Effects of External Application of Malic Acid on Key Genes of Anthocyanin Synthesis in Purple Cabbage (Brassica campestris ssp. chinensis var. comunis)
CHEN Lu, LI Jia-Ming, ZHOU Xiang-Zhu, XU Ru, LIN Yi-Zhang, ZHONG Feng-Lin*
College of Horticulture, Fujian Agricultural and Forestry University, Fuzhou 350002, China
Abstract：Purple cabbage (Brassica campestris ssp. chinensis var. comunis) leaves are purple on the front and green on the back, rich in anthocyanins and have high nutritional value. Organic acids are important components of the flavor and quality of fruits and vegetables, and play a role as signal molecules in the biosynthesis of anthocyanins.To this end, teh present study set the applied malic acid concentration of 0, 0.5, 1.5, 3, 5, 10 mg/L to determine the color parameters and anthocyanin content of purple cabbage. The BcMYB2 (v-myb avian myeloblastosis viral oncogene homolog 2), BcMYB12 and BcMYB111 genes were cloned by RT-PCR, bioinformatics analysis was carried out, and the effects of different concentrations of malic acid on the expression of BcMYB2, BcMYB12 and BcMYB111 in purple cabbage leaves were analyzed by qRT-PCR, in order to clarify the effects of external application of malic acid on the coloration and anthocyanin synthesis of purple cabbage from physiological and molecular levels. The results showed that external application of malic acid had a significant effect on the color and anthocyanin content of purple cabbage. With the increasing of external application of malic acid, the purple degree of leaves went from light to deep to light, and the anthocyanin content showed a trend of increasing and then decreasing. The gene BcMYB2, BcMYB12 and BcMYB111 were cloned by RT-PCR, and the ORF sequences were 758, 1 102, 963 bp, encoding 248, 365 and 321 amino acids, respectively. The amino acid sequences encoded by the 3 genes all had R2 and R3 conserved domain, belonging to the R2R3-MYB gene family, and had close genetic relationship with cabbage (Brassica rapa) and Arabidopsis thaliana. All 3 genes were unstable hydrophilic proteins, no signal peptides and no transmembrane regions. qRT-PCR results showed that the relative expression of BcMYB2 and BcMYB12 in the leaves of purple cabbage were the highest in 12 d, and the relative expression of BcMYB2 and BcMYB12 increased first and then decreased with the increasing of malic acid concentration. However, the relative expression of BcMYB111 was relatively low during the whole treatment period, and there was no significant difference among treatments. Above results indicated that appropriate concentration of applied malic acid could promote BcMYB2 and BcMYB12 expression in the leaves of purple cabbage, thereby deepening the leaf color, increasing the anthocyanin content and nutritional quality of purple cabbage. This study provides basic data for further exploration of the biological mechanism of purple cabbage.
1 陈露, 李家明, 林志强, 等, 2019. 紫色小白菜有机酸的提取优化及UPLC定量分析[J]. 天然产物研究与开发, 31(6): 1038-1045. (Chen L, Li J M, Lin Z Q, et al.2019. Optimization of organic acids and UPLC analysis of purple cabbage[J]. Natural Product Research and Development, 31(6): 1038-1045.) 2 杜杨梅. 2017. 紫花椰菜MYB-TT8-TTG1转录因子调控烟草花青素积累的研究[D]. 硕士学位论文,西南大学, 导师: 宋洪元, pp: 4-14. (Du Y M.2017. Study anthocyanin accumulation in tobacco by constitutively expression MYB2, TT8, TTG1 transcription factors from purple cauliflower[D]. Thesis for M.S., Southwest University, Supervisor: Song H Y, pp: 4-14.) 3 冯露, 吴际洋, 鞠易倩, 等. 2017. 紫叶紫薇呈色生理及光合特性研究[J]. 北京林业大学学报, 39(12): 93-101. (Feng L, Wu J Y, Ju Y Q, et al.2017. Leaf color characteristics and photosynthetic characteristics of purple-leafed Lagerstroemia indica 'Ebony Ember'[J]. Journal of Beijing Forestry University, 39(12): 93-101.) 4 何琼. 2018. 紫心大白菜花青素合成和积累的分子机理研究[D]. 