CULLIN (CUL) protein is a subtype of E3 ubiquitin ligase that is involved in a variety of biological processes and responses to stress in plants. In Uncaria rhynchophylla, the CUL gene family has not been identified and its role in plant development, stress response and secondary metabolite synthesis has not been studied. In this study, 12 UrCUL gene members all contained the typical N-terminal domain and C-terminal domain identified from the U. rhynchophylla genome and were classified into four subfamilies based on the phylogenetic relationship with CULs in Arabidopsis thaliana. They were unevenly distributed on eight chromosomes but had a similar structural composition in the same subfamily, indicating that they were relatively conserved and potentially had similar gene functions. An interspecific and intraspecific collinearity analysis showed that fragment duplication played an important role in the evolution of the CUL gene family. The analysis of the cis-acting elements suggests that the UrCULs may play an important role in various biological processes, including the abscisic acid (ABA) response. To investigate this hypothesis, we treated the roots of U. rhynchophylla tissue-cultured seedlings with ABA. The expression pattern analysis showed that all the UrCUL genes were widely expressed in roots with various expression patterns. The co-expression association analysis of the UrCULs and key enzyme genes in the terpenoid indole alkaloid (TIA) synthesis pathway revealed the complex expression patterns of 12 UrCUL genes and some key TIA enzyme genes, especially UrCUL1, UrCUL1-likeA, UrCUL2-likeA and UrCUL2-likeB, which might be involved in the biosynthesis of TIAs. The results showed that the UrCULs were involved in the response to ABA hormones, providing important information for elucidating the function of UrCULs in U. rhynchophylla. The mining of UrCULs in the whole genome of U. rhynchophylla provided new information for understanding the CUL gene and its function in plant secondary metabolites, growth and development.

Uncaria rhynchophylla, a medicinal plant extensively used in traditional Chinese medicine, is an important plant source of terpenoid indole alkaloids (TIAs), but the mechanism of TIA biosynthesis at molecular level remains unclear. Geraniol synthase (GES) serves as a crucial enzyme in catalyzing the formation of geraniol from geranyl pyrophosphate (GPP) in various plants, but the functional characterization of the GES gene in U. rhynchophylla has not been investigated. In this study, a GES was identified and characterized through genome mining and bioinformatic analysis. Functional validation was performed via a protein catalysis experiment, transient expression in Nicotiana benthamiana, and methyl jasmonate (MeJA) induction experiments. The full-length UrGES gene was 1761 bp, encoding a protein product of 586 amino acids with an estimated 67.5 kDa molecular weight. Multiple sequence alignments and phylogenetic analysis placed UrGES within the terpene synthase g (TPS-g) subfamily, showing high similarity to known GESs from other plants. Enzymatic assays confirmed that recombinant UrGES catalyzed GPP conversion to a single product of geraniol. The transient expression of UrGES resulted in geraniol accumulation in N. benthamiana, further confirming its function in vivo. UrGES expression was observed in leaves, stems, and roots, where leaves had the highest transcript levels. Moreover, MeJA treatment significantly upregulated UrGES expression, which positively correlated with an increase in alkaloid content. This study functionally characterizes UrGES as a geraniol synthase in U. rhynchophylla, contributing to the current knowledge of the TIA biosynthetic pathway. These findings may offer insights for future metabolic engineering aiming to enhance TIA yields for pharmaceutical and industrial applications.

Plant U-box E3 ubiquitin ligases (PUBs) are involved in various biological processes in response to abiotic stress. Recent studies have shown that E3 ubiquitin ligases can regulate the production of important compounds in medicinal plants by targeted degradation of transcription factors. Abscisic acid (ABA), a plant stress response hormone, can cause changes in the content of the medically important monoterpene indole alkaloids (MIAs) in U. rhynchophylla. In this study, we explored the relationship between UrPUB gene expression and MIA content. ABA was applied to tissue-cultured seedlings of U. rhynchophylla, resulting in consistent changes in the content of four MIAs. Seventy-three UrPUB genes were identified and bioinformatically characterized from the genome sequence of U. rhynchophylla. The expression levels of transcription factors involved in regulating the biosynthesis pathway of MIAs and UrPUB genes with higher RNA transcript levels in the roots were determined. Co-expression association analysis revealed that UrPUB17, UrPUB40, UrPUB41, UrPUB44 and UrPUB55 negatively correlate with UrGATA8 and UrWRKY37 under ABA stimulation. Based solely on these correlation data, we hypothesize that these UrPUBs might regulate MIA biosynthesis via ubiquitination of UrGATA8 and UrWRKY37, but direct evidence (protein interaction, ubiquitination, degradation, or genetic manipulation) is lacking. This study identified the UrPUB gene family in the U. rhynchophylla genome and proposes this ubiquitination model as a testable hypothesis, not a demonstrated mechanism. These findings provide new insights into the biological function of the PUB family in response to ABA.