褪黑素对番茄衰老叶片抗氧化系统的影响

doi:10.16420/j.issn.0513-353x.2023-0964

摘要/Abstract

摘要：

为了探讨褪黑素（melatonin，MT）对番茄（Solanum lycopersicum L.）叶片衰老的调控机理，以‘金棚1号’番茄以及MT合成关键基因L–色氨酸脱羧酶编码基因SlTDC过量表达和敲除的番茄为试材，以黑暗模拟衰老，研究MT对番茄衰老相关基因Senescence-associated gene 12（SAG12）表达、活性氧（ROS）积累、抗氧化系统的影响。结果表明，随着黑暗处理时间的延长，番茄叶片的SAG12 mRNA表达量、过氧化氢（H₂O₂）和超氧阴离子积累量显著增加，超氧化物歧化酶（Superoxide dismutase，SOD）、过氧化物酶（peroxidase，POD）和抗坏血酸过氧化物酶（ascorbate peroxidase，APX）活性及其基因表达量和氧化还原物质抗坏血酸（reduced ascorbic acid，AsA）、还原型谷胱甘肽（glutathione，GSH）含量随着黑暗处理时间的延长亦趋于上升。与清水对照相比，外源MT处理的SAG12 mRNA表达量、H₂O₂和$\mathrm{O}_{2}^{\bar{·}}$含量显著下降，而SOD、POD和APX活性及其基因表达和氧化还原物质含量显著增加；SlTDC过量表达同样可以显著下调黑暗处理下番茄叶片的SAG12 mRNA表达量及H₂O₂和$\mathrm{O}_{2}^{\bar{·}}$含量，增强抗氧化能力，而抑制或敲除SlTDC则上调了黑暗下番茄叶片的衰老基因表达和活性氧（ROS）的积累，加速叶片衰老；田间应用研究发现，外源MT亦可以显著下调日光温室番茄衰老叶片（叶龄 > 35 d）衰老基因表达和ROS的积累，延缓叶龄增大导致的叶片衰老。综上说明，MT可通过下调衰老基因表达和上调抗氧化能力，加速ROS的清除，延缓黑暗或依赖于叶龄的番茄叶片衰老。

关键词: 番茄, 褪黑素, 叶片衰老, 活性氧, 抗氧化系统, SAG12, SlTDC

Abstract:

To explore the regulation mechanism of exogenous melatonin（MT）on leaf senescence of tomato（Solanum lycopersicum L.）,‘Jinpeng 1'tomato and SlTDC（L-tryptophan decarboxylase encoding gene，a key gene for MT synthesis）overexpression and knockout transgenic tomato plants were used as experimental materials and dark condition was used to simulate senescence，the effects of MT on senescence-related gene（SAG12）expression,reactive oxygen species（ROS）accumulation and antioxidant system were studied in this paper. The results showed that the mRNA abundance of SAG12，the accumulation of hydrogen peroxide（H₂O₂）and superoxide anion superoxide anion，the activities of superoxide dismutase（SOD），peroxidase（POD）and ascorbate peroxidase（APX）and their gene expression，and the content of ascorbic acid（AsA）and glutathione（GSH）in tomato leaves increased significantly with the prolongation of dark treatment time. Compared with the control（H₂O），the tomato treated with exogenous MT showed lower SAG12 mRNA abundance，the content of H₂O₂and superoxide anion and higher activities of SOD，POD，APX as well as their gene expression and the content of redox substances. Meanwhile，overexpression of SlTDC also significantly down-regulated the mRNA abundance of SAG12 and the content of H₂O₂and superoxide anion，and enhanced antioxidant capacity，while inhibition or knockout of SlTDC up-regulated the expression of senescence genes and the accumulation of reactive oxygen species（ROS）in tomato leaves under dark treatment，which accelerated leaf senescence. Moreover，field application studies showed that exogenous MT could also significantly down-regulate the expression of senescence genes and accumulation of ROS in tomato senescent leaves（leaf age > 35 d）in solar greenhouse，and delay leaf senescence caused by increasing leaf age. In summary，MT can accelerate the removal of ROS by down-regulating the expression of senescence genes and up-regulating antioxidant capacity，and then delay the senescence of dark or leaf-age-dependent tomato leaves.

