ABA和乙烯互作调控葡萄VlMybA1和VlMybA2表达并促进果皮着色

doi:10.16420/j.issn.0513-353x.2022-0919

摘要/Abstract

摘要：

以‘巨峰’葡萄（Vitis vinifera × V. labrusca‘Kyoho’）为试材，研究了不同发育阶段果实花青素和ABA积累和乙烯释放的变化模式，及其与其合成或调控关键基因表达量的变化。基因表达、启动子响应和相对丰度分析表明，外源ABA处理上调了VlMybA1和VlMybA2表达，其中VlMybA1显著表达，促进果皮着色；外源ACC处理显著上调了VlMybA2表达，促进果皮着色。ABA和乙烯存在互作，100 μmol · L^-1的外源ABA处理能够大幅度诱导内源乙烯的释放，500 μmol · L^-1的外源ACC处理能够显著提高内源ABA含量；外源ABA处理能通过提高果皮内源乙烯释放水平增加VlMybA2的相对丰度。总之，ABA能直接调控VlMybA1表达，通过乙烯间接调控VlMybA2表达，促进葡萄果皮着色。

关键词: 葡萄, ABA, 乙烯, VlMybA1, VlMybA2, 花青素

Abstract:

The patterns of anthocyanin and ABA accumulation and ethylene production as well as the changes in expression of the key genes associated with their synthesis or regulation during berry development were studied using Kyoho grapes（Vitis vinifera × V. labrusca‘Kyoho’）. Analysis of gene expression，promoter response and relative mRNA abundance showed that exogenous ABA treatment upregulated the expression level of VlMybA1 and VlMybA2，in which VlMybA1 was significantly expressed and promoted berry skin pigmentation；In contrast，exogenous ACC treatment increased the VlMybA2 expression and anthocyanin accumulation. The interaction between ABA and ethylene was found，100 μmol · L^-1ABA largely induced the production of endogenous ethylene，and 500 μmol · L^-1ACC significantly increased ABA content. Additionally，exogenous ABA treatment increased the relative mRNA abundance of VlMybA2 via ethylene. Collectively，ABA promoted berry skin pigmentation by upregulating the VlMybA1 expression directly and the VlMybA2 expression indirectly via ethylene.

Key words: grape, ABA, ethylene, VlMybA1, VlMybA2, anthocyanins

孙玉帅, 王菲, 管雪强, 郗慧茹, 姚玉新. ABA和乙烯互作调控葡萄VlMybA1和VlMybA2表达并促进果皮着色[J]. 园艺学报, 2023, 50(11): 2323-2336.

SUN Yushuai, WANG Fei, GUAN Xueqiang, CHI Huiru, YAO Yuxin. ABA and Ethylene Enhance the Expression VlMybA1 and VlMybA2 and Promote Pigmentation in the Berry Skin via Their Interaction[J]. Acta Horticulturae Sinica, 2023, 50(11): 2323-2336.

图/表 7

表1 本研究中使用的引物序列

Table 1 Primer sequence used in this study

目的基因 Target gene	登录号 Accession number	上游引物（5′-3′） Forward primer sequence	下游引物（5′-3′） Reverse primer sequence
VlMybA1，VlMybA2，VlMybA3（qRT-PCR）	AB427165，AB073013， AB073012	GAGGGTGATTTTCCATTTGAT	CAAGAACAACTTTTGAACTTAAACAT
VlMybA2（qRT-PCR）	AB073013	GTTCCAAAGTTGTTTAATCTTAG	ATCCACTGCTCACATCCAT
VlUFGT（qRT-PCR）	AF000372^*	GGGATGGTAATGGCTGTGG	ACATGGGTGGAGAGTGAGTT
VlNCED1（qRT-PCR）	XM_002277318^*	ATACATAGCTGCTTGGTGTCAGA	CTGTAGAGAGTTTGGAGATTCCG
VlNCED4（qRT-PCR）	XM_002270125^*	GGCTTCCATGGATTGTTTGT	CCTAATTCGAGCTTGCTTTCC
VlACS（qRT-PCR）	XM_002278453^*	CTCAGGTAATTGTCGGATGTG	GATCAAAACGAGAATCCGGT
VlACO（qRT-PCR）	XM_002273394^*	CTGTTGTCGTTCTAGTGTTGGTA	CATCAAACCATACCTTAGGAGCT
VlUbiquitin1（qRT-PCR）	BN000705^*	GTGGTATTATTGAGCCATCCTT	AACCTCCAATCCAGTTATCTAC
VlMybA1（promoter）	AB427165	TGCCATCTCCATGCAGC	GAGTCAACTCAACACAAGAGACC
VlMybA2（promoter）	AB073013	GCGATAAATTCACAAATAGAGGA	GAGTCAACTCAACACAAGAGACC

