Cytological and Transcriptomic Analyses Reveal Important Roles of <i>CLE19</i> in Pollen Exine Formation

Abstract

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 <i>CLE19</i> gene, exhibited significantly reduced anther size and pollen grain number and abnormal pollen wall formation in Arabidopsis (<i>Arabidopsis thaliana</i>). Interestingly, the <i>DN</i>-<i>CLE19</i> pollen grains showed a more extensively covered surface, but <i>CLE19-OX</i> 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 <i>cle19</i>, <i>DN</i>-<i>CLE19</i>, and <i>CLE19-OX</i> plants, we demonstrated that <i>CLE19-OX</i> plants produced highly vacuolated and swollen <i>aborted microspores</i> (<i>ams</i>)-like tapetal cells, lacked lipidic tapetosomes and elaioplasts, and had abnormal pollen primexine without obvious accumulation of sporopollenin precursors. Moreover, <i>CLE19</i> is important for the normal expression of more than 1,000 genes, including the transcription factor gene <i>AMS</i>, 280 <i>AMS</i>-downstream genes, and other genes involved in pollen coat and pollen exine formation, lipid metabolism, pollen germination, and hormone metabolism. In addition, the <i>DN-CLE19(+/+) ams(-/-)</i> plants exhibited the <i>ams</i> anther phenotype and <i>ams(+/-)</i> partially suppressed the <i>DN-CLE19</i> transgene-induced pollen exine defects. These findings demonstrate that the proper amount of CLE19 signal is essential for the normal expression of <i>AMS</i> and its downstream gene networks in the regulation of anther development and pollen exine formation.