Key words: tomato, melatonin, leaf senescence, reactive oxygen species, antioxidant system, SAG12, SlTDC

王雅楠, 刘绪涛, 景桐彤, 柴亚婷, 张晓伟, 艾希珍, 毕焕改. 褪黑素对番茄衰老叶片抗氧化系统的影响[J]. 园艺学报, 2024, 51(11): 2594-2606.

WANG Yanan, LIU Xutao, JING Tongtong, CHAI Yating, ZHANG Xiaowei, AI Xizhen, BI Huangai. Effect of Melatonin on Antioxidant System of Tomato Senescent Leaves[J]. Acta Horticulturae Sinica, 2024, 51(11): 2594-2606.

图/表 10

表1 RT-qPCR引物序列

Table 1 The primer sequences of real-time qPCR

基因 Gene	登录号 Accession number	Solyc基因号 Solyc gene number	引物序列（5′-3′） Primer sequence
ß-Actin	XM_004243253.4	Solyc07g054860.1.1	F：TTTGCTGGTGATGATGCC
ß-Actin	XM_004243253.4	Solyc07g054860.1.1	R：CCTTAGGGTTGAGAGGTGCTT
SAG12	XM_004233006	Solyc02g076910.3.1	F：GGAGCTCTCAATTAGCTTCATC
SAG12	XM_004233006	Solyc02g076910.3.1	R：GTACCCTTTATCTTCTCCTGCAT
SOD	NM_001347093	Solyc08g079830.4.1	F：CTTCTTCGCAGTTTGAACG R：AACACAGCCTTGACACGA
POD	NM_001247203	Solyc01g006300.3.1	F：GGTCCAACATGGCAAGTTCT R：ACATCTTGCCCTTCCAAATG
APX	NM_001306170	Solyc06g060260.3.1	F：AGCTTAAGATTCGAAATTG R：GATCTCCCCAAAGTGTGAGCC

图1 外源褪黑素（MT）对黑暗条件下番茄叶片SAG12表达量的影响不同小写字母表示差异显著（P < 0.05）。下同。

Fig. 1 Effects of exogenous melatonin（MT）on the expression of SAG12 in tomato leaves under dark condition Different letters after data indicate significant differences（P < 0.05）. The same below.

图2 外源褪黑素（MT）对黑暗条件下番茄叶片ROS含量影响

Fig. 2 Effects of exogenous melatonin（MT）on ROS content in tomato leaves under dark condition

图3 外源褪黑素（MT）对黑暗条件下番茄叶片抗氧化酶活性影响

Fig. 3 Effects of exogenous melatonin（MT）on the activities and gene expression of antioxidant enzymes in tomato leaves in darkness

图4 外源褪黑素（MT）对黑暗番茄叶片氧化还原物质的影响

Fig. 4 Effects of exogenous melatonin（MT）on redox substances in dark tomato leaves

图5 SlTDC1对黑暗下番茄叶片SAG12表达量的影响 WT：野生型植株；OE14、OE10：过量表达植株；RNAi4、RNAi7：干扰植株；Crispr1：敲除植株。下同。

Fig. 5 Effect of SlTDC1 on the relative expression of SAG12 in tomato leaves under darkness WT：Wild-type plants；OE14，OE10：Overexpression plants；RNAi4，RNAi7：Interference plants；Crispr1：Knockout plants. The same below.

图6 SlTDC1对黑暗下番茄叶片抗氧化酶活性及其基因表达量的影响

Fig. 6 Effects of SlTDC1 on antioxidant enzyme activities and gene expression in tomato leaves under darkness

图7 SlTDC1对黑暗下番茄叶片氧化还原物质含量的影响

Fig. 7 Effects of SlTDC1 on redox substances contents in tomato leaves under darkness

图8 外源褪黑素（MT）对不同叶龄番茄叶片SAG12表达量的影响

Fig. 8 Effects of exogenous melatonin（MT）on SAG12 expression in tomato leaves at different leaf ages

图9 外源褪黑素（MT）对不同叶龄番茄叶片ROS含量的影响

Fig. 9 Effects of exogenous melatonin（MT）on ROS content in tomato leaves at different leaf ages

