RALF4/19 are Autocrine Signals to Maintain Pollen Tubes Integrity

Receptor-like kinases BUPS1/2 coordinate with ANX1/2 to control pollen tubes integrity possibly via depending on ligands from pollen tubes, and RALF peptides are included as the candidates. To screen the ligand of BUPS1/2–ANX1/2 complex, we first performed transcriptome analysis of RALF gene family and found 17 RALF peptides, of which RALF4 and RALF19 were focused on in this chapter, showing high or specific transcript in pollen. RALF4/19 were mainly expressed in pollen grains and pollen tubes via GUS staining, and their coding proteins accumulated in pollen grains/tubes. We adopted CRISPR/Cas9 technology to knock out RALF4/19 and successfully generated a series of mutants with RALF4 and RALF19 mutated separately/simultaneously. Through the phenotypical analyses, ralf4 ralf19 double mutants, other than any single mutants, showed defects in plant fertility. These defects were male-specific and caused by the abolished cell integrity of pollen tubes. ralf4 ralf19 pollen tubes ruptured prematurely in vitro. Aniline blue staining determined that the pollen grains of ralf4 ralf19 could normally germinate on the stigma, but the pollen tubes arrested their growth shortly after penetrating the transmitting tracts. Given that this phenotype highly resembles that of bups1 bups2 or anx1 anx2, pull-down experiments and MST assays were performed and uncovered that RALF4/19 could not only bind to BUPS1/2 but also show high affinity to ANX1/2. Therefore, we believe that pollen tube-secreted RALF4/19 peptides are the ligands of BUPS1/2–ANX1/2 complex on the membrane of pollen tubes, to maintain pollen tubes integrity during the polar growth to the ovules in Arabidopsis.