NtCycB2 negatively regulates tobacco glandular trichome formation, exudate accumulation, and aphid resistance.

KEY MESSAGE: NtCycB2 negatively regulates the initiation of tobacco long stalk glandular trichomes and influences the expression of diterpenoid biosynthesis- and environmental stress resistance-related genes. Many asterid plants possess multicellular trichomes on their surface, both glandular and non-glandular. The CycB2 gene plays a key role in multicellular trichome initiation, but has distinct effects on different types of trichomes; its mechanisms remain unknown. In tomato (Solanum lycopersicum), SlCycB2 negatively regulates non-glandular trichome formation, but its effects on glandular trichomes are ambiguous. In this study, we cloned the SlCycB2 homolog of Nicotiana tabacum, NtCycB2, and analyzed its effect on three types of trichomes, long stalk glandular trichomes (LGT), short stalk glandular trichomes (SGT), and non-glandular trichomes (NGT). Knocking out NtCycB2 (NtCycB2-KO) promoted LGT formation, while overexpression of NtCycB2 (NtCycB2-OE) decreased LGT density. SGT and NGT were not significantly influenced in either NtCycB2-KO or NtCycB2-OE plants, indicating that NtCycB2 regulated only LGT formation in tobacco. In addition, compared with NtCycB2-OE and control plants, NtCycB2-KO plants produced more trichome exudates, including diterpenoids and sugar esters, and exhibited stronger aphid resistance. To further elucidate the function of NtCycB2, RNA-Seq analysis of the NtCycB2-KO, NtCycB2-OE, and control plants was conducted. 2,552 and 1,933 differentially expressed genes (DEGs) were found in NtCycB2-KO and NtCycB2-OE plants, respectively. Gene Ontology analysis of the common DEGs revealed that ion transport, carbohydrate and amino acid metabolism, photosynthesis, and transcription regulation processes were significantly enriched. Among these DEGs, diterpenoid biosynthesis genes were upregulated in NtCycB2-KO plants and downregulated in NtCycB2-OE plants. Two MYB transcription factors and several stress resistance-related genes were also identified, suggesting they may participate in regulating LGT formation and aphid resistance.