The Camellia oil tree (Camellia oleifera Abel.) is an important nonwood forest species in China, and the majority of its cultivars are late-acting self-incompatibility (LSI) types. Although several studies have examined the mechanism of LSI, the process is quite complicated and unclear. In this study, pollen tube growth and fruit setting of two Camellia oil tree cultivars Huashuo (HS) and Huajin (HJ) were investigated after non and self-pollination, and transcriptomic analysis of the ovaries was performed 48 h after self-pollination to identify the potential genes implicated in the LSI of Camellia oil trees. The results showed that the fruit set of HS was significantly higher than that of HJ after self-pollination. Transcriptomic analysis revealed that plant hormone signal transduction, the phosphatidylinositol signaling system, ATP-binding cassette (ABC) transporters, reactive oxygen species (ROS) metabolism, and Ca²⁺ signaling were mainly contributed in the LSI of reaction of Camellia oil tree. Moreover, nine RNase T2 genes were identified from the transcriptome analysis, which also showed that CoRNase7 participated in the self-incompatibility reaction in HS. Based on phylogenetic analysis, CoRNase6 was closely related to S-RNase from coffee, and CoRNase7 and CoRNase8 were closely related to S-RNase from Camellia sinensis. The 9 RNase T2 genes successfully produced proteins in prokaryotes. Subcellular localization indicated that CoRNase1 and CoRNase5 were cytoplasmic proteins, while CoRNase7 was a plasma membrane protein. These results screened the main metabolic pathways closely related to LSI in Camellia oil tree, and SI signal transduction might be regulated by a large molecular regulatory network. The discovery of T2 RNases provided evidence that Camellia oil tree might be under RNase-based gametophytic self-incompatibility.

Flowering is a key agronomic trait that directly influences the yield of the tea-oil tree (Camellia oleifera). Floral initiation, which precedes flower bud differentiation, represents a critical developmental stage affecting the flowering outcomes. However, the molecular mechanisms underlying floral initiation in C. oleifera remain poorly understood. In this study, buds from five key developmental stages of a 12-year-old C. oleifera cultivar ‘changlin53’ were collected as experimental samples. Scanning electron microscopy was employed to identify the stage of floral initiation. UPLC-MS/MS was used to analyze endogenous gibberellin (GA) concentrations, while transcriptomic analysis was performed to reveal the underlying transcriptional regulatory network. Six GA types were detected during floral initiation and petal development. GA₄ was exclusively detected at the sprouting stage (BII), while GA₃ was present in all samples but was significantly lower in BII and the flower bud primordium formation stage (BIII) than in the other samples. A total of 64 differentially expressed genes were concurrently enriched in flower development, reproductive shoot system development, and shoot system development. Weighted gene co-expression network analysis (WGCNA) identified eight specific modules significantly associated with different developmental stages. The magenta module, containing Unigene0084708 (CoFT) and Unigene0037067 (CoLEAFY), emerged as a key regulatory module driving floral initiation. Additionally, GA20OX1 and GA2OX8 were identified as candidate genes involved in GA-mediated regulation of floral initiation. Based on morphological and transcriptomic analyses, we conclude that floral initiation of C. oleifera is a continuous regulatory process governed by multiple genes, with the FT-LFY module playing a central role in the transition from apical meristem to floral meristem.

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.