Identification And Functional Analysis Of GhIm And COBRA Like(COBL) Genes Related To Secondary Wall Biosynthesis In Cotton

Cotton is a major source of natural textile fiber.Cotton fibers originate from the epidermis of ovules,and are highly elongated and thickened single-celled trichomes.There exists more than 90%crystalline cellulose in mature fibers.Thus,screening and study of genes related to cotton fiber development is not only beneficial to clarify the mechanism of cellulose synthesis,but also can provide some reference for improving cotton quality through modern molecular breeding technology.In this paper,we identified the candidate gene GhlmA,related to immature fiber mutant(im)with secondary wall development defect and explored the molecular mechanism.Moveover,we detected a highly differentially expressed gene between im mutant and TM-1,belonging to COBRA-Like(COBL)genes.Using the whole genome data,we identified COBL genes in cotton and screened for COBL9(A subgenome)related to secondary wall formation and conducted functional analysis by transgenic approach.1.Identification and functional analysis of cotton immature fiber(im)mutantMature cotton fiber exhibited fluffy state after the seeds capsule dehisced with fiber cells quickly dehydrated;while immature fiber(im)mutant displayed the tight lock bolls phenotype.Here,we also found that compared to TM-1,the NIL of im mutant,the flowering time of im mutant was also delayed and a portion of pollens failed to pollinate.To mapping im gene,we identified a polymorphic site between im mutant and three materials(TM-1,CSIL028 and 14005)by map-based cloning,call SNPs/InDels and sequence amplification.To confirm the results above,we developed polymorphic marker amplifying the InDel locus in three genetic populations including 342,1837 and 737 F2:3 or F2 individuals from the cross of CSIL028 × im,14005 × im and TM-1 × im.The genotyping of polymorphic marker was consistent with the fiber phenotypes and no cross-overs were detected between the genotyping and phenotypes in these three segregation population.Finally,im gene was identified as Gh_A03G0489,and named as GhImA gene with its homologs Gh_D03G1048(GhImD).Sequence analysis found that the full-length of GhIm gene(Both GhImA and GhImD)was 3072 bp and without introns existing.In TM-1,there were 95 SNP loci between GhImA and GhImD,of which contained 49 non-synonymous sites.Both GhImA and GhImD genes displayed constitutive expression patterns in different tissues of cotton.Domain analyze found that GhIm had 24 P(PPR)motifs and one S(Short)motif,belonging to the P subgroup of PPR proteins.In the im mutant,GhImA gene had a 22 bp-deletion,leading to early termination after 184 amino acids.Examining GhIm expression patterns in TM-1 and im mutant fibers found that expression levels of GhImA in TM-1 increase rapidly in 22 DPA fibers,during which the transcripts of GhImA in im mutant were lower than that in TM-1.While in 25 DPA fibers,the expression levels of GhImD was higher in im mutant than that in TM-1,resulted almost the same amount of the sum levels of GhIm in TM-1 and im mutant.Subcellular localization analysis showed that GhIm protein targeted into mitochondria.To explore the downstream mitochondria gene of GhIm protein,RNA-Seq data of TM-1 and im were mapped to mitochondria genome and to call SNPs/InDels.Meanwhile,with the aid of semi-quantitative technology,find mitochondrial genes with different transcriptional levels.Among 35 genes related to functional proteins,only seven were predicted to have SNPs/InDels between TM-1 and im mutant.Sequence amplify and analysis showed that only cox2 mature transcript was dramatically changed in im mutant:Except integral cox2 mRNA,various types of cox2 transcripts were detected in im mutant,which were shorter than in TM-1.RNA-Protein pull down assay found that GhIm protein could bind stably to the cox2 mRNA.The mitochondrial cox2 gene encoded subunit ? of respiratory electron transport chain Complex ?