RNA-seq And Metabolic Analysis Revealed The Mechanism Of Promoted Cotton Fiber Elongation By Flavonoid Eriodictyol

Cotton fiber is an elongated single cell seed trichome that develops from the outer protective protodermal cells of cotton seed coat.Flavonoids are commonly found in nature and play various roles which assist plants in maintaining growth and functions.Flavonoids biosynthesis,secondary metabolic pathway,is vital in modulating various biological functions.The flavonoid biosynthesis pathway modulate pigmentation in flowers is well conserved in plants.However,genetic modification of flavonoid biosynthesis pathway for production of higher quality cotton fiber is not out of scope.During the recent years,molecular mechanisms modulating fiber development have been flawlessly revealed through functional genomics and conventional breeding tools.While in cotton,flavonoids have decisively regulated fiber development.In vivo fiber growth was found suppressed when naringenin（NAR）flavonoid was accumulated in developing cotton fiber（Tan et al.,2013a）.But even in high-quality fiber specie,the flavonoid pathway is active,so we want to know the effect of each flavonoid on fiber development in detail.First,in vitro fiber development was examined through culturing 0 DPA ovules with different concentration of NAR,dihydrokaempferol（DHK）,dihydroquerectin（DHQ）and eriodictyol（ERI）and optimal concentration for promoted in vitro fiber growth was determined.The flavonoid ERI（20 μM）was found to promote fiber growth during fiber initiation and elongation stages.To further dissect transcriptional regulatory networks and molecular mechanism underlying flavonoid based efficient fiber development,whole transcriptome RNA sequencing was performed for samples collected from the control and ERI treatment at fiber initiation（1 DPA: Control₁,ERI₁）,early（5 DPA: Control₅,ERI₅）and late fiber elongation（10 DPA: Control₁0 and ERI₁0）stages.About 190 DEGs were detected at fiber initiation（160 Up and 30 Down with ERI₁/Control₁）,604 at early elongation（245 Up and 359 Down with ERI₅/Control₅）and 2474（1517 Up and 957 Down with ERI₁0/Control₁0）at late fiber elongation stage.Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）analysis revealed that pathways like carbohydrate metabolism（9.67%）,biosynthesis of secondary metabolites（7.22%）and plant hormonal signal transduction（7.09%）were most enriched.The RNA-seq and q RT-PCR results showed that flavonoid pathway genes like CHI and CHS were up-regulated at 1-,5-,10-DPA,while F3 H wad up-regulated at 1-,5-DPA,but at 10-DPA down regulated in response ERI treatment.Then we measure the contents of the flavonoid and IAA,in context to redrawn flavonoid pathway in cotton,our results showed that both ERI and DHQ positively related to fiber development while NAR and DHK could not trigger the in vitro fiber growth.Moreover,ERI was found to positively modulate in vitro fiber development in presence of auxin.Exogenous ERI application positively regulated fiber development through modulating flavonoid metabolic and auxin signaling pathways.Because NAR negatively regulated fiber development but ERI positively,we have got the F3 H transgenic cotton and identified the F3 H function.So we further want to elucidate the functions of F3’H and F3’5’H those are downstream of NAR and ERI.Seven F3’H and four F3’5’H proteins from Swisprot database were used as query to “blastx” against G.raimondii,G.arboreum,G.barbadense and G.hirsutum genomes.Overall,36 candidate genes were identified in Gossypium genomes where 7 and 5 in two diploid cotton（G.arboreum and G.raimondii）,while 11 and 13 were identified in two cultivated tetraploids（G.hirsutum and G.barbadense）.Comprehensive annotation framework predicted that about 7 gene could act as CYP75A（F3’5’H）metabolic functions while remaining 29 were like CYP75 B which were predicted to act as F3’H in four cotton genomes.Phylogenetic analysis showed that genes associated to two different isoform categories were distinctly clustered i.e.,CYP75A（F3’5’H）and CYP75B（F3’H）.Moreover,the candidate genes associated to two CYP75 isoforms categories demonstrated distinct features in two tetraploid cotton genomes（G.hirsutum and G.barbadense）.The expression profiles of putative F3’H and F3’5’H gene families were compared in different vegetative and fiber tissues for two lines.i.e.,YZ1 and Pima 3-79.Both digital expression and q RT-PCR suggested that F3’H and F3’5’H candidate genes were differentially expressed in ovule and fiber tissues between the two cultivated tetraploids.In F3H-OX and F3H-RNAi transgenic lines the expression levels of Gh_A12G2650 / Gh_D12G1798 were the most regulated by F3 H,which should be mainly responsible for metabolic contributions as F3’H in G.hirsutum.Thereafter,F3’H RNAi constructs were genetically transferred to a standard line YZ1 and phenotypic evaluation was performed.Phenotype analysis of F3’H-RNAi showed that downregulating Gh F3’H significantly affected fiber elongation compared to standard line YZ1.