SPATULA and ALCATRAZ confer female sterility and fruit cavity via mediating pistil development in cucumber

Fruits and seeds play essential roles in plant sexual reproduction and the human diet. Successful fertilization involves delivery of sperm in the pollen tube to the egg cell within the ovary along the transmitting tract (TT). Fruit cavity is an undesirable trait directly affecting cucumber (Cucumis sativus) commercial value. However, the regulatory genes underlying fruit cavity formation and female fertility determination remain unknown in crops. Here, we characterized a basic Helix-Loop-Helix (bHLH) gene C. sativus SPATULA (CsSPT) and its redundant and divergent function with ALCATRAZ (CsALC) in cucumber. CsSPT transcripts were enriched in reproductive organs. Mutation of CsSPT resulted in 60% reduction in female fertility, with seed produced only in the upper portion of fruits. Csspt Csalc mutants displayed complete loss of female fertility and fruit cavity due to carpel separation. Further examination showed that stigmas in the double mutant turned outward with defective papillae identity, and extracellular matrix contents in the abnormal TT were dramatically reduced, which resulted in no path for pollen tube extension and no ovules fertilized. Biochemical and transcriptome analysis showed that CsSPT and CsALC act in homodimers and heterodimers to confer fruit cavity and female sterility by mediating genes involved in TT development, auxin-mediated signaling, and cell wall organization in cucumber. Two bHLH transcription factors interact with each other to redundantly define stigma morphology and the pollen tube extension path in pistils, thus maintaining female fertility and carpel fusion in cucumber.