Abstract：The growth and development of foliar glandular hairs in tobacco (Nicotiana tabacum) is related to the aroma quality of tobacco leaves. In order to investigate the relationship between Ent-copalyl diphosphate synthase 2 (NtCPS2) and the growth and development of glandular hair and the synthesis of glandular hair secretion in tobacco, the wild-type strain (WT), NtCPS2 homozygous editing strain (KD) and overexpression strain (OE) of '8306' were used as materials to observe the phenotype of glandular hair and the density of glandular hair. Content of cyperane substances was determined and the expression of key synthetic genes was analyzed. The results showed that compared with WT, the density of leaf secretory glandular hairs in KD strains increased by about 75%, and the secretion of leaf glandular hairs increased significantly (P<0.05), while the secretion of leaf glandular hairs in OE strains was significantly lower than that of WT strains (P<0.05), and decreased by about 50% compared with WT strains (P<0.05). The expression level of cyclin B2 (NtCycB2) gene in the negative regulation of glandular hair gene was significantly lower than that in WT and KD strain, and the expression level of NtCycB2 gene in OE and KD strains showed an opposite trend, and the expression level of NtCycB2 gene was opposite to that of glandular hair phenotype. The gibberellin content of KD tobacco plant was significantly higher than that of WT strain (P<0.05). The gibberellin content of OE strains was significantly lower than that of WT strains (P<0.05). The content of gibberellin in KD strains increased, the density of secretory glandular hairs increased, and the content of cyperane substances, the main glandular hair secretions in the foliar strain increased. The OE strain showed an opposite trend to the KD strain. The results suggested that the NtCPS2 gene might promote the increase of gonadal hair density and foliar glandular hair secretion by positively regulating gibberellin biosynthesis. This study provides a new research direction for the regulation of the synthesis of tobacco glandular hair and tobacco aroma compounds by NtCPS2 gene through gibberellin pathway.
[1] 冯琦, 王永, 武东玲, 等. 2013. 外源MeJA诱导烟草叶面防御反应[J]. 中国烟草科学, 34(05): 83-88. (Feng Q, Wang Y, Wu D L, et al.2013. Exogenous MeJA induces foliar defense response in tobacco[J]. Chinese Tobacco Science, 34(05): 83-88.) [2] 高秀华, 傅向东. 2018, 赤霉素信号转导及其调控植物生长发育的研究进展[J]. 生物技术通报, 34(07): 1-13. (Gao X H,Fu X D.2018. Research progress on gibberellin signaling and its regulation of plant growth and development[J]. Biotechnology Bulletin, 34(07): 1-13.) [3] 宫沛文, 吴秀双, 孟淑真, 等. 2022. 过量表达NtCAF1基因对烟草腺毛密度的影响[J]. 种子, 41(11): 17-24. (Gong P W, Wu X S, Meng S Z, et al.202. Effect of overexpression of NtCAF1 gene on densities of tobacco glandular hairs[J]. Seeds, 41(11): 17-24.) [4] 贺凌霄, 薛刚, 孙聚涛, 等. 2021. 外源赤霉素对烤烟叶面腺毛和香气物质的影响[J]. 河南农业科学, 50(01): 52-59. (He L X, Xue G, Sun J T, et al.2021. Effect of exogenous gibberellin on leaf glandular hair and aroma substances of flue-cured tobacco[J]. Henan Agricultural Sciences, 50(01): 52-59.) [5] 贺凌霄, 何雷, 李洪臣等. NtCPS2基因对顺-冷杉醇与赤霉素合成代谢途径的影响[J]. 中国农业大学学报, 2021,26(06): 33-41. (He L X, He L, Li H C, et al.2021. NtCPS2 gene pairs cis-flitaxel and gibberellinEffects of anabolic pathways[J]. Journal of China Agricultural University, 26(06): 33-41. [6] 韩文龙, 郑聪, 曹可心, 等. 2023. NtCPS2基因对烟草腺毛发育及其分泌物合成的影响[J]. 河南农业科学, 52(03):48-55. (Han W L, Zheng C, Cao K X, et al.2023. Effect of NtCPS2 gene on glandular hair development and secretion synthesis in tobacco[J]. Henan Agricultural Sciences, 52(03): 48-55.) [7] 刘一华. 2018. miR414和TOE1调控植物表皮毛及NbGIS调控烟草腺毛发育的功能研究[D]. 博士论文, 浙江大学, 导师: 甘银波, pp. 71-74. (Liu Y H, 2018. Study on the function of miR414 and TOE1 in regulating plant epidermal hair and NbGIS in regulating the development of tobacco glandular hair[D]. Thesis for Ph.D., Zhejiang University, supervisor: Gan Y B, pp. 71-74.) [8] 娄亚楠, 王召军, 杨欣玲, 等. 2018. 茉莉酸甲酯对烟草分泌型和非分泌型腺毛形态发生的影响[J]. 