Molecular Mechanism Of Apple SUMO E3 Ligase MdSIZ1 Regulates Cuticular Wax Biosynthesis By SUMOylating Transcription Factor MdMYB30

Plant cuticle represents a crucial adaptation for plant survival in terrestrial environments.Plant cuticles are composed of hydrophobic cuticular waxes and cutin.Cuticular wax plays an important role in regulating epidermal permeability,preventing the loss of non-stomatal water,preventing organ fusion,and resisting biological and abiotic stresses.In fleshy fruit,cuticular wax has effect on the quality of the apple fruit,which are closely associated with shelf life,post-harvest storage,mechanical damage and the glossiness of the fruit.The formation of epidermal wax is strictly controlled by growth,development,and environmental conditions,which makes plants adapt to the changing environment during the whole life cycle.In recent years,studies have shown that the biosynthesis of cuticular wax is regulated by the level of transcription,post-transcription and translation.However,at the level of post-translation,there are few studies on the biosynthesis and regulation of cuticular wax.As ubiquitin,SUMO is attached to protein targets through sequential reactions catalyzed by E1 SUMO-activating enzyme,E2 SUMO-conjugating enzyme,and E3 SUMO ligase,thus affecting the transcription activity,interaction,stability,and localization of target proteins.In apple,the SUMO E3 ligase MdSIZ1 is involved in multiple biological processes,including anthocyanin accumulation,iron homeostasis,and the formation of lateral roots,however,at the post-translation level whether MdSIZ1-mediated SUMOylation regulates cuticular wax biosynthesis remains unclear.To further investigate the molecular mechanism of cuticular wax biosynthesis in apple,apple seedlings and apple fruits were used as materials to reveal the molecular mechanism by which MdSIZ1-mediated post-translational modification of MdMYB30 regulated the formation of cuticular wax via molecular biology techniques and genetic transformation.The results are as follows:1.MdSIZ1 positively regulates SUMO conjugates in apple and Arabidopsis.Protein SUMOylation examine revealed that the levels of protein SUMOylation,including SUMO conjugates and free SUMO,were higher in MdSIZ1-OE plants than in WT plants.The the SUMO conjugate profile in a total protein immunoblot,an increase in SUMO conjugates was observed in 35S::MdSIZ1-Myc ectopically expressed Arabidopsis,and the SUMO conjustes were slightly weaker in siz1-2 than in Col plants.This results indicates that MdSIZ1 promotes the total protein SUMOylation.2.MdSIZ1 positively regulates cuticular wax biosynthesis and alters leaf cuticular membrane permeability.Scanning electron microscopy(SEM)and transmission electron microscopy(TEM)analysis of the leaf cuticular wax crystals,thickness of the cuticle,and thickness of epidermal cells wall were higher on MdSIZ1-OE leaves than on WT leaves.The GC-MS analysis of total wax of apple seedlings showed that MdSIZ1 increased the total wax content and promote the accumulation of apple leaf cuticular wax by regulating the composition changes of alkanes,alcohol,fatty acid,triterpene,and ester.Water droplet attachment and toluidine blue(TB)staining experiment indicated the cuticular membrane permeability of MdSIZ1-OE leaves decreases compared with WT.Overall,MdSIZ1 might alter wax accumulation by promoting SUMOylation to affected leaf cuticle permeability.3.MdSCE1 physically interacts with MdMYB30.The wax synthesis related factor MdMYB30 interacting with SUMO E2 conjugating enzyme MdSCE1 was screened by Y2 H.Pull-down,Bi FC and LUC complementary assays verify the interaction between MdMYB30 and MdSCE1.Analyzing the biological function of MdMYB30,it was found that MdMYB30 positive regulation cuticular wax load.4.MdSIZ1 mediates the SUMOylation of MdMYB30.In vitro and in vivo SUMOylation assays indicated that MdSIZ1 SUMOylated MdMYB30.The protein degradation assay of MdMYB30 indicating that MdSIZ1-mediated SUMOylation increased the protein stability of MdMYB30 and blocked its degradation via the 26 S proteasome pathway.5.To demonstrate the MdSIZ1 positively regulates cuticle wax biosynthesis in an MdMYB30-dependent manner,a viral vector-based transformation method in apple fruit peel was performed.In addition,siz1-2,35S::MdSIZ1-Myc,35S::MdMYB30-GFP,35S::MdMYB30-GFP/siz1-2 and 35S::MdSIZ1-Myc/35S::MdMYB30-GFP transgenic Arabidopsis were obtain to analyzed their SUMO conjugate profile and wax deposition.The decrease in SUMO conjugates and wax deposition in siz1-2 mutant was restored to near the level observed in 35S::MdMYB30-GFP by ectopic expression of MdMYB30 in the siz1-2mutant.SUMO conjugate levels and wax accumulatin were higher in 35S::MdMYB30-GFP and35S::MdSIZ1-Myc than in Col and lower in 35S::MdMYB30-GFP and 35S::MdSIZ1-Myc than in 35S::MdSIZ1-Myc/35S:: MdMYB30-GFP.These results suggested that MdSIZ1 positively regulates cuticle wax biosynthesis in an MdMYB30-dependent manner and At SIZ1 upstream of MdMYB30 and modulates wax accumulation by SUMOylating MdMYB30.6.MdMYB30 bind to the promoter of MdKCS1 gene to activate its expression,promoting wax accumulation.Chromatin immunoprecipitation(Ch IP)-q PCR,Y1 H,and electrophoretic mobility shift assay(EMSA)suggested that MdKCS1 is a direct target of MdMYB30.Dualluciferase reporter assay revealed that the expression of wax-related structural gene MdKCS1 was directly activated.The above results indicate that SUMO E3 ligase MdSIZ1-mediated sumoylation of MdMYB30 positively regulates cuticular wax biosynthesis in apple.The MdSIZ1-MdMYB30-MdKCS1 module positively regulates cuticular wax biosynthesis in apples.This study provide theoretical foundation for the regulation networks of cuticular wax biosynthesis and candidate genes for genetic improvement of apple fruit quality and resistance.