Molecular Mechanisms Of SERKs In Regulating Arabidopsis Integument Development

As the seed precursor,the ovule is essential for plant propagation.In angiosperms,the female gametophyte（or embryo sac）is produced within the ovule and the subsequent double fertilization also occurs in it.The ovule contains the nucellus,integuments,and funiculus.The nucellus is the most important structure where embryo sac forms and develops into a multicellular structure consisting of an egg cell,a central cell,two synergid cells,and three antipodal cells.Female gametes,including the egg cell and the central cell,participate in double fertilization with assistance of the synergid cells.The funiculus mounts an ovule to the placenta,physically supporting the ovule and transmitting nutrients into the ovule.The integuments,including two layers of outer integument and two layers of inner integument,emerge sequentially from the integument primordium at the early stages of ovule development.After emergence,the integuments grow quickly and enwrap the embryo sac gradually,providing physical protection and chemical nutrition for the embryo sac.Therefore,normal morphogenesis of the integuments is key to ensure the development of the embryo sac.However,the molecular mechanisms regulating integument development have not been well understood.For example,what signals regulate the development process of the integuments?What receptors perceive these signals?How are the signals perceived by the receptors transuced to regulate the development process of the integuments?In angiosperms,there are numerous receptor-like protein kinases（RLKs）that can sense the signals from the surrounding cells and the environment to regulate various developmental processes and defence responses.Among them,the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE（SERK）subfamily members have been intensively investigated.SERK subfamily consists of five members that work usually as co-receptors and form complexes with other RLKs to perceive many signaling molecules in regulating plant growth,development,and stress responses,such as stomatal patterning,anther development,embryogenesis,and innate immunity.In this study,we found that SERKs play critical roles during integument development in Arabidopsis.The serk1/2/3 triple mutant showed arrested integuments and abnormal embryo sacs.Two independent triple mutants,serk1/2/3 and serk1/2/3-2,showed similar ovule defects.In addition,the expression of either SERK1,SERK2,or SERK3 complemented the ovule defects of serk1/2/3,indicating that the defects in serk1/2/3 ovules were caused by loss-of-fucntion of these three SERK genes.Expression analysis showed that SERKs were expressed in the integuments and nucellus epidermis.Similar to serk1/2/3,the er erl1/2^/+mutant also generated defective integuments and abnormal embryo sac.ERECTA family（ERf）genes,including ER,ERL1,and ERL2,were also expressed in the integuments and nucellus epidermis.Ovules of the sextuple mutant serk1/2/3 er erl1/2 showed defects similar to those of er erl1 erl1 and serk1/2/3.Moreover,results of yeast two-hybrid analyses,bimolecular fluorescence complementation,and co-immunoprecipitation assays demonstrated that SERKs interact with ERf,suggesting that SERKs and ER may function together to regulate integument development.In addition,ovules of both brassinosteroid（BR）receptor mutant bri1 brl1/3 and BR biosynthetic mutant cpd also displayed abnormal integuments and exposed nucellus.The sextuple mutant bri1 brl1/3 er erl1/2^/+showed ovule defects similar to serk1/2/3.These results suggested that SERKs may regulate ovule development together with two other groups of RLKs,namely ERf and BRI1,BRL1,and BRL3,respectively.Previous studies showed that a group of cysteine-rich peptides encoded by EPFL family genes regulate stomatal development as ligands of ERf.Our results show that,EPFL6 overexpression or treatment with mature peptide MEPFL4/6 could enhance the interaction between SERKs and ER,indicating that EPFL6 may act as a ligand of the ER–SERK complex to regulate integument development.In addition,EPFL2/3/4/5/6were expressed specifically in the region where the inner integuments initiated and extended,while EPFL1 was expressed in the outer integuments.The sextuple mutant epfl1/2/3/4/5/6 created by the CRISPR/Cas9 technology showed ovule defects similar to both of serk1/2/3 and er erl1/2.Taken together,this study demonstrated that ERf–SERK-mediated EPFL signaling and BRI1/BRL1/3–SERK-mediated BR signaling orchestrated the development of the female gametophyte and the surrounding sporophytic integuments.