The Molecular Mechanism Of Mepiquat Chloride Inhibiting Internode Elongation Of Cotton Seedlings

Cotton(Gossypium hirsutum L.)is an important textile crop and edible oil crop.Mepiquat Chloride(MC)can effectively inhibit the growth of cotton main stems,reduce the plant height,and produce compact plant architecture through shortening the internodes and reducing the number of plant internodes;however the molecular mechanism of MC inhibiting cotton growth remains unknown.Exploring the metabolic pathways and understanding the molecular mechanism of MC regulation on the growth of cotton internodes could improve the present MC control technology,and provide a theoretical basis for extending MC application on other crops.In this study,cotton cultivar Zhong Mian 49 was used to study the effect of MC application on seedlings.Plant phenotypic characteristics,cell structure and number were detected in the 2nd internod of cotton seedling after MC treatment.In addition,RNA-Seq and LC-MS were employed to detect transcriptional responsive and phytohormone changes to MC treatment,respectively.The main conclusions are as follows:1.MC affects the morphology of cotton seedlings.MC treatment shortened cotton seedling height by inhibitingr the internode elongation.Compared with control plants,plant height was significantly decreased by 19.7%,29.5%,35.8%,and 36.6% at the 4th day,6th day,8th day,and 10 day after MC treatment(P<0.01),respectively.The length of first,second,third,and fourth internodes(from stem bottom to top)was significantly decreased by 46.0%,55.1%,51.8%,and 83.3% at ten days after MC treatment(P<0.01),respectively.The elongation of the second internode was more severerly inhibited than that of the first internode after MC application.The length of the second internode was reduced by 48.7%,59.7%,and 57.4% at the 6th day,8th day,and 10 th day after MC treatment(P<0.01),respectively.2.Using light microscope slices,cell length and cell number along the vertical axes of the 2nd internode were examined at the 10 th day after MC treatment.The results show that the size of longitudinal internode cells in cortex and pith with MC treatment was significantly smaller than that of control.The longitudinal cell length of MC-treated internode was 67.15 ?m(internode length / cell length)and significantly reduced by 27% compared with control(91.54 ?m)(P<0.01).The cell number in the internode with MC treatment was 447,significantly decreased by 21% compared with the control(564)(P<0.01).These data indicated that MC inhibited the elongation of internode through restraining both cell elongation and cell division.3.Transcriptional response to MC in the 2nd internode of cotton seedlings was analyzed using RNA-seq.Compared with controls(0 h),1378 differentially expressed genes(DEGs)(645 upregulated and 733 downregulated),2751 DEGs(1086 upregulated and 1665 downregulated),and 3459 DEGs(1479 upregulated and 1980 downregulated)were identified at 48 h,72 h and 96 h after MC treatment,respectively.DEGs number increased with the time after MC treatment.Twenty DEGs were random selected for q RT-PCR and the quantitative PCR results were consistent with that of sequencing data,indicated the reliability of the transcriptome sequencing results.We further checked the functions of DEGs by GO enrichment analysis,674,786,and 543 GO terms were categorized from the pairwise comparisons between 48 vs 0h,72 vs 0h,and 96 vs 0h,respectively.The most significant GO terms were “single-organism process”,“response to stimulus”,“binding”,and “chloroplast part”.For the KEGG pathway enrichment analysis,89,97,and 108 pathways were categorized from the pairwise comparisons between 48 vs 0h,72 vs 0h,and 96 vs 0h,respectively.The pathways with the highest number of DEGs were “Metabolic pathways”,followed by “Biosynthesis of secondary metabolites”,and “Plant hormone signal transduction” for each comparison.The annotation of DEGs showed that the DEGs were main related to phytohormone metabolism and signal transduction(155 DEGs),transcription factors(273 DEGs),secondary metabolites(Lignin and Flavonoid)(54 DEGs),cell cycle and division(58 DEGs),and cell wall(75 DEGs).The results indicated that MC inhibited cotton internode elongation via altering multiple metabolic pathways.4.LC-MS was used to measure the contents of auxin(IAA),cytokinin(CTK),gibberellin(GA)and brassinosteroid(BR)in the 2nd internode at the 6th day after MC treatment.The results showed that MC treatment significantly reduced the contents of GA1,GA3,GA4,IAA,and BL by 78%,57%,29%,46%,and 36%(P<0.01),respectively;while,the CTK(trans-zeatin)content increased by 95%(P <0.01).Together,these results indicated that the MC-mediated phytohormones changes,like GA,IAA,BR,and CTK,are responsible for the growth inhibition of MC on cotton internode.