图1 VlMybA1、VlMybA2和VlMybA3的cDNA部分序列比对红色字母为PCR扩增区域，下划线表示引物位置，星号表示相同的序列，tga为终止密码子，蓝色字母代表3个VlMybA之间不同的碱基，用于区分VlMybA1、VlMybA2和VlMybA3。

Fig. 1 Alignment of VlMybA1，VlMybA2和VlMybA3 fragments The region used for PCR amplification is indicated with red letters，the primers are highlighted with underlined letters，asterisk means the same letter for the three genes，tga is the stop codonh，the blue letters represent the bases that were different between the three VlMybAs and were utilized to differentiate VlMybA1，VlMybA2和VlMybA3.

图2 不同发育阶段葡萄果实成熟参数（A，B），ABA和乙烯（C）及其合成关键基因表达（D，E）的变化

Fig. 2 Changes in grape berry ripening-related parameters（A，B），ABA and ethylene（C），and the expression of the genes（D，E） involved in biosynthesis of ABA and ethylene

图3 葡萄果粒ABA和ACC处理不同时间对其花青素含量及其合成相关基因表达的影响各处理分别与对照进行差异显著性分析，* 表示在0.05水平上显著差异，** 表示在0.01水平上显著差异，下同。

Fig 3 Effects of ABA and ACC treatment time on anthocyanin and anthocyanin-related gene expression The significant differences was analyzed between each treatment and control group was analyzed，* indicates a significant at the 0.05 level，**indicates a significant difference at the 0.01 level. The same below.

图4 VlMybA1和VlMybA2基因启动子的生物信息（A），ABA或ACC处理转基因愈伤的GUS染色和GUS活性测定（B，C）

Fig. 4 Promoter analysis of VlMybA1 and VlMybA2 genes（A），GUS staining and GUS activity measurement of VlMybA1/2 promoter:: GUS overexpressed by ABA or ACC treatment（B，C）

图5 ABA或不同ACC浓度处理对ABA和乙烯释放速率及其相应基因表达量的影响

Fig. 5 Effects of treatment with ABA or different ACC concentrations on ABA and ethylene release rates and corresponding gene expression

图6 ABA或ACC处理不同时长对VlMybA2相对丰度的影响图C，D显著性分析为同一基因不同处理间比较，不同小写字母代表差异显著（P < 0.05）。

Fig. 6 Effects of ABA or ACC treatment time on the relative abundance of VlMybA2 Figure C，D significance analysis shows the comparison of the same gene among different treatments. Different lowercase letters indicate significant differences at 0.05 level.

