Molecular Mechanism Of Vegetative Growth Control By A Gibberellin Biosynthesis Inhibitor DPC In Cotton (Gossypium Hirsutum L.)

Mepiquat chloride （N, N-dimethylpiperidinium chloride, DPC） is the widely used plant growth retardant to control excessive vegetative growth in cotton production. Several literatures hypothesize that DPC inhibits the activities of ent-copalyl diphosphate synthase （CPS） and ent-kaurene synthase （KS） involved in gibberellin early biosynthesis, leading to the growth inhibition of plant. In the study, cotton （Gossypium hirsutum L.） seedlings at the third leaf stage were used to investigate the roles of genes encoding gibberellin （GA） metabolism and DELLA repressor protein in the growth inhibition by DPC. In order to discover if there were other action mechanisms of DPC, we screened an Arabidopsis activation tagging mutant library to obtain DPC-insensitive mutants. Mutant181was obtained and analyzed. The main results were as follow:1. DPC reduced endogenous GA levels through downregulating the expressions of GA early and late biosynthesis genes, which caused the growth inhibition by decreasing the expression levels of GhEXP and GhXTH2. The expression of GhCPS and GhKS in apical bud, leaf and internode were inhibited by DPC. The expression levels of both genes in the internode were downregulated for a longer time compared with that in apical bud and leaf. In addition, DPC treatment significantly decreased the expression levels of Gh20oxs and GhGA3ox in consistent with the changes of GhCPS and GhKS in time. Feedback upregulations of GhCPS, GhKS, Gh20oxs and GhGA3ox were observed in the internode from3to24h after DPC application. The expressions of GhGA2oxs and DELLA genes （GhGAI4a and GhGAI4b） were feedforward and feedback-downregulated by DPC from2to10d after treatment, respectively. DPC decreased endogenous GA content. The cell size of internode in the transverse and longitudinal sections was decreased by DPC through downregulating the expression levels of GhEXP and GhXTH2, leading to a reduction in diameter and length of internode. Exogenous GA3could completely relieve the growth inhibition caused by DPC.2. Four DPC-insensitive mutants were screened from Arabidopsis aetivation tagging library. Among them, mutant181was further studied. Sequencing TAIL-PCR and BLAST on NCBI revealed that two T-DNA sequences were found to be inserted side by side into the3’U’TR of At1g01070on chromosome1. The expression levels of At1g01070and At1g01060were enhanced in mutant181. The increase in expression level of At1g01070was two-fold of that of At1g01060. Atlg01070was impired in SALK₁47481mutant. However, no obvious difference in response to DPC was observed between wild type and SALK₁47481mutant, suggesting the protein encoded by At1g01070could be functional redundancy. Compared with wild type, mutant181showed late-flowering phenotype and had higher plant height during the late growth period. Overexpression vector of At1g01070were successfully constructed and was used to transform Arabidopsis. The promoter of At1g01070transcriptionally fused to the GUS reporter gene was constructed, which was used to transform Arabidopsis. At1g01070has two alternatively spliced forms At1g01070.1and At1g01070.2. At1g01070.1is the main mRNA of At1g01070. At1g01070.1was mainly expressed in hypocotyls and roots. Analysis of the hydrophobicity profile of At1g.01070.1and At1g01070.2predicted ten and eight potential alpha helical transmembrane-spanning domains, respectively.