| Cotton,which produces the most prevalent natural fibers used in the textile industry,is one of the mainstays of the global economy,and allotetraploid upland cotton(Gossypium hirsutum,Gh)accounts for more than 90%of cultivated cotton worldwide.Plant JAZ(Jasmonate ZIM-domain)proteins play versatile roles in multiple aspects of plant development and defense.However,little is known about the functions of JAZ family in allotetraploid upland cotton.In this study,30 non-redundant JAZ genes were identified and their expression and interaction pattern have been identified.One of GhJAZ genes,GhJAZ11-D,has been studied the function in cotton fiber elongation.In addition,the mechanisms of GhXND1 in negative regulation xylem development have been characterized.1.Identification of GhJAZ genes and their expression and phylogenetic relationship in cotton tissues30 non-redundant candidate GhJAZ genes were identified in upland cotton through genome-wide screening,including 15 JAZs from A genome and 15 JAZs from D genome.Phylogenetic analysisindicated that cotton JAZs are more closely related to those of Arabidopsis,and we designated the 30 putative JAZ genes as GhJAZ1a-A/D to GhJAZ12-A/D.GhJAZ proteins were classified into five distinct groups(?,?,?,? and ?),and members in the same group share highly conserved motif structures.Quantitative RT-PCR analysis indicated that GhJAZs display different expression patterns in cotton tissues,and most of them could be induced by Jasmonic(JA).Furthermore,some GhJAZ genes preferentially expressed in cotton ovules and fibers implied they may function in cotton fiber initiation and elongation.2.The interaction pattern of GhJAZ proteinsSubcellular localization assay indicated the GhJAZ proteins localized in the cell nucleus.We have selected nine GhJAZ proteins as candidates to elucidate the interaction pattern of GhJAZs in yeast,and we found GhJAZ1a-A,GhJAZ1b-D,GhJAZ2-D,GhJAZ5-A,GhJAZ11-D and GhJAZ12-D could interact widely with other cotton JAZ proteins,while GhJAZ3-D and GhJAZ7-A only interacted with one or two GhJAZ proteins,implying that the GhJAZ proteins may display isoform selectivity in formation of heterodimers and homodimers.Most of GhJAZs could interact with the core jasmonic acid signalling proteins,GhCOI1,GhMYC2,GhMYC3 and GhNINJA,suggesting that GhJAZ proteins may recruit GhNINJA,and function as repressors of JA signaling pathway by interacting with GhMYC2 and GhMYC3,and they may be targeted for proteasome degradation by SCFCOI1 and activated the JA responses by releasing GhMYC2 and GhMYC3.3.GhJAZ proteins may function in regulating cotton fiber initiationWe have chosen 10 GhJAZ genes to test the transcription level difference between cotton cultivar wild type(Xuzhou142)and its fiberless mutant(fl)in 0 DPA ovules with initiating fiber cells,and 8 of these GhJAZ genes showed the similar variation trend in Xuzhou142 and fl ovules.To further investigate whether GhJAZs could interact with the key transcription factors to control fiber development,we used yeast two-hybrid assay to explore interactions of the 9 GhJAZ proteins with major members of cotton fiber initiation factors including GhMYB2,GhMYB23(GL1 homolog),GhMYB25 and GhMYB25-like,and GhDEL65(GL3 homolog).Y2H and BiFC assay demonstrated the interaction between GhJAZla-A and GhMYB23,GhJAZlb-D and GhMYB23/25-like.These results revealed that GhJAZ proteins may function in regulating cotton fiber initiation.4.GhJAZ11-D function as positive regulator in cotton fiber elongationTo investigate the functional importance of GhJAZll-D in fiber development,constitutive CaMV35S promoter-driven overexpresion and RNA interference(RNAi)constructs were introduced into cotton by Agrobacterium-mediated transformation.SEM assay revealed GhJAZ11-D has not participated in regulating cotton fiber initiation.Phenotypic observation and statistical analysis indicated that GhJAZ11-D overexpression plants produced longer fibers,whereas GhJAZ11-D-silenced lines displayed no difference,compared with this of wild type.Furthermore,fiber strength and micronaire value were also changed in some transgenic cotton lines,compared with those of wild type.In vitro ovule culture assay also indicated GhJAZ11-D positive regulating fiber elongation.GhEIN3,which could interact with GhJAZl 1-D,has been identified by yeast two-hybrid screen.The expression of GhERF genes and GhEXP1 were down-regulation in 10DPA fibers of GhJAZ11-D overexpression plants.The interaction between GhJAZ11-D and GhEIN3 resulted in ET and JA synergistic regulating cotton fiber elongation.5.GhXND1 is involved in negative regulation of plant xylem developmentIn this study,a gene(designated as GhXND1)encoding a NAC transcription factor was identified in cotton.Sequence analysis indicated that GhXND1 gene contains two introns inserted in its open reading frame.GhXNDl protein was localized in the cell nucleus,and displayed the transactivation activity.GhXND1 transcripts were mainly detected in cotyledons,petals,roots,hypocotyls and stems,but little or no signals of GhXNDl expression were found in the other tissues.Ectopic expression of GhXND1 in Arabidopsis resulted in a reduction in the number of xylem vessel cells and cell wall thickness of interfascicular fibers in the transgenic plants,compared with those of wild type.And expression of some cell wall biosynthesis-related genes was down-regulated in the GhXND1 transgenic plants.Collectively,the data presented in this study suggested that GhXND1 may be involved in regulation of plant xylem development. |
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