参考文献 51

[1]	Amandeep K S, Dennis J G, Lu J, Gu L W. 2011. Effects of exogenous abscisic acid on antioxidant capacities,anthocyanins,and flavonol contents of muscadine grape (Vitis rotundifolia) skins. Food Chemistry, 126 (3):982-988. doi: 10.1016/j.foodchem.2010.11.105 URL
[2]	Ban T, Ishimura M, Kobayashi S, Shiozaki S, Goto-Yamamoto N, Horiuchi S. 2003. Abscisic acid and 2,4-dichlorophenoxyacetic acid affect the expression of anthocyanin biosynthetic pathway genes in‘Kyoho’grape berries. The Journal of Horticultural Science and Biotechnology, 78:586-589. doi: 10.1080/14620316.2003.11511668 URL
[3]	Bonaventure G, Schuck S, Baldwin I T. 2011. Revealing complexity and specificity in the activation of lipase-mediated oxylipin biosynthesis:a specific role of the Nicotiana attenuata GLA 1 lipase in the activation of jasmonic acid biosynthesis in leaves and roots. Plant Cell and Environment, 34 (9):1507-1520. doi: 10.1111/pce.2011.34.issue-9 URL
[4]	Boss P K, Davies C, Robinson S P. 1996. Expression of anthocyanin biosynthesis pathway genes in red and white grapes. Plant Molecular Biology, 32:565-569. pmid: 8980508
[5]	Chervin C, El-Kereamy A, Roustan J P, Latche A, Lamon J, Bouzayen M. 2004. Ethylene seems required for the berry development and ripening in grape,a non-climacteric fruit. Plant Science, 167:1301-1305. doi: 10.1016/j.plantsci.2004.06.026 URL
[6]	Chervin C, Tira-Umphon A, Chatelet P, Jaunmau A, Boss P K, Tesniere C. 2009. Ethylene and other stimuli affect expression of the UDP glucose-flavonoid 3-O-glucosyltransferase in a non-climacteric fruit. Vitis, 48 (1):11-16.
[7]	Chervin C, Tira-umphon A, Terrier N, Zouine M, Severac D, Roustan J P. 2008. Stimulation of the grape berry expansion by ethylene and effects on related gene transcripts,over the ripening phase. Physiologia Plantarum, 134:534-546. doi: 10.1111/ppl.2008.134.issue-3 URL
[8]	Coombe B G. 1992. Research on development and ripening of the grape berry. American Journal of Enology and Viticulture, 43:101-110. doi: 10.5344/ajev.1992.43.1.101 URL
[9]	de Santis D, Bellincontro A, Forniti R, Botondi R. 2021. Time of Postharvest ethylene treatments affects phenols,anthocyanins,and volatile compounds of Cesanese red wine grape. Foods, 10 (2):doi:10.3390/foods10020322.
[10]	Deluc L G, Grimplet J, Wheatley M D, Tillett R, Quilici D R, Osborne C, Schooley D A, Schlauch K A, Cushman J C, Cramer G R. 2007. Transcriptomic and metabolite analyses of Cabernet Sauvignon grape berry development. BMC Genomics, 8:doi:10.1186/1471-2164-8-429.
[11]	El-Kereamy A, Chervin C, Roustan J P, Cheynier V, Souquet J M, Moutounet M, Raynal J, Ford C, Latché A, Pech J C, Bouzayen M. 2003. Exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis in grape berries. Physiologia Plantarum, 119:175-182. doi: 10.1034/j.1399-3054.2003.00165.x URL
[12]	Ferrara G, Mazzeo A, Matarrese A M S, Pacucci C, Punzi R, Faccia M, Trani A, Gambacorta G. 2015. Application of abscisic acid (S-ABA) and sucrose to improve colour,anthocyanin content and antioxidant activity of cv. Crimson Seedless grape berries. Australian Journal of Grape and Wine Research, 21:18-29. doi: 10.1111/ajgw.2015.21.issue-1 URL
