Timing a switch in tissue integrity: In plants, sperm cells travel through the pollen tube as it grows toward the ovule. Successful fertilization depends on the pollen tube rupturing to release the sperm cells (see the Perspective by Stegmann and Zipfel). Ge et al. and Mecchia et al. elucidated the intercellular cross-talk that maintains pollen tube integrity during growth but destroys it at just the right moment. The signaling peptides RALF4 and RALF19, derived from the pollen tube, maintain its integrity as it grows. Once in reach of the ovule, a related signaling peptide, RALF34, which derives from female tissues, takes over and causes rupture of the pollen tube. Science , this issue p. 1596 , p. 1600 ; see also p. 1544

In recent years, the rapid alkalinization factor (RALF) family of cysteine-rich peptides has been reported to be crucial for several plant signaling mechanisms, including cell growth, plant immunity and fertilization. RALF4 and RALF19 (RALF4/19) pollen peptides redundantly regulate the pollen tube integrity and growth through binding to their receptors ANXUR1/2 (ANX1/2) and Buddha’s Paper Seal 1 and 2 (BUPS1/2), members of the Catharanthus roseus RLK1-like (CrRLK1L) family, and, thus, are essential for plant fertilization. However, the signaling mechanisms at the cellular level that follow these binding events remain unclear. In this study, we show that the addition of synthetic peptide RALF4 rapidly halts pollen tube growth along with the excessive deposition of plasma membrane and cell wall material at the tip. The ratiometric imaging of genetically encoded ROS and Ca²⁺ sensors-expressing pollen tubes shows that RALF4 treatment modulates the cytoplasmic levels of reactive oxygen species (ROS) and calcium (Ca²⁺) in opposite ways at the tip. Thus, we propose that pollen RALF4/19 peptides bind ANX1/2 and BUPS1/2 to regulate ROS and calcium homeostasis to ensure proper cell wall integrity and control of pollen tube growth.

Mildew Locus O ( MLO ) genes, initially identified as powdery mildew susceptibility factors, are increasingly recognized as multifunctional regulators implicated in diverse processes, including plant reproduction, root thigmotropism, and interactions with beneficial microbes. Recent evidence shows that MLO proteins can act as Ca ²⁺ -permeable channels in response to Rapid Alkalinization Factors (RALF) peptides in reproductive cells, pointing to broader roles in Ca ²⁺ -mediated signalling. In this study, we investigate the symbiotic clade IV member LjMLO4 in the model legume Lotus japonicus , focusing on its role in root development and responsiveness to LjRALF34 peptides. We show that LjMLO4 expression is strongly induced in root cells colonized by arbuscular mycorrhizal (AM) fungi, yet loss-of-function mutants exhibit only subtle AM-associated phenotypes. Instead, we uncover a previously uncharacterized function of LjMLO4 as a regulator of primary root growth and lateral root formation, acting even in the absence of AM fungal colonization and in a Ca ²⁺ -dependent manner. Heterologous expression in E. coli confirms that LjMLO4 facilitates Ca ²⁺ transport, while genetic and physiological assays demonstrate its contribution to LjRALF34-triggered root growth responses and Ca ²⁺ signalling. Together, these findings identify LjMLO4 as a molecular hub between peptide signalling, Ca ²⁺ transport and root system architecture, highlighting how MLO proteins integrate developmental, nutritional and symbiotic cues.

Mildew Locus O (MLO) genes, initially identified as powdery mildew susceptibility factors, are increasingly recognized as multifunctional regulators implicated in diverse processes, including plant reproduction, root thigmotropism, and interactions with beneficial microbes. Recent evidence shows that MLO proteins can act as Ca2+-permeable channels in response to Rapid Alkalinization Factors (RALF) peptides in reproductive cells, pointing to broader roles in Ca2+-mediated signalling. In this study, we investigate the symbiotic clade IV member LjMLO4 in the model legume Lotus japonicus, focusing on its role in root development and responsiveness to LjRALF34 peptides. We show that LjMLO4 expression is strongly induced in root cells colonized by arbuscular mycorrhizal (AM) fungi, yet loss-of-function mutants exhibit only subtle AM-associated phenotypes. Instead, we uncover a previously uncharacterized function of LjMLO4 as a regulator of primary root growth and lateral root formation, acting even in the absence of AM fungal colonization and in a Ca2+-dependent manner. Heterologous expression in E. coli confirms that LjMLO4 facilitates Ca2+ transport, while genetic and physiological assays demonstrate its contribution to LjRALF34-triggered root growth responses and Ca2+ signalling. Together, these findings identify LjMLO4 as a molecular hub between peptide signalling, Ca2+ transport and root system architecture, highlighting how MLO proteins integrate developmental, nutritional and symbiotic cues.