Actin-Depolymerizing Factor Subgroup I Members Negatively Regulate Pollen Tube Growth in Fortunella hindsii

Citrus is one of the most economically important horticultural crops worldwide due to its edible fruits. Larger fruit size and higher yields depend on successful pollination and fertilization, which in turn rely on efficient pollen germination and pollen tube growth. Current research on sexual reproduction primarily focuses on pollen-stigma interactions; little is known about pollen tube growth in Citrus. Actin-depolymerizing factors (ADFs) are key regulators of actin cytoskeleton dynamics and play critical roles in pollen tube growth. However, their functions in major horticultural crops remain poorly understood. Here, we characterized two pollen-abundant ADF genes, FhADF1 and FhADF2, in Hongkong kumquat (Fortunella hindsii). CRISPR/Cas9-generated Fhadf1/2 double knockout mutants exhibited accelerated pollen tube elongation and reduced sensitivity to the actin-depolymerizing drug LatB. Examination of actin filaments in pollen tubes revealed decreased fluorescence intensity and density, indicating reduced abundance of actin filaments in Fhadf1/2 mutants compared to WT. This growth-accelerating phenotype was conserved in tomato (Solanum lycopersicum). The depletion of SlADF1, a homologous gene of FhADF1 and FhADF2, also promoted pollen tube growth and enhanced the tolerance to LatB. Furthermore, the sladf1 mutant maintained a relative growth advantage under short-term acute heat stress. These findings suggest that ADF1 is a conserved negative regulator of pollen tube growth in crops and may help plants respond to heat stress during pollen development. Our study expands new insights into subgroup I ADF family members on pollen tube growth regulation and high temperature adaptation in economically important horticultural species.