[13]	Ford C M, Boss P K, Høj P B. 1998. Cloning and characterization of Vitis vinifera UDP-glucose:flavonoid 3-o-glucosyltransferase,a homologue of the enzyme encoded by the maize Bronze-1 locus that may primarily serve to glucosylate anthocyanidins in vivo. Journal of Biological Chemistry, 273:9224-9233. doi: 10.1074/jbc.273.15.9224 pmid: 9535914
[14]	Gagné S, Cluzet S, Mérillon J M, Gény L. 2011. ABA initiates anthocyanin production in grape cell cultures. Journal of Plant Growth Regulation, 30:1-10. doi: 10.1007/s00344-010-9165-9 URL
[15]	Gutha L R, Casassa L F, Harbertson J F, Naidu R A. 2010. Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L.) leaves. BMC Plant Biology,doi:10.1186/1471-2229-10-187.
[16]	Jefferson R A, Kavanagh T A, Bevan M W. 1987. GUS fusions,beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. The EMBO Journal, 6 (13):3901-3907. doi: 10.1002/embj.1987.6.issue-13 URL
[17]	Jeong S T, Goto-Yamamoto N, Kobayashi S, Esaka M. 2004. Effects of plant hormones and shading on the accumulation of anthocyanins and the expression of anthocyanin biosynthetic genes in grape berry skins. Plant Science, 167:247-252. doi: 10.1016/j.plantsci.2004.03.021 URL
[18]	Jiang Tianmei, Yin Xueren, Wang Ping, Sun Chongde, Xu Changjie, Li Xian, Chen Kunsong. 2011. Research advance in regulation of ethylene during ripening and senescence of non-climacteric fruit. Acta Horticulturae Sinica, 38 (2):371-378. (in Chinese)
	蒋天梅, 殷学仁, 王平, 孙崇德, 徐昌杰, 李鲜, 陈昆松. 2011. 乙烯调控非跃变型果实成熟衰老研究进展. 园艺学报, 38 (2):371-378.
[19]	Jiang Y M, Joyce D C. 2003. ABA effects on ethylene production,PAL activity,anthocyanin and phenolic contents of strawberry fruit. Plant Growth Regulation, 39:171-174. doi: 10.1023/A:1022539901044 URL
[20]	Kobayashi S, Goto-Yamamoto N, Hirochika H. 2004. Retrotransposon- induced mutations in grape skin color. Science, 304 (5673):982. doi: 10.1126/science.1095011 pmid: 15143274
[21]	Kojima K. 1996. Distribution and change of endogenous IAA and ABA in asparagus spear and orange fruit. Chemical Regulation Plants, 31:68-71.
[22]	Koyama K, Sadamatsu K, Goto-Yamamoto N. 2010. Abscisic acid stimulated ripening and gene expression in berry skins of the Cabernet Sauvignon grape. Functional & Integrative Genomics, 10:367-381.
[23]	Koyama R, Roberto S R, de Souza R T, Borges W F S, Anderson M, Waterhouse A L, Cantu D, Fidelibus M W, Blanco-Ulate B. 2018. Exogenous abscisic acid promotes anthocyanin biosynthesis and increased expression of flavonoid synthesis genes in Vitis vinifera × Vitis labrusca table grapes in a subtropical region. Frontiers in Plant Science,doi:10.3389/fpls.2018.00323.
[24]	Kuhn N, Guan L, Dai Z W, Wu B H, Lauvergeat V, Gomès E, Li S H, Godoy F, Arce-Johnson P, Delrot S. 2014. Berry ripening:recently heard through the grapevine. Journal of Experimental Botany, 65:4543-4559. doi: 10.1093/jxb/ert395 URL
[25]	Lai B, Li X J, Hu B, Qin Y H, Huang X M, Wang H C, Hu G B. 2014. LcMYB 1 is a key determinant of differential anthocyanin accumulation among genotypes,tissues,developmental phases and ABA and light stimuli in Litchi chinensis. PLoS ONE, 9 (1):e86293. doi: 10.1371/journal.pone.0086293 URL
[26]	Li D D, Shi W, Deng X X. 2002. Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene. Plant Cell Report, 21:153-156.