硕士学位论文, 西北农林科技大学, 导师: 张鲁刚, pp: 7-17. (He Q.2018. Mechanism of anthocyanin biosynthesis and accumulation in purple-head Chinese cabbage[D]. Thesis for M.S., Northwest A＆F University, Supervisor: Zhang L G, pp: 7-17.) 5 刘厚诚, 黄琴, 陈日远, 等. 2003. 高温条件下芥蓝菜薹色泽的形成[J]. 中国蔬菜, 1(6): 12-14. (Liu H C, Huang Q, Chen R Y, et al.2003. Formation of kale color under high temperature conditions[J]. Chinese Vegetables, 1(6): 12-14.) 6 刘金, 魏景立, 刘美艳, 等. 2012. 早熟苹果花青苷积累与其相关酶活性及乙烯生成之间的关系[J]. 园艺学报, 39(7): 1235-1242. (Liu J, Wei J L, Liu M Y, et al.2012. The relationships between the enzyme activity of anthocyanin biosynthesis, ethylene release and anthocyanin accumulation in fruits of precocious apple cultivars[J]. Acta Horticulturae Sinica, 39(7): 1235-1242.) 7 刘羽. 2018. 调控杏果实花色素苷合成MYB转录因子的克隆及表达分析[D]. 硕士学位论文,西南大学, 导师:席万鹏, pp: 7-12. (Liu Y.2018. Cloning and functional analysis of an MYB trascription factor involved in regulating anthocyanin production in apricot fruit[D]. Thesis for M.S., Southwest University, Supervisor: Xi W P, pp: 7-12.) 8 刘玉莲, 车飞, 王海, 等. 2016. 苹果果实中糖、酸和花青苷的组分及含量特征分析[J]. 西北林学院学报, 31(6): 236-242. (Liu Y L, Che F, Wang H, et al.2016. Characteristics of the components and contents of soluble sugars,organic aicds and anthocyanins in apple fruit[J]. Journal of North West Forestry University, 31(6): 236-242.) 9 李鹏, 黄晓慧, 王克磊, 等. 2017. 李果实生长发育过程中糖酸和花青苷含量的变化及相关分析[J]. 热带作物学报, 38(2): 269-277. (Li P, Huang X H, Wang K L, et al.2017. Changes of sugar, organic acid and anthocyanin content and the correlation analysis in fruit growth and development pf plum[J]. Chinese Journal of Tropical Crops, 38(2): 269-277.) 10 邵建辉, 马春花, 祖艳群, 等. 2017. 转色初期喷施BTH对‘红地球’葡萄着色和果实品质的影响[J]. 果树学报, 34(12): 1556-1565. (Shao J H, Ma C H, Zu Y Q, et al.2017. Effect of BTH application at veraison on berry coloration and quality of 'Red Globe' berries[J]. Journal of Fruit Science, 34(12): 1556-1565.) 11 唐容, 黄泽素, 王海, 等. 2016. 紫红叶甘蓝型油菜叶片的色素变化及光合生理特性[J]. 西南农业学报, 29(06): 1270-1275. (Tang R, Huang Z S, Wang H, et al.2016. Pigment changes and photosynthetic physiological characteristics in rapesees with purple-red leaf[J]. Southwest China Journal of Agricultural Sciences, 29(06): 1270-1275.) 12 王美玲, 艾希珍, 郑楠. 2008. 紫甘蓝不同叶位叶片色素含量及需光特性的研究[J]. 西北农业学报, 17(01): 221-225. (Wang M L, Ai X Z, Zheng N.2008. Study of pigment content and light-requiring characteristics of different leaf-position leaves in purple cabbage[J]. Acta Agriculturae Boreali-occidentalis Sinica, 17(01): 221-225.) 13 巫伟峰. 2017. 李果实苹果酸转运体的克隆表达及其有机酸的关联性分析[D]. 硕士学位论文, 福建农林大学,导师: 陈发兴, pp: 5-8. (Wu W F.2017. Cloning and expression analysis of malate transporter in plum fruit and analysis of the correlation of organic aicds[D]. Thesis for M.S., Fujian Agriculture and Foresity University, Supervisor: Chen F X, pp: 5-8.) 14 许海峰, 王楠, 姜生辉, 等. 2016. 新疆红肉苹果杂种一代4个株系类黄酮含量及其合成相关基因表达分析[J].中国农业科学, 49(16): 3174-3187. (Xu H F, Wang N, Jiang S H, et al.2016. Content and analysis of biosynthesis-related genes of flavonoid among four strains of Malus sieversii f. neidzwetzkyana F1 population[J]. Scientia Agricultura Sinica, 49(16): 3174-3187.) 15 谢银鹏. 2018. 苹果MdMYB88和MdMYB124转录因子在低温和干旱胁迫中的作用机理研究[D]. 硕士学位论文, 西北农林科技大学, 导师: 管清美, pp: 11-13. (Xie Y P.2018. Molecular mechanisms of MdMYB88 and MdMYB124 in response to cold and drought stresses in apple[D]. Thesis for M.S., Northwest A＆F University, Supervisor: Guan Q M, pp: 11-13.) 16 杨捷, 张星, 彭梦笛, 等. 2018. 百合转录因子MYB12的克隆与表达分析[J]. 植物科学学报, 36(06): 42-53. (Yang J, Zhang X, Peng M D, et al.2018. Cloning and expression analysis of MYB12 in lilium oriental hybrid ‘Sorbonne'[J]. Plant Science Journal, 36(06): 42-53.) 17 杨碧云, 钟凤林, 林义章, 等. 2018. 干旱对紫色小白菜光合特性及营养品质的影响[J]. 西北植物学报, 38(05): 912-921. (Yang B Y, Zhong F L, Lin Y Z, et al.2018. Nutritional quality and photosynthetic characteristics of purple cabbage under drought stress[J]. Acta Botanica Boreali-Occidentalia Sinica, 38(05): 912-921.) 18 周相助. 2018. 氮素形态对尖叶莴苣生长及有机酸代谢影响研究[D]. 