(cytochrome c oxidase).In im mutant,the activity of complex ? was reduced seriously.Gene Ontology analysis indicated that differential expression genes related to respiratory metabolism were enriched between TM-1 and im mutant:Genes involved in detoxification of copper ion and alternative oxidases were activated,ATP synthase were greatly inhibited in im mutant.At the same time,lots of DEGs involved in cell wall/cellulose synthesis was also enriched.Defective respiratory metabolism caused ROSburst and further triggerred biological processes like stress response and hormone balance.The heterological tetraploid cotton TM-1 contains four copies of Ghlm gene(AADD)and im mutant contained two copies(aaDD).To clarify the roles of different copies of GhIm protein,we obtained GhIm-sileneced plants(aadd)by virus-induced gene silencing(VIGS).The TM-1 plants could grow up from seed germination,seedling,flowering and ripening.Im mutant grew up to vegetative growth stage as TM-1,but displayed delayed flowering time,poor self-fertility and delayed fiber development,immature fiber phenotypes.When Ghlm gene was completely silenced,the plants appeared to stop growing.Meanwhile,the activity of Complex IV,transcription of ATP synthase also reduced with the decreasing of Ghlm copies,also with the alternative oxidases were activated.Correspondingly,respiration-induced ROS also increased when Ghlm gene copies decreased.Therefore,we speculated that GhIm protein could regulate plant growth and development,and displayed the dosage-dependent effects.2.Identification of COBRA-Like(COBL)Genes in cotton and transgenic verification for candidate geneUsing the whole genome data of cotton,we identified 19,18 and 33 COBL genes in G.raimondii(Diploid D-genome),G.arboreum(Diploid A-genome)and G.hirsutum acc.TM-1(Tetraploid cotton),respectively.Evolutionary analysis indicated that COBLs were descendants of an ancient duplication that occurred even before the separation of monocots and dicots.COBL members had a conserved N-terminal signal peptide,carbohydratebinding domain(CBM),CCVS domain and C-terminal GPI hydrophobic domain.These genes could be divided into two groups and members in each group showed their own obvious characteristics:COBL genes in Group I had more introns;While COBL genes in Group II had less intron and shared with an unknown motif and a longer C-terminal GPI tail.Meanwhile,COBL genes showed different expression patterns at different stages of fiber development.In addition to GhCOBL3,GhCOBL8,GhCOBLIO and GhCOBL16 expressed at all stages and maintained higher accumulation levels,the other GhCOBLs displayed relatively division of roles in the life cycle of fiber formation.GhCOBL9 and GhCOBL13 had higher expression accumulation in fibers during the stages of secondary cell wall biosynthesis and displayed a significantly high coexpression pattern with GhCESA4,GhCESA7 and GhCESA8,which were involved in cellulose formation of secondary cell wall.GhCOBL9 was co-located with the QTLs related to fiber quality traits:fiber strength(FS),fiber micronaire(FM),fiber elongation(FE)and fiber uniformity(FU);GhCOBL13 was co-located with the QTLs related to fiber length(FL),FS,FM and FE.Sequence analysis showed that the D sub-genome of GhCOBL9 was terminated prematurely in TM-1 but normal in Hai7124.By exploring SNP marker,we detected polymorphism genotyping of GhCOBL9 in upland cotton natural populations.Correlation analysis revealed that genotype with normal GhCOBL9Dt locus had significantly positive correlations with several fiber quality traits than that with prematurely terminated locus.Besides,both of these two COBLs had higher expression levels in Hai7124 which showed the superior fiber quality than in TM-1.Hence,we could conclude that GhCOBL9 and GhCOBL13 played positive roles in fiber formation and fiber quality traits.To verify the roles of GhCOBL9 and GhCOBL13,we overexpressed GhCOBL9(A subgenome)in Arabidopsis Col-0(WT)and the growth potential of transgenic plants was superior to that of WT.In detail,the lengths of root and hypocotyl of transgenic plants were longer that of WT.Further analysis found that:GhCOBL9 could promote cell elongation mainly by promoting cellular polarity elongation.Besides,the lengths of siliques and plants were longer in transgenic plants than those in WT.Determination of crystalline cellulose content showed the cellulose contents in transgenic plants were higher than those in WT.Staining analysis of microstructure found that increasing cellulose contents was not only in the vascular fibers,but in the interstitial fibers and marrow,and the cell wall was significantly thicker.During the harvest period,the numbers of siliques on inflorecence stem,cauline stem and rosette stem in over-expression plants were much more than in WT.GhCOBL9 was transferred into the mutant of Arabidopsis homologous gene AtCOBL4.Although the plant height and cellulose content of transgenic plants were superior to Atcobl4 mutant,the vessel cells still showed a certain degree of collapse and of inflorecence stem was brittle like Atcobl4 mutant.At the same time,the other two homologous genes of AtCOBL4,GhCOBL4 and GhCOBL13 were also transferred into Atcobl4 mutant and failed to recover the phenotype of Atcobl4 mutant.This suggested that AtCOBL4 homologous genes have diverse function in cotton and Arabidopsis.