[27]	Liu M Y, Song C Z, Chi M, Wang T M, Zuo L L, Li X L, Zhang Z W, Xi Z M. 2016. The effects of light and ethylene and their interaction on the regulation of proanthocyanidin and anthocyanin synthesis in the skins of Vitis vinifera berries. Plant Growth Regulation, 79 (3):377-390. doi: 10.1007/s10725-015-0141-z URL
[28]	Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-∆∆CTmethod. Methods, 25:402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[29]	Lu Suwen, Zheng Xuan’ang, Wang Jiayang, Fang Jinggui. 2021. Research progress on the metabolism of flavonoids in grape. Acta Horticulturae Sinica, 48 (12):2506-2524. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0558 URL
	卢素文, 郑暄昂, 王佳洋, 房经贵. 2021. 葡萄类黄酮代谢研究进展. 园艺学报, 48 (12):2506-2524. doi: 10.16420/j.issn.0513-353x.2021-0558 URL
[30]	Ma W Y, Xu L L, Gao S W, Lyu X N, Cao X L, Yao Y X. 2021. Melatonin alters the secondary metabolite profile of grape berry skin by promoting VvMYB14-mediated ethylene biosynthesis. Horticulture Research,doi:10.1038/s41438-021-00478-2.
[31]	Merchante C, Vallarino J G, Osorio S, Araguez I, Villarreal N, Ariza M T, Martinez G A, Medina-Escobar N, Civello M P, Fernie A R, Botella M A, Valpuesta V. 2013. Ethylene is involved in strawberry fruit ripening in an organ-specific manner. Journal of Experimental Botany, 64 (14):4421-4439. doi: 10.1093/jxb/ert257 pmid: 24098047
[32]	Mou Wangshu. 2019. Effects and mechanism of abscisic acid and abscisic acid-ethylene interaction on tomato fruit ripening[Ph. D. Dissertation]. Hangzhou: Zhejiang University. (in Chinese)
	牟望舒. 2019. 脱落酸及脱落酸-乙烯互作调控番茄果实成熟的效应与机理[博士论文]. 杭州: 浙江大学.
[33]	Neff M, Chory J. 2003. Genetic interactions between phytochrome A,phytochrome B,and Cryptochrome 1 during Arabidopsis development. Plant Physiology, 118:27-36. doi: 10.1104/pp.118.1.27 URL
[34]	Palapol Y, Ketsa S, Lin-Wang K, Ferguson I B, Allan A C. 2009. A MYB transcription factor regulates anthocyanin biosynthesis in mangosteen (Garcinia mangostana L.) fruit during ripening. Planta, 226:1323-1334. doi: 10.1007/s00425-007-0568-1 URL
[35]	Peppi M C, Fidelibus M W, Dokoozlian N. 2006. Abscisic acid application timing and concentration affect firmness,pigmentation,and color of ‘Flame seedless’grapes. HortScience, 41:1440-1445. doi: 10.21273/HORTSCI.41.6.1440 URL
[36]	Peretz Y, Mozes-Koch R, Akad F, Tanne E, Czosnek H, Sela I. 2007. A universal expression/Silencing vector in plants. Plant Physiology, 145:1251-1263. doi: 10.1104/pp.107.108217 pmid: 17905866
[37]	Qi Yingwei, Liu Xuanshuo, Ding Yuduan, Jiang Yonghua, Zhang Yujie, Liu Jia, Jiang Zitao, Ren Xiaolin. 2021. Effect of abscisic acid treatment on apple fruit ripening and expression of genes related to ethylene biosynthesis and ethylene signal transduction. Food Science, 42 (17):225-232. (in Chinese) doi: 10.7506/spkx1002-6630-20200813-180
	戚英伟, 刘悬烁, 丁毓端, 姜永华, 张玉洁, 刘佳, 蒋紫洮, 任小林. 2021. 脱落酸处理对苹果成熟、乙烯合成及其信号转导基因表达的影响. 食品科学, 42 (17):225-232.
[38]	Ré D A, Raud B, Chan R L, Baldwin I T, Bonaventure G. 2012. RNAi-mediated silencing of the HD-Zip gene HD20 in Nicotiana attenuate affects benzyl acetone emission from corollas via ABA levels and the expression of metabolic genes. BMC Plant Biology, 12:60-75. doi: 10.1186/1471-2229-12-60
