Objective To investigate the role of Sapindus mukorossi SmCYCD3;2 gene in flower sex differentiation, to provide valuable reference genes for improving the male-to-female ratio of Sapindus mukorossi flowers, and then to lay a solid theoretical foundation for improving S. mukorossi fruit yields through molecular design breeding.

Methods The SmCYCD3;2 gene of S. mukorossi was cloned, subjected to multiple sequence comparison and phylogenetic evolutionary analysis, and its expression pattern in different organs was analyzed by real-time fluorescence quantitative PCR (RT-qPCR). We constructed overexpression vectors and heterologously transformed Arabidopsis thaliana to compare its growth phenotypes, stained its mature anthers with Alexandrium staining, observed pollen viability and in vitro pollen germination using the pollen germination assay, and observed the morphology and surface features of anthers and pollen with the aid of scanning electron microscopy.

Results (1) The full length of SmCYCD3;2 gene sequence was 1 092 bp, encoding 364 amino acids, and it is closely related to Populus trichocarpa. (2) RT-qPCR analysis revealed that the expression of SmCYCD3;2 gene was highest in female flowers of S. mukorossi, followed by leaves, and the lower expression was found in male flowers. (3) Compared with wild-type plants, transgenic Arabidopsis exhibited a series of significant growth phenotypic changes: growth rate was slowed down, plant height was significantly reduced, and leaves appeared curled and slightly bent at each stem node. In addition, the floral organs phenotype also underwent significant changes, which were characterized by elongated pistils, reduced and shorter stamens, reduced pollen activity, and ultimately shorter fruit pods. (4) In transgenic Arabidopsis plants, the expression of cell cycle-related genes (AtCYCDs) and genes promoting gynoecium elongation and development (AtCRC) were significantly up-regulated, while the expression of genes related to stamen development (AtAP3, AtPI, AtAMS, and AtDEL) as well as genes related to stem and leaf development (AtPIN) were significantly down-regulated.

Conclusion The SmCYCD3;2 gene is able to regulate the development of stem and leaf, promote pistil elongation and inhibit the stamen development, which plays an important role in sex differentiation. The results of the study provide new insights into the functional resolution of CYCD3 gene, and lay a pre-theoretical foundation for improving the female-to-male ratio of flowers and increasing fruit yield of S. mukorossi.