硕士学位论文,福建农林大学, 导师: 钟凤林, pp: 2-6. (Zhou X Z.2018. Effect of nitrogen forms on the growth and organic acid metabolism of lettuce[D]. Thesis for M.S., Fujian Agriculture and Foresity University, Supervisor: Zhong F L, pp: 2-6.) 19 Baudry A, Heim M, Dubreucq B, et al.2004. Tt2, tt8 and ttg1 synergistically specify the expression of banyuls and proanthocyanidin biosynthesis in Arabidopsis thaliana[J]. Plant Journal, 39(3): 366-380. 20 Yang B Y, Zhou X Z, Xu R, et al.2016. Comprehensive analysis of photosynthetic characteristics and quality improvement of purple cabbage under different combinations of monochromatic light[J]. Frontiers in Plant Science, 7: 1 21 Chiu L, Zhou X, Burke S, et al.2010. The purple cauliflower arises from activation of a MYB transcription factor[J]. Plant Physiology, 154(3): 1470-1480. 22 Cone K C, Cocciolone S M, Burr B.1993. Maize anthocyanin regulatory gene P1 is a duplicate of C1 that fuctions in plant[J]. Plant Cell, 5(12): 1795-1805. 23 Dubos C, Stracke R, Grotewold E, et al.2010. MYB transcription factors in Arabidopsis[J]. Trends in Plant Science, 15(10): 573-581. 24 Feller A, Machemer K, Braun E L, et al.2011. Evolutionary and comparative analysis of myb and bhlh plant transcription factors[J]. Plant Iournal, 66(1): 94-116. 25 Hale K L, Tufan H A, Pickering I J, et al.2002. Anthocyanins facilitate tungsten accumulation in Brassica[J]. Physiologia Plantarum, 116(3): 351-358. 26 Jin H L, Martin C.1999. Multifunctionality and diversity within the plant myb-gene family[J]. Plant Molecular Biology, 41(5): 577-585. 27 Koes R, Verweij W, Quattrocchio F.2005. Flavonoids: A colourful model for the regulation and evolution of biochemical pathways[J]. Trends in Plant Science, 10(5): 236-242. 28 Masakazu H, Karin O, Kyoko H.2003. Enhancement of anthyanin biosythesis by sugar in radish[J]. Hypocotyl Plant Science, 164(2): 259-265. 29 Mol J, Grotewold E, Koes R.1998. How genes paint flowers and seeds[J]. Trends in Plant Science, 3(6): 212-217. 30 Yamagishi M, Shimoyamada Y, Nakatsuka T, et al.2010. Two R2R3-MYB genes homologs of Petunia AN2 regulate anthocyanin biosyntheses in flower tepals, tepal spots and leaves of Asiatic Hybrid Lily[J]. Plant and Cell Physiology, 51(3): 463-474. 31 Noro S, Kudo N, Kitsuwa T.1989. Differences in sugar and organic acid contents between bagged and unbagged fruits of the yellow apple cultivars, and the effect on development of anthocyanin[J]. Journal of the Japanese Society for Horticultural Science, 58(1): 17-24. 32 Pabo C O, Sauer R T.1992. Transcription factors-structural families and principles of DNA recognition[J]. Annual Review of Biochemistry, 61: 1053-1095. 33 Petroni K, Tonelli C.2011. Recent advances on the regulation of anthocyanin synthesis in reproductive organs[J]. Plant Science, 181(3): 219-229. 34 Spelt C, Quattrocchio F, Mol J N, et al.2000. Anthocyaninl of petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes[J]. The Plant Cell Online, 12(9): 1619-1631. 35 Wang Y F, Johnson-Cicalese J, Singh A P, et al.2017. Characterization and quantification of flavonoids and organic acids over fruit development in american cranberry (Vaccinium macrocarpon) cultivars using HPLC and APCI-MS/MS[J]. Plant Science, 262: 91-102. 36 Wang Y, Zhou B, Sun M, et al.2012. UV-A light induces anthocyanin biosynthesis in a manner distinct from synergistic blue plus UV-B light and UV-A/blue light responses in different parts of the hypocotyls in turnip seedlings[J]. Plant and Cell Physiology, 53(8): 1470-1480. 37 Wrolstad R E, Durst R W, Lee J.2005. Tracking color and pigment changes in anthocyanin products[J]. Trends in Food Science & Technology, 16(9): 0-428. 38 Yuan Y X, Chiu L W, Li L.2009. Transcription regulation of anthocyanin biosynthesis in red cabbage[J]. Planta, 230(6): 1141-1153. 39 Zbinovsky V, Burris R H.1952. Metabolism of infiltrated organic acids by tobacco leaves[J]. Plant Physiology, 27(2): 240-250.