参考文献 47

[1]	An R H, Liu X S, Luo S F, Li G F, Hu H L, Li P X. 2022. Taxifolin delays the degradation of chlorophyll in pakchoi(Brassica rapa L subsp. chinensis)by regulating the ascorbate-glutathione cycle. Postharvest Biology and Technology, 191:111982.
[2]	Arnao M B, Hernández-Ruiz J. 2009. Protective effect of melatonin against chlorophyll degradation during the senescence of barley leaves. Journal of Pineal Research, 46 (1):58-63. doi: 10.1111/j.1600-079X.2008.00625.x pmid: 18691358
[3]	Arnao M B, Hernández-Ruiz J. 2019. Melatonin:a new plant hormone and/or a plant master regulator? Trends in Plant Science, 24 (1):38-48.
[4]	Byeon Y, Park S, Lee H Y, Kim Y S, Back K. 2014. Elevated production of melatonin in transgenic rice seeds expressing rice tryptophan decarboxylase. Journal of Pineal Research, 56 (3):275-282. doi: 10.1111/jpi.12120 pmid: 24433490
[5]	Chen Xian, Yang Yong, Liu Fengquan. 2020. Research progress of plant melatonin. Jiangsu Agricultural Sciences, 48 (24):17-24. (in Chinese)
	陈贤, 杨勇, 刘凤权. 2020. 植物褪黑素的研究进展. 江苏农业科学, 48 (24):17-24.
[6]	Desclos-Théveniau M, Coquet L, Jouenne T, Etienne P. 2015. Proteomic analysis of residual proteins in blades and petioles of fallen leaves of Brassica napus. Plant Biology, 17 (2):408-418. doi: 10.1111/plb.12241 pmid: 25294336
[7]	Fu M N, Yuan C S, Song A H, Lu J, Wang X J, Sun S L. 2019. AtWDS1 negatively regulates age-dependent and dark-induced leaf senescence in Arabidopsis. Plant Science, 285:44-54.
[8]	Gong B, Yan Y Y, Wen D, Shi Q H. 2017. Hydrogen peroxide produced by NADPH oxidase:a novel downstream signaling pathway in melatonin-induced stress Solanum lycopersicum. Physiologia Plantarum, 160:396-409.
[9]	Gong Biao, Shi Qinghua. 2017. Research progress of melatonin in horticultural crops. Scientia Agricultura Sinica, 50 (12):2326-2337. (in Chinese)
	巩彪, 史庆华. 2017. 园艺作物褪黑素的研究进展. 中国农业科学, 50 (12):2326-2337. doi: 10.3864/j.issn.0578-1752.2017.12.013
[10]	Guo Mingxin, Liu Jiajia, Hou Linlin, Zhang Xiaotian, Liu Mianxiao. 2021. Research progress on the production and scavenging mechanism of reactive oxygen species in plants. Science & Technology Vision,(8):104-106. (in Chinese)
	郭明欣, 刘佳佳, 侯琳琳, 张笑天, 刘含笑. 2021. 植物体内活性氧的产生及清除机制研究进展. 科技视界,(8):104-106.
[11]	Guo P R, Li Z H, Huang P X, Li B S, Fang S, Chu J F, Guo H W. 2017. A tripartite amplification loop involving the transcription factor WRKY75,salicylic acid,and reactive oxygen species accelerates leaf senescence. The Plant Cell, 29 (11):2854-2870.
[12]	Guo Y, Cai Z, Gan S. 2004. Transcriptome of Arabidopsis leaf senescence. Plant,Cell and Environment, 27:521-549.
[13]	He Zhichong. 2019. Study on the senescence process and regulation of Ginkgo biloba leaves[M. D. Dissertation]. Yangzhou: Yangzhou University. (in Chinese)
	何智冲. 2019. 银杏叶片衰老转色进程及调控研究[硕士论文]. 扬州: 扬州大学.
[14]	James M, Poret M, Masclaux-Daubresse C, Marmagne A, Coquet L, Jouenne T, Chan P, Trouverie J, Etienne P. 2018. SAG12,a major cysteine protease involved in nitrogen allocation during senescence for seed production in Arabidopsis thaliana. Plant & Cell Physiology, 59 (10):2052-2063.
