Screening Of CLE Polypeptides Determining Cell Differentiation In Arabidopsis Early Anther

During the development of early anthers in higher flowering plants,a variety of leucinerich repeat receptor-like protein kinases are involved in the regulation of the division and differentiation of archesporial cells into the endothecium,middle layer,tapetum and microspore mother cells.It has been known that BAM1/2 are important regulators of cell fate determination in early anther development,and CIKs act as the co-receptor of BAM1/2 to mediate the signaling pathway.As a receptor on the cell membrane,BAM1/2 sense the signal from the extracellular ligand to activate the activity of the intracellular kinase domain,and finally transduce the signal through phosphorylation.However,the extracellular ligand sensed by BAM1/2 to control cell fate determination during early anther development remains unknown.In this study,in order to screen for the extracellular ligands sensed by BAM1/2,we cloned the CDS sequences of all CLEs expressed during reproductive development and further constructed transgenic plants harboring pUBQ10::CLE in wild type.Phenotypic analysis results indicated that overexpression of genes in subfamilies of CLE12 and CLE16 produced thinner and longer anthers.Alexander staining results showed that the pollen sacs of these transgenic plants cannot produce active pollen grains.Opposite to the loss-of-function phenotype of the bam1/2 null mutant,semi-thin sections showed that both groups of transgenic plants generated more anther wall cell layers.Because the signaling pathway mediated by BAM1/2 is completely blocked and no endothecium can be generated,the bam1/2 null mutant cannot be used for ligand screening.Luckily,cik1/2/3 is a weak mutant in which the CIK-mediated signaling pathway is only partially impaired and some endothecium cells can be generated.Therefore,cik1/2/3 mutants were used in this study as the background to screen for the potential ligand perceived by BAM1/2 to regulate anther cell fate.All CLEs possibly involved in reproductive development were overexpressed in cik1/2/3 mutants.The results showed that overexpression of CLE12 and its homologous genes can specifically restore the anther withdrawal phenotype of cik1/2/3,but increased expression of CLE16 and its homologous genes cannot.Because of defective cell differentiation of anther wall,the microspore mother cells dramatically expand and are surrounded only by the epidermis in cik1/2/3 early anthers.These defects can be partially restored by overexpression of CLE12,and the microspore mother cells are surrounded by three layers of anther wall cell although no middle layer can be generated in the transgenic plants.Alexander staining further indicated that overexpression of CLE12 and its homologous genes in cik1/2/3 can produce more active pollen grains.Our data indicated that increased expression of CLE12 can only specifically regulate archesporial cell division controlled by the interaction between CIKs and BAM1/2,but cannot regulate the formation of middle layer controlled by CIKs and RPK2.To further explore the biological functions of CLE12 and its homologues,the CRISPR/Cas9 technique was employed to create null mutants of these CLE genes.Possibly because of gene redundancy,anther development of single cle mutants did not exhibit any obvious defects.We will create high-order mutants of these CLE genes to further determine whether they are ligands of BAM1/2 during anther development.