中国烟草学报, 24(02): 24-29. (Lou Y N, Wang Z J, Yang X L, et al.2018. Effect of methyl jasmonate on the morphogenesis of secretory and non-secretory glandular hairs in tobacco[J]. Journal of Chinese Tobacco Science, 24(02): 24-29.) [9] 马旭东, 王召军, 闫筱筱, 等. 2021. 分泌型烟草腺毛形态和叶面化学成分的比较[J]. 烟草科技, 54(01): 10-16. (Ma X D, Wang Z J, Yan X X, et al.2021. Comparison of morphological and foliar chemical components of secretory tobacco glandular hairs[J]. Tobacco Science and Technology, 54(01): 10-16.) [10] 孟盈, 闫筱筱, 王召军, 等. 2019. NtCycB2基因表达对烟草腺毛发生的影响[J]. 中国烟草学报, 25(02): 85-92, 98. (Meng Y, Yan X X, Wang Z J, et al.2019. Effect of NtCycB2 gene expression on glandular hair occurrence in tobacco[J]. Journal of Chinese Tobacco Society, 25(02): 85-92, 98.) [11] 石琰璟, 沙广利, 束怀瑞. 2006. 赤霉素生物合成及其分子机理研究进展[J]. 西北植物学报, (07): 1482-1489. (Shi Y J, Sha G L, Shu H R. 2006. Research progress on gibberellin biosynthesis and molecular mechanism[J]. Northwest Botany, (07): 1482-1489.) [12] 吴秀双, 宫沛文, 黄璐, 等. 2022. 过表达NtHD20降低烟草腺毛密度[J]. 分子植物育种, 20(16): 5332-5338. (Wu X S, Gong P W, Huang L, et al.2022. Molecular Plant Breeding, 20(16): 5332-5338.) [13] 王关林, 方宏均. 1998. 植物基因工程技术原理与技术[M].北京: 科学出版社, pp. 229-609. (Wang G L, Fang H J.1998. Principles and techniques of plant genetic engineering[M]. Science Press, Beijing, China, pp. 229-609.) [14] 王伟, 孔光辉, 李佛琳, 等. 2007. 烤烟烟叶腺毛及其分泌物研究进展[J]. 中国农学通报, 152(02): 251-254. (Wang Wei, Kong G H, Li F L, et al.2007. Research progress on flue-cured tobacco leaf glandular hairs and their secretions[J]. Chinese Agricultural Science Bulletin, 152(02): 251-254.) [15] 岳阳, 梁巧兰, 魏列新, 等. 2021. 苜蓿花叶病毒和白三叶草花叶病毒复合侵染对本氏烟中5种激素含量的影响[J]. 草业科学, 38(11): 2255-2265. (Yue Y, Liang Q L, WeI L X, et al.2021. Effects of combined infection of alfalfa mosaic virus and white clover mosaic virus on the contents of five hormones in N. benthamiana[J]. Pratacultural Science, 38(11): 2255-2265.) [16] 张思琦, 何佳, 贺凌霄, 等. 2020. 基于CRISPR/Cas9技术的烟草CPS2基因敲除及功能分析[J]. 烟草科技, 53(05):17-25. (Zhang S Q, He J, He L X, et al.2020. Tobacco CPS2 gene knockout and functional analysis based on CRISPR/Cas9 technology[J]. Tobacco Science and Technology, 53(05):17-25.) [17] Chien J C, Sussex I M.1996. Differential regulation of trichome formation on the adaxial and abaxial leaf surf aces by gibberellins and photoperiod in Arabidopsis thaliana(L.) Heynh[J]. Plant Physiology, 111(4): 1321-1328. [18] Lin Y, Wagner G J.1994. Rapid and simple method for estimation of sugar esters[J]. Journal of Agricultural & Food Chemistry, 42(8): 1709-1712. [19] Barja M V, Rodriguez-Concepcion M.2021. Plant geranylgeranyl diphosphate synthases: Every (gene) family has a story[J]. aBIOTECH, 2(3): 1-10. [20] Perazza D, Vachon G, Herzog M.1998. Gibberellins promote trichome formation by up-regulating GLABRO US1 in Arabidopsis[J]. Plant Physiology, 117(2):375-383. [21] Sallaud C, Giacalone C, Töpfer R, et al.2012. Characterization of two genes for the biosynthesis of the labdane diterpene Z-abienol in tobacco (Nicotiana tabacum) glandular trichomes[J]. The Plant Journal: For Cell and Molecular Biology, 72(1): 52-64. [22] Stratmann J W, Bequette C J.2016. Hairless but no longer clueless: Understanding glandular trichome development[J]. Journal of Experimental Botany, 67(18): 5285-5287. [23] Tooker J, Peiffer M, Luthe D S, et al.2010. Trichomes as sensors[J]. Plant Signaling & Behavior, 5(1): 73-75. [24] Telfer A, Bollman K M, Poething R S.1997. Phasechange and the regulation of trichome distribution in Arabidopsis thaliana[J]. Development, 124(3): 645-654. [25] Wagner G J.1991. Secreting glandular trichomes: More than just hairs[J]. Plant Physiology, 96: 675-679. [26] Wang Z J, Yan X X, Zhang H Y, et al.2021. NtCycB2 negatively regulates tobacco glandular trichome formation, exudate accumulation, and aphid resistance[J]. Plant Molecular Biology, 108(1-2): 65-76. [27] Wang E M, Wagner G J.2003. Elucidation of the functions of genes central to diterpene metabolism in tobacco trichomes using posttranscriptional gene silencing[J]. Planta, 216(4): 686-91.
