Cytological and Transcriptomic Analyses Reveal Important Roles of CLE19 in Pollen Exine Formation

The CLAVATA3/ESR-RELATED (CLE) peptide signals are required for cell-cell communication in several plant growth and developmental processes. However, little is known regarding the possible functions of the CLEs in the anther. Here, we show that a T-DNA insertional mutant, and dominant-negative (DN) and overexpression (OX) transgenic plants of the CLE19 gene, exhibited significantly reduced anther size and pollen grain number and abnormal pollen wall formation in Arabidopsis (Arabidopsis thaliana). Interestingly, the DN-CLE19 pollen grains showed a more extensively covered surface, but CLE19-OX pollen exine exhibited clearly missing connections in the network and lacked separation between areas that normally form the lacunae. With a combination of cell biological, genetic, and transcriptomic analyses on cle19, DN-CLE19, and CLE19-OX plants, we demonstrated that CLE19-OX plants produced highly vacuolated and swollen aborted microspores (ams)-like tapetal cells, lacked lipidic tapetosomes and elaioplasts, and had abnormal pollen primexine without obvious accumulation of sporopollenin precursors. Moreover, CLE19 is important for the normal expression of more than 1,000 genes, including the transcription factor gene AMS, 280 AMS-downstream genes, and other genes involved in pollen coat and pollen exine formation, lipid metabolism, pollen germination, and hormone metabolism. In addition, the DN-CLE19(+/+) ams(-/-) plants exhibited the ams anther phenotype and ams(+/-) partially suppressed the DN-CLE19 transgene-induced pollen exine defects. These findings demonstrate that the proper amount of CLE19 signal is essential for the normal expression of AMS and its downstream gene networks in the regulation of anther development and pollen exine formation.