Leaf surface chemicals improvement through molecular regulation of glandular trichome formation of tobacco
Tobacco (Nicotiana tabacum) plant is covered with dense trichomes distinguished in morphology structure and secretion ability. Long-stem glandular trichome is the most important type of tobacco trichome. It could specifically synthesize and secrete diterpene and sucrose esters, composing the main constituents of tobacco leaf chemicals, and contribute greatly to plant resistance and leaf quality. B-type cyclins, which play important roles in the transition of G2-to-M have been reported to participate in reproductive organ development and trichome formation. In this study, tobacco B-type cyclin gene, NtCycB2, was cloned and transferred to the cultivated variety K326. Transgene plants analysis showed that NtCycB2 overexpression caused the obvious decrease of glandular trichome density and abnormal development of lateral roots. On the contrary, knockout of NtCycB2 through CRISPR-Cas9 technique could promote the formation of glandular trichomes, most of them branched with more than one glandular head. NtCycB2 gene knockout plants proved to have many advantages, compared to NtCycB2 gene overexpression plants, with resistance to aphid attack and drought, cold and UVB stress. This suggested their great potential use in tobacco variety improvement. Therefore, several homozygous gRNA knockout mutant strains with expression vector deleted were selected and cultivated in the field and the agronomy and leaf chemistry characteristics were comparatively analyzed. Except for trichome density changes, no other morphological differences were discovered between the NtCycB2 knockout plants and the wild control. Detection of leaf surface chemical components by GC/MS indicated that the production of diterpene and sucrose ester compounds were significantly increased in NtCycB2 knockout plants, which are supposed to benefit leaf aroma quality and plant resistance. These results indicate that NtCycB2 plays a critical role in glandular trichome initiation, leaf surface chemical accumulation and defense in the tobacco plant.