Tomato pollen tube integrity is critical for pollen function at high temperature and is controlled by RALF signaling.

Reproductive success at high temperature (HT) depends on the ability of pollen to germinate a pollen tube that delivers sperm for double fertilization. We recently found that tomato cultivars capable of setting fruit at HT produce pollen tubes that continue to grow under heat stress. Our goal is to define the molecular basis of reproductive thermotolerance, the capacity to sustain cellular function at elevated temperature. Using quantitative live imaging, transcriptomic and proteomic profiling, and genetic analysis, we identified mechanisms distinguishing thermotolerant from thermosensitive tomato cultivars. HT accelerates pollen activation by increasing cell rupture in thermosensitive Heinz, whereas thermotolerant Tamaulipas and Nagcarlang maintain pollen tube integrity and elongation. Germination triggers broad transcriptional reprogramming, but corresponding proteomic changes are modest. We identify four pollen-expressed RALF peptides and show that RALF1, RALF3, and RALF4 repress germination at control temperature but signaling is antagonized by HT. Loss of SlRALF1 enhances pollen tube integrity at HT in Heinz but delays successful germination in Tamaulipas, revealing cultivar-specific tuning of individual RALF function. HT and RALF signaling jointly modulate pectin organization at the tube tip, linking cell wall mechanics to reproductive resilience and establishing a mechanistic framework for generating heat-resilient crop plants.