Age-Dependent Heteroblastic Development Of Leaf Hairs In Arabidopsis

After germination,Arabidopsis thaliana needs to go through a juvenile and an adult phase before entering the reproductive phase.The transition from the juvenile to the adult phase is referred to as vegetative phase transition.Vegetative phase transition is usually accompanied with a series of physiological and biochemical changes,such as leaf shape,leaf size,leaf margin incision,cell size,number of trichomes,cell reproductive capacity and accumulation of secondary metabolites.In Arabidopsis,the juvenile leaves are rounder,with fewer incisions at the leaf margin,lighter color,larger leaf cells and with only adaxial trichomes compared with adult leaves,adult leaves are longer and narrower,with more incisions,darker color,smaller leaf cells than juvenile leaves and have both adaxial and abaxial trichomes.The initiation of abaxial trichomes is a key morphological marker of vegetative phase transition in Arabidopsis.Based on this morphological marker,previous studies have shown that the transition from the juvenile to adult phase is regulated by a conserved miR156-SPL pathway in plants.During vegetative development,the expression of miR156 decreases gradually,and its target genes-SPLs-increase gradually.How the production of abaxial trichomes in Arabidopsis thaliana is regulated by the miR156-SPL pathway is still unclear.We recovered a mutant with an accelerated juvenile-to-adult phase transition phenotype in a forward genetic screen of an EMS-mutagenized population.We studied the underlying mechanism for the precocious phenotype using this mutant.The main results are as follows:1.Genetic analysis showed that the mutant is a dominant mutant,therefore we named it pre1-D?precocious juvenile-to-adult phase transition mutant 1,dominant?.Under short day conditions,pre1-D mutant had a significant earlier production of abaxial trichomes compared with WT.,while there was no significant change in leaf shape.This result suggests that pre1-D only affects leaf epidermal trait development.2.qRT-PCR analysis indicated that pre1-D didn't influence the expression of genes in the miR156-SPL pathway.3.Map-based cloning narrowed the pre1-D mutation down to a region between 10162 Kb and 11051Kb on chromosome 3,the physical distance was 889 Kb.The whole genome resequencing showed that there were 6 mutations in the candidate region,and one of the mutation was located in the non-coding region at 867nt downstream of GL1?a key regulate gene of trichomes initiation?stop codon.4.To prove the phenotype of pre1-D is due to GL1 mutation,we cloned the genomic sequence of GL1from both WT and pre1-D and transformed them into WT to generate gGL1^WT and gGL1^pre1-D transgenic lines.Phenotypic analysis showed that the gGL1^pre1-D transgenic lines produced abaxial trichomes significantly earlier than gGL1^WT transgenic lines.Therefore,the mutation in GL1 was responsible for the pre1-D phenotype.Gene expression analysis indicated that GL1 expression was significantly higher in pre1-D in contrast to that in WT.5.AthaMap analysis suggested that there was a conserved AP2-like transcription factors?TOE1,TOE2,TOE3,AP2,SMZ,SNZ?binding site at 866-875nt downstream of GL1 gene.In pre1-D,the G-to-A substitution was coincidentally located at this conserved binding site.6.AP2-like transcription factors function as negative regulators of abaxial trichomes production,therefore we determined the GL1 expression level in 35S::TOE1 and toe1 toe2.The results showed that GL1 expression was down-regulated in 35S::TOE1,and was up-regulated in toe1 toe2.ChIP?Chromatin immunoprecipitation?analysis showed that TOE1 could directly bind to the AP2-like TFs binding motifs downstream of the GL1 gene,while the binding was abolished in the pre1-D background.ChIP and the gene expression results demonstrated that TOE1 could directly bind to the AP2-like TFs binding motif downstream of GL1 gene to inhibit its expression.7.Genetic analysis showed that pre1-D could significantly rescued the delayed abaxial trichome production in 35S::TOE1 and 35S::MIR156A,this suggests that pre1-D was epistatic to 35S::TOE1 and35S::MIR156A.Based on an age-dependent trichomes initiation model defective mutant pre1-D,we revealed that TOE1which located downstream of miR156-SPL pathway could directly bind to the downstream region of the key trichome initiation gene GL1 and resulted in the age-dependent abaxial trichomes phenotype by inhibiting GL1 expression.In pre1-D mutant,a single-base mutation occurred in the TOE1 binding motif at the downstream region of GL1,so that TOE1 could not bind to inhibit the expression of GL1 and resulted in the phenotype of early abaxial trichomes initiation.Based on the above results,we presented an age-dependent abaxial trichomes initiation mechanism as follows:during the process of vegetative development,miR156 declines gradually to upregulate SPL transcription factors such as SPL9 to activate miR172 expression.The activated miR172 represses AP2-like transcription factors.Thus,the expression of the key trichome initiation gene GL1 is derepressed to promote abaxial trichomes initiation.