[39]	Shen X J, Zhao K, Liu L, Zhang K, Yuan H Z, Liao X, Wang Q, Guo X W, Li F, Li T H. 2014. A role for PacMYBA in ABA-regulated anthocyanin biosynthesis in red-colored sweet cherry cv. Hong Deng(Prunus avium L.). Plant Cell and Physiology, 55 (5):862-880. doi: 10.1093/pcp/pcu013 URL
[40]	Sun L, Zhang M, Ren J, Qi J X, Zhang G J, Leng P. 2010. Reciprocity between abscisic acid and ethylene at the onset of berry ripening and after harvest. BMC Plant Biology, 10:257. doi: 10.1186/1471-2229-10-257 pmid: 21092180
[41]	Sun Y L, Liu Q Z, Xi B, Dai H J. 2019. Study on the regulation of anthocyanin biosynthesis by exogenous abscisic acid in grapevine. Scientia Horticulturae, 250:294-301. doi: 10.1016/j.scienta.2019.02.054 URL
[42]	Walker A R, Lee E, Bogs J, McDavid D A J, Thomas M R, Robinson S P. 2007. White grapes arose through the mutation of two similar and adjacent regulatory genes. Plant Journal, 49:772-785. doi: 10.1111/j.1365-313X.2006.02997.x pmid: 17316172
[43]	Wang P P, Ge M Q, Yu A S, Song W, Fang J G, Leng X P. 2021. Effects of ethylene on berry ripening and anthocyanin accumulation of‘Fujiminori’grape in protected cultivation. Journal of the Science of Food and Agriculture, 102 (3):1124-1136. doi: 10.1002/jsfa.v102.3 URL
[44]	Wang P P, Yu A S, Yu X L, Mu Q, Haider M S, Wei R N, Leng X P, Fang J G. 2022. Transcriptome and metabolite integrated analysis reveals that exogenous ethylene controls berry ripening processes in grapevine. Food Research International, 155:111084. doi: 10.1016/j.foodres.2022.111084 URL
[45]	Wheeler S, Loveys B, Ford C, Davies C. 2009. The relationship between the expression of abscisic acid biosynthesis genes,accumulation of abscisic acid and the promotion of Vitis vinifera L. berry ripening by abscisic acid. Australian Journal of Grape and Wine Research, 15:195-204. doi: 10.1111/ajgw.2009.15.issue-3 URL
[46]	Yang S, Hoffman N. 1984. Ethylene biosynthesis and its regulation in higher plants. Annual Reviev of Plant Physiology and Plant Molecular Biology, 35:155-189.
[47]	Yao Y X, Li M, Liu Z, Hao Y J, Zhai H. 2007. A novel gene,screened by cDNA-AFLP approach,contributes to lowering the acidity of fruit in apple. Plant Physiology and Biochemistry, 45:139-145. doi: 10.1016/j.plaphy.2007.01.010 URL
[48]	Zaharah S S, Singh Z, Symons G M, Reid J B. 2013. Mode of action of abscisic acid in triggering ethylene biosynthesis and softening during ripening in mango fruit. Postharvest Biology and Technology, 75:37-44. doi: 10.1016/j.postharvbio.2012.07.009 URL
[49]	Zhang M, Leng P, Zhang G, Li X. 2009a. Cloning and functioning analysis of 9-cis-epoxycarotenoid dioxygenase (NCED) genes encoding a key enzyme during abscisic acid biosynthesis from peach and grape fruits. Journal of Plant Physiology, 166:1241-1252. doi: 10.1016/j.jplph.2009.01.013 URL
[50]	Zhang M, Yuan B, Leng P. 2009b. The role of ABA in triggering ethylene biosynthesis and ripening of tomato fruit. Journal of Experimental Botany, 60:1579-1588. doi: 10.1093/jxb/erp026 URL
[51]	Zhu Ziguo, Zhang Qingtian, Han Zhen, Li Bo, Li Guodong, Li Xiujie. 2021. VvMYB6,an R2R3-MYB transcription factor,is involved in anthocyanin biosynthesis of grapevine. Acta Horticulturae Sinica, 48 (3):465-476. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-0468 URL
	朱自果, 张庆田, 韩真, 李勃, 李国栋, 李秀杰. 2021. 欧洲葡萄VvMYB6正向调控花青素合成. 园艺学报, 48 (3):465-476. doi: 10.16420/j.issn.0513-353x.2020-0468 URL