| Served as the first waterproof barrier between plant and the outside world,the cuticle layer on plant surface protects plant from the internal and external environment stress,and limits non-stomatal transpiration.Wax biosynthesis is regulated by various extrinsic environment changes to enhance plant adaptation and survival.Therefore,it is of great significance to explore the regulation mechanism of cuticle for improving plant stress resistance and crop yield.In this study,we identified two MYB-SHAQKYF transcription factors,MYS1 and MYS2,which are co-expressed with wax biosynthetic genes.Using CRISPR/Cas9 system,we constructed double mutants of MYS1 and MYS2.The wax loads were decreased and the epidermal permeability was significantly increased in the double mutants.Whereas the single mutants showed the same phenotype as wild type.Overexpression of MYS1 or MYS2 decreased chlorophyll leaching rate and significantly elevated leaf wax loads,suggesting that MYS1 and MYS2 function redundantly in positively regulating wax biosynthesis and drought tolerance.Transcription activity assays confirmed that MYS1 and MYS2 were transcription repressors containing EAR motifs.Among all detected wax synthetic genes,the expression of DEWAX,a negative regulator of wax biosynthesis,was significantly increased and the expression of alkane synthetase CER1(ECERIFERUM 1),a direct target of DEWAX,was significantly decreased in mys1 c mys2 double mutants.Together with further molecular and biochemical experiments,such as dual-LUC assays,EMSAs and Ch IP-q PCR,demonstrated that MYS1 and MYS2 indirectly affect CER1 expression through directly transcriptional regulation on DEWAX,thus promoting wax biosynthesis.At the genetic level,MYS1 and MYS2 act upstream of the DEWAX-SPL9 module to regulate CER1 expression in wax biosynthesis.Interestingly,MYS1 and MYS2 showed the opposite expression patterns to DEWAX under the diurnal and light/dark changes.DEWAX expression was significantly higher in mys1 c mys2 double mutant under light/dark changes,demonstrating that MYS1 and MYS2 are involved in diurnal wax biosynthesis by regulating DEWAX expression.In addition,we unexpectedly discovered the abnormal stomatal morphology of mys1 c mys2 plants.Through cell biological and plant physiological experiments,such as microscopic observation,immunofluorescence staining and stomatal conductance measurement,we found that the stomata of mys1 c mys2 plants exhibited lower density,larger volume,abnormal pore formation,smaller pore size,and impaired movement,which might result from the elevated methylesterified pectin in guard cell wall.These results indicated that MYS1 and MYS2 influence the structure of guard cell wall via altering pectin methylesterification,thus regulating stomatal morphology and movement.Plants loss water from both the cuticle and the stomata.In drought stress experiments,mys1 c mys2 double mutant showed drought-sensitivity,while MYS1 or MYS2 overexpressed plants were drought-tolerant.Knockdown of DEWAX or overexpression of SPL9 restores the drought-sensitivity of mys1 c mys2 double mutant.However,function research of stomata indicated that mys1 c mys2 plants exihibted less stomatal water loss than Col-0,which further confirmed that wax defect-controlled nonstomatal water loss plays a major role in drought sensitivity of mys1 c mys2 plants.Our findings elucidate the roles of two noval transcription regulatory components in Arabidopsis leaf wax biosynthesis,and shed light on the mechanism of wax accumulations during light/dark cycles.Meanwhile,we also explored their function in stomatal development and stomatal movement,which is significanct for research in stomatal function from the aspect of guard cell wall.It can better explain how plant leaves coordinate the function of stomata and the cuticle to improve plant adaptability and survival.Moreover,our findings also suggest the potential of transcription factors for the improvement of plant stress tolerance through modulation of cuticle and stomata. |
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