Their Regulation Mechanism In Fiber Development Of Cotton(Gossypium Hirsutum)

Basic helix-loop-helix/helix-loop-helix(bHLH/HLH)transcription factors play important roles in plant development.Many reports have suggested that bHLH/HLH proteins participate in brassinosteroid(BR)hormone signaling pathways to regulate cell elongation.Cotton fibers are single-cells and derived from seed surface.To explore the roles of bHLH/HLH proteins in cotton fiber development progress by modulating BR signaling pathway,we performed a systematic analysis of the bHLH/HLH gene family in upland cotton(Gossypium hirsutum)genome in this study.Among these bHLH/HLH genes,an atypical HLH transcription factor GhFP2 was identified as a BR responding gene.The functions of GhFP2 in regulating cotton fiber elongation were studied.And GhFP2's interacting protein GhACE1,which is a typical bHLH protein,was identified and studied.The molecular mechanisms of the two bHLH/HLH proteins antagonistically regulating fiber elongation was illustrated.The main findings are showed as follows:1.Characterization of bHLH/HLH genes that are involved in brassinosteroid(BR)signaling in fiber development of cotton(Gossypium hirsutum)In this study,we identified 437 bHLH/HLH genes in upland cotton(G.hirsutum)genome.Phylogenetic analysis revealed that GhbHLH/HLH proteins were split into twenty six clades in the tree.These GhbHLH/HLH genes are distributed unevenly in different chromosomes of cotton genome.Segmental duplication is the predominant gene duplication event and the major contributor for amplification of GhbHLH/HLH gene family.The GhbHLH/HLHs within the same group have conserved exon/intron pattern and their encoding proteins show conserved motif composition.Based on transcriptome data,we identified 77 GhbHLH/HLH candidates that are expressed at relatively high levels in cotton fibers.As adding exogenous BR(brassinolide,BL)or brassinazole(Brz,a BR biosynthesis inhibitor),expressions of these GhbHLH/HLH genes were up-regulated or down-regulated in cotton fibers.2.GhFP2 was negatively regulated by GhBZR1 via BR signalingBZR1 is known as a key transcription factor in BR signaling pathway.Our Previous study indicated that GhBZR1 proteins participate in the regulation of fiber elongation by modulating BR signaling.In Arabidopsis BZR1 could directly inhibit the expression of HLH genes to promote plants growth.The promoter sequences of GhHLH/HLH genes was isolated from cotton genome,and one BRRE(BR-response element)and three E-boxes(CANNTG)were found at the GhFP2 promoter sequence.Yeast one-hybrid assay and ChIP-qPCR analysis suggested that ChBZR1 could directly bind to the promoters of GhFP2.Additionally,dual-luciferase assay system was employed to examine how GhBZRl influences the expression of GhFP2.The level of the luciferase activity controlled by GhFP2 promoter was reduced remarkably when GhBZRl was expressed.Thus,GhBZRl is supposed to directly bind to the GhFP2 promoter via the BRRE and E-box to repress the expression of GhFP2.3.GhFP2 negatively regulates fiber elongation and may act as a transcriptional repressorGhFP2 overexpression and RNA interference(RNAi)vectors were constructed under the control of fiber specific promoter GhRDL1p,and transformed into cotton by Agrobacterium-mediated cotton transformation.Quantitative RT-PCR analysis showed that the expression values of GhFP2 in 9 DPA fibers of GhFP2 overexpression cotton were 2-to 5-fold higher than that in wild type,and its expression levels in 9 DPA fibers of GhFP2 RNAi cotton were 2-to 8-fold lower than that in wild type.Phenotypic observations indicated that the general morphology and total plant height were nearly similar.The most prominent feature was that GhFP2 overexpression plants exhibited markedly shorter fibers that those in controls,whereas GhFP2 RNAi lines displayed significantly longer fibers than those in controls.The results of scanning electron microscopy revealed that fiber initiation(density)in GhFP2 transgenic cotton showed no significant difference compared with that in wild type.However,fiber early elongation in GhFP2 overexpression plants was retarded severely,whereas was promoted in GhFP2 RNAi plants.In vitro ovule culture experiments also confirmed that up-regulation of GhFP2 suppressed fiber elongation,but down-regulation of GhFP2 promoted fiber elongation.Furthermore,fiber cell elongation was partially restored on GhFP2 overexpression ovules by adding 1 ?M BL to the culture medium.Collectively,above results suggested that GhFP2 plays negative roles in cotton fiber elongation via modulating BR signaling.The results of transcriptomic analysis in 9 DPA fibers of GhFP2 overexpression lines and wild type showed that the expression level of fiber elongation related genes were reduced in GhFP2 overexpression fibers.GhFP2 protein was localized in the cell nucleus,but GhFP2 without containing DNA binding domain lacked the activity of transcriptional activation.The above results indicated that GhFP2 can't directly regulate expressions of downstream genes and may interfere with the transcriptional activation activity of other positive transcription factors through physical interaction to suppress fiber cell elongation.4.GhFP2 interacted with GhACE1GhACE1 was identified as a GhFP2 interacted protein by yeast two-hybrid(Y2H)screens(a homologous proteins of AtACE1).We confirmed the interaction by conducting bimolecular fluorescence complementation(BiFC)assay and Co-IP assay in vivo.GhACE1 protein were localized in the cell nucleus,and GhACE1 had the activity of transcriptional activation.Additionally,GhACE1 was preferentially expressed in elongating fibers.These results demonstrated that GhACE1 may act as a positive transcription factors in regulating fiber elongation.5.GhACE1 promotes fiber elongation by directly activating the transcription of fiber elongation related genes,but GhFP2 impairs these activationsWe prepared GhACEl overexpression vector under the control of fiber specific promoter GhRDL1p,and the vector was transformed into cotton by Agrobacterium-mediated cotton transformation.Quantitative RT-PCR analysis showed the expression values of GhACEl in 9 DPA fibers of GhACEl overexpression cotton were 1.5-to 3-fold higher than that in wild type.In vitro ovule culture experiments showed that GhACEl overexpression results in longer fibers.Besides,mature fibers of the GhACEl overexpression transgenic lines were still remarkably longer than those of controls.These results suggested that GhACE1 positively regulates cotton fiber elongation.The expression levels of GhEXP4/8,GhKCS1/2,GhPIP2;7,and GhCYP187A3 were significantly increased in fibers of GhACE1 overexpression cotton plants.Bioinformatics analysis showed that"E-box" cis-acting elements existed in the GhPIP2;7 and GhEXP8 promoters.EMSA and ChIP-qPCR assay revealed that GhACE1 could directly bind to the "E-box" elements in promoter regions of GhPIP2;7 and GhEXP8.Dual-luciferase assay confirmed that GhACE1 could activate the transcriptional activity of GhPIP2;7 and GhEXP8 promoters.However,this activation was impaired observably when GhFP2 was coexpressed with GhACE1.These data indicated that GhACE1 may form homodimers to activate expressions of GhEXP8 and GhPIP2;7,but GhFP2 may repress GhACE1's functions through forming heterodimers with GhACE1.