Study Of Jasmonates Signaling On Stomatal Development In The Adaxial Epidermis Of Arabidopsis Thaliana

Stomatal development is strictly regulated genetically by a complex pathway which was extensively studied. The well-established stomatal development regulation module (SDD/STOMAGEN/CHAL-TMM/ERL-YODA/MAPK3,6-MAPKK4,5,7,9-SPCH/MUTE/FAMA) contains all the signaling components from the intercellular signal to the membrane receptors, followed by intracellular phosphate cascade system and downstream transcription factors. In addition, several hormones have also been reported to involve in the regulation of stomatal development, such as auxin, brassinoids, abscisic acid and gibberellic acid. This study aimed to study the function of another kind of hormone, Jasmonates, in stomatal development. Cell biological, genetical, molecular biological and biochemistry methods were used to analysis the stoamtal development of JA biosynthesis and signaling mutants to explore how JA regulates stomatal development in the epidermis of cotylen in Arabidopsis. The main results were as follows:1. Exogenous MeJA inhibites the stomatal development in the epidermis of Arabidopsis thaliana cotyledons. Stomatal density of adaxial epidermis of cotylen decreased after treatment with MeJA companied by patterning defect of stomatal cluster formation.2. The potential function site of MeJA on stomatal development module was between upstream ligand-receptor and downstream transcriptional factors. Exogenous MeJA treatment weakened the big stoma cluster phenotype in sdd-1(ligand mutant) and tmm-1(receptor mutant), while had no effect on the stomatal phenotype of spch-1and fama-1(transcriptional factor mutants).3. JA receptor mutant coi1-1and signaling mutants myc234have increased stomatal density and form abnormal stomatal clusters, which indicated that JA have negative effect on stomatal development.4. Stomatal development key stage markers, such as pTMM:TMM-GFP (all the stomatal development stages, stomatal lineage cell marker, expressed in all the stomatal lineage cells), pSPCH:SPCH-GFP (initiation stage of stomatal development, early stem cell marker, expressed in Mother of Meristemoids and Meristemoids), pFAMA:FAMA-GFP (late stage of stomatal development, befor and after symmetric divison of GMC, expressed in GMCs and GCs) and E1728:erYFP (final stage of stomatal development, GC differentiation, expressed in mature GCs) were introduced into coil-1and myc24background. And their changed expression in these background compared to wild type indicate that SPCH may be a key point of JA pathway on stomatal development by the MYC2and its homologes binding with SPCH.5. Biochemical experiments confirmed that MYC2and its two homologes, MYC3and MYC4, can interact with SPCH to form protein complex in Yeast two hybrid (Y2H) and bimolecular fluorescence (BiFC) experiments.6. Genetical relationship analyses of double mutans (JA signaling vs. stomatal development pathway) further proved that MYC2/4acts upstream of SPCH genetically, as deduced by the stomatal phenotype of double mutant coil-Ifama-l and myc2myc4spch-1, which phenocopied fama-1and spch-1respectively.