Genome-Wide Identification and Functional Analysis of RNase T2 Family Genes in Camellia oleifera

Camellia oleifera Abel. is a typical self-incompatible plant, yet its molecular mechanisms have not been comprehensively elucidated, which constitutes the principal cause underlying the relatively low natural fruit set rate in C. oleifera. S-RNase belongs to the RNase T2 gene family and represents a class of glycoproteins specifically expressed in the style with certain “cytotoxicities” capable of degrading ribonucleic acid (RNA) in their own pollen tube cells, thereby inducing programmed cell death in pollen tube cells. To identify pistil S genes participating in the self-incompatibility response of C. oleifera, nine RNase T2 family genes were identified based on transcriptomic and whole-genome sequencing data of the camellia oil tree and designated CoRNS1–CoRNS9. Systematic evolutionary analysis revealed that CoRNS5, CoRNS7, and CoRNS8 exhibit close phylogenetic relationships with S-RNases of the Camelliaceae family, whereas CoRNS6 shows closer affinity with S-RNases of the Solanaceae family. Analysis of promoter cis-acting elements revealed that RNase T2 family genes are regulated by multiple hormones, including abscisic acid, methyl jasmonate, cytokinin, auxin, salicylic acid, and gibberellin, and possess MYB transcription factor-binding sites. Expression analysis revealed that CoRNS6 is expressed exclusively in the ovary; CoRNS5 is expressed at the highest level in the style, followed by lower levels in the petals, anthers, filaments, receptacle, and ovary; CoRNS1 is expressed in all tissues except anthers; and the other genes are expressed across various floral tissues. Ex vitro pollen culture system experiments demonstrated that the CoRNS1 and CoRNS5 recombinant proteins significantly inhibited the elongated growth of autogamous pollen tubes, whereas CoRNS7 significantly reduced the pollen germination rate. Fluorescence labeling revealed that treatment of autogamous pollen tubes with the recombinant proteins CoRNS1, CoRNS5, and CoRNS7 induced microfilament skeleton depolymerization and increased the Ca2+ concentration within the pollen tubes. Additionally, treatment with the CoRNS5 recombinant protein increased the reactive oxygen species (ROS) levels in autogamous pollen tubes. These findings suggest that RNase T2 family genes are involved in the self-incompatibility response in C. oleifera, with CoRNS1, CoRNS5, and CoRNS7 playing pivotal roles. Overall, our results not only offer a theoretical foundation for elucidating the regulatory network governing self-incompatibility in C. oleifera, but also furnish valuable genetic resources for future molecular breeding programs targeting improved self-compatibility and increased fruit yield.