| The plant cuticle is a hydrophobic structure covering the surface of cell wall,mainly composed of long-chain fatty acids.As a physical barrier between plant and the surrounding environment,the cuticle plays a critical role to restrict transpiration water loss,defend against potential pathogens,protect plants against UV radiation,and regulate organogenesis.During organogenesis,the cuticle can prevent neighboring organs and tissues from adhering together.In the early stages of embryo development,a discontinuous nascent cuticle covers the surface of embryo,and part of the syncytial endosperm adhered to surface of the embryo with other spreads to the surrounding inner integument,which drives seed coat expansion and determines the final seed size.When the embryo develops to the heart stage,the intact embryonic cuticle covers the surface of embryo as a diffusion barrier to prevent the embryo from adhering with the adjacent cellularized endosperm and maternal tissues.Actually,the embryo itself plays critical roles in regulating embryonic cuticle integrity through a bidirectional molecular dialogue with the endosperm.In the early stages of embryo development,the sulfated TWISTED SEED1(TWS1)peptide precursor generated in the embryo can diffuse to the surrounding endosperm where it is cleaved by ABNORMAL LEAF SHAPE1(ALE1)and becomes an active mature form.The activated mature TWS1 returns to the embryo where it is perceived by leucine-rich repeats(LRRs)receptor-like protein kinases(RLKs)GASSHO1(GSO1)and GSO2 to start the downstream signaling and guide the formation of an intact embryonic cuticle.However,the early signaling events after TWS1 is perceived by GSO1/2 are still unknown.In this study,we found that serk1/2/3 mutants exhibit defective cotyledon epidermis,such as adhered cotyledons,torn epidermal cells,protruded mesophyll cells,and defective embryos abnormally adhered to the endosperm.Based on toluidine blue and fluorochrome fluorol yellow 088(FY)staining and transmission electron microscopy results,it was concluded that the cuticle integrity in the serk1/2/3 mutant was affected.Then,the quadruple mutants ale1 serk1/2/3 and zou serk1/2/3 and the quintuple mutant gso1/2 serk1/2/3 were generated by genetic crossing.These highorder mutants also produced shrivelled seeds with short cotyledons adhering to the maternal tissues and highly permeable to toluidine blue,which is very similar to the serk1/2/3 mutant,suggesting that SERKs probably act in the same genetic pathway with ZOU,ALE1,and GSO1/2 to regulate embryonic cuticle integrity.Furthermore,mb SUS yeast two-hybrid,Bi FC,in vitro pull-down,and co-immunoprecipitation results showed that SERKs can interact with GSO1/2,and the in vivo interactions can be enhanced significantly when TWS1 was exogenously applied,suggesting that SERKs function as coreceptors of GSO1/2 mediating TWS1 signaling to regulate the embryonic cuticle integrity.Finally,the SERK kinases can phosphorylate the GSO1 inactive kinase in an in vitro kinase assay.In vivo phosphorylation assay results indicated that the phosphorylation levels of GSO1/2 were drastically elevated when treated with TWS1,but the phosphorylation levels of GSO1 were greatly attenuated in the serk1/2/3 mutant background,suggesting that the phosphorylation of GSO1 upon TWS1 treatment relies on SERKs.When SERKs were co-expressed with GSO1/2,the phosphorylation levels increased dramatically after the treatment of TWS1,demonstrating that the phosphorylation of SERKs responding to TWS1 relies on GSO1/2.Therefore,we provided evidence to demonstrate that the phosphorylation of SERKs and GSO1/2responding to the sulfated peptide TWS1 in planta depend on each other.In summary,this study utilized methods and techniques of cell biology,genetics,and biochemistry demonstrated that SERKs function with ZOU,ALE1,and GSO1/2 in a common pathway,and act as coreceptors of GSO1/2 in responding to TWS1,thereby to activate downstream signaling which monitors the integrity of the embryonic cuticle. |
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