The Molecular Reglualtory Mechanism Of Vegetative Phase Change By KANADI1 In Arabidopsis

The juvenile-to-adult phase change(vegetative phase change) is one of the pivotal developmental processes in plants. It regulates the onset of reproductive development, plant biomass, fruit yield, plant architecture, biotic and abiotic response, and secondary metabolite production. Recent studies indicated that the mi R156-SPLs pathway functions as a conserved and master regulatory pathway to regulate vegetative phase change in plants; however, the function of upstream and downstream components of mi R156-SPLs pathway in vegetative phase change still awaits to be explored.KANADI1(KAN1) has been shown to function as an abaxial polarity gene to promote abaxial cell fate and to suppress adaxial cell fate in plants. KAN1 loss of function mutant(kan1-12) exhibits an accelerated vegetative phase change phenotype in Arabidopsis thaliana; therefore, KAN1 has dual roles in polarity development and vegetative phase change. However, as a polarity gene, how KAN1 is involved in vegetative phase change remains unknown. To explore the function of KAN1 in vegetative phase change, Experiments including phenotype characterization, gene expression analyses, genetic analysis, GUS histochemical staining, gene ectopic expression analysis, and Ch IP experiments were conducted in this study, and the major results are as follows: 1. kan1-12 mutant exhibited an accelerated vegetative phase change phenotype with early abaxial trichome initiation on the abaxial side of leaves, and it is also early flowering; 2. q RT-PCR showed that SPL9 and its target gene, mi R172, were up-regulated in kan1-12 mutant; 3. GUS histochemical staining of KAN1(p KAN1::GUS) and SPL9(p SPL9::r SPL9-GUS) reporter lines indicated that KAN1 transcripts exhibited an abaxial expression pattern, whereas SPL9 exhibited an adaxial expression pattern; kan1-12 mutant resulted in higher and extopic expression of SPL9 transcripts on the abaxial side of leaves; 4. Genetic analysis showed that mi R156 over-expression or SPL9 loss-of-function mutant could partially rescue the kan1-12 mutant phenotype, together with the fact that SPL9 is upregulated in kan1-12, indicating that SPL9 acts downstream of KAN1; 5. Phenotypic characterization of transgenic lines ectopically expressing SPL9, KAN1, and mi R156 showed that p KAN1::r SPL9 exhibited an accelerated vegetative phase change phenotype, whereasp SPL9::KAN1 and p KAN1::mi R156 transgenic lines exhibited a delayed vegetative phase change phenotype, these results suggested that the repression of SPL9 by KAN1 on the abaxial side of leaves is required for normal vegetative phase change in plants; 6. In order to understand if KAN1 regulates SPL9 by direct binding, a p KAN1::3×Flag-KAN1/kan1-12 transgenic rescue line was generated, and p KAN1::3×Flag-KAN1 construct could partially or fully rescue the phenotype of kan1-12, indicating that the fusion 3×Flag-KAN1 protein functioned normally in plants. Ch IP analysis using FLAG antibody showed that KAN1 directly binds to the-469 bp site within the promoter region of SPL9.In summary, our study indicated that KAN1 could directly bind to the promoter region of SPL9 to repress the expression of SPL9 on the abaxial side of leaveas. A model was proposed based on our results: During the juvenile stage, SPL9 is transcriptionally and post-transcriptionally repressed by KAN1 on the abaxial side of leaves and by mi R156 on both sides of leaves. As plants age, mi R156 expression declines, and SPL9 is released from the posttranscriptional repression of mi R156 resulting in the expansion of SPL9 expression to the abaxial side of leaves to promote the formation of abaxial trichomes to initiate adult phase. Our study discovered a link between leaf polarity development and vegetative phase change in plants, and provide a starting point to study the relationship between leaf development and developmental timing in plants, and provide a base about regulation of plant biomass, fruit yield, plant architecture, biotic and abiotic response, and secondary metabolite production through mi R156-SPLs genes and polarity genes.