[15]	Jia Le,Han Yanchao,Fang Xiangjun,Chen Hangjun,Gao Haiyan. 2021. Effects of melatonin treatment on postharvest quality and active oxygen metabolism of Lentinula edodes. Food Science, 42 (23):229-236. (in Chinese)
	贾乐, 韩延超, 房祥军, 陈杭君, 郜海燕. 2021. 褪黑素处理对香菇采后品质及活性氧代谢的影响. 食品科学, 42 (23):229-236.
[16]	Jing T T, Liu K, Wang Y N, Ai X Z, Bi H G. 2022. Melatonin positively regulates both dark- and age-induced leaf senescence by reducing ROS accumulation and modulating abscisic acid and auxin biosynthesis in cucumber plants. International Journal of Molecular Sciences, 23:3576.
[17]	Kang K, Kim Y S, Park S, Back K. 2009. Senescence-induced serotonin biosynthesis and its role in delaying senescence in rice leaves. Plant Physiology, 150 (3):1380-1393. doi: 10.1104/pp.109.138552 pmid: 19439571
[18]	Khanna-Chopra R. 2012. Leaf senescence and abiotic stresses share reactive oxygen species-mediated chloroplast degradation. Protoplasma, 249 (3): 469-481. doi: 10.1007/s00709-011-0308-z pmid: 21805384
[19]	Li Hesheng, Sun Qun, Zhao Shijie, Zhang Wenhua. 2000. Plant physiological and biochemical experimental principles and techniques. Beijing:Higher Education Pres:164-167. (in Chinese)
	李合生, 孙群, 赵世杰, 章文华. 2000. 植物生理生化实验原理和技术. 北京:高等教育出版社:164-167.
[20]	Li Junming, Xiang Chaoyang, Wang Xiaoxuan, Guo Yanmei, Huang Zejun, Liu Lei, Li Xin, Du Yongchen. 2021. Current situation of tomato industry in China during“The Thirteenth Five-year Plan”period and future prospect. Chinese Vegetables,(2):13-20. (in Chinese)
	李君明, 项朝阳, 王孝宣, 国艳梅, 黄泽军, 刘磊, 李鑫, 杜永臣. 2021. “十三五”我国番茄产业现状及展望. 中国蔬菜,(2):13-20.
[21]	Lim J, Park J H, Jung S, Hwang D, Nam H G, Hong S. 2018. Antagonistic roles of PhyA and PhyB in far-red light-dependent leaf senescence in Arabidopsis thaliana. Plant & Cell Physiology, 59 (9):1753-1764.
[22]	Liu F J, Zhang X W, Cai B B, Pan D Y, Fu X, Bi H G, Ai X Z. 2020. Physiological response and transcription profiling analysis reveal the role of glutathione in H₂S-induced chilling stress tolerance of cucumber seedlings. Plant Science, 291:110363.
[23]	Liu J L, Wang W X, Wang L Y, Sun Y. 2015. Exogenous melatonin improves seedling health index and drought tolerance in tomato. Plant Growth Regulation, 77 (3):317-326.
[24]	Liu K, Jing T T, Wang Y N, Ai X Z, Bi H G. 2022. Melatonin delays leaf senescence and improves cucumber yield by modulating chlorophyll degradation and photoinhibition of PSⅡ and PSⅠ. Environmental and Experimental Botany, 200:104915.
[25]	Liu X T, Wang Y N, Feng Y Q, Zhang X W, Bi H G, Ai X Z. 2023. SlTDC1 overexpression promoted photosynthesis in tomato under chilling stress by improving CO₂ assimilation and alleviating photoinhibition. International Journal of Molecular Sciences, 24 (13):11042.
[26]	Mao Jingjing. 2020. Study on the mechanism of exogenous melatonin delaying the senescence of Arabidopsis thaliana leaves[M. D. Dissertation]. Sichuan: Sichuan Agricultural University. (in Chinese)
	毛晶晶. 2020. 外源褪黑素延缓拟南芥叶片衰老的机制探讨[硕士论文]. 四川: 四川农业大学.
[27]	Nakano Y, Asada K. 1987. Purification of ascorbate peroxidase in spinach chloroplasts; its inactivation in ascorbate-depleted medium and reactivation by monodehydroascorbate radical. Plant & Cell Physiology, 28 (1):131-140.
[28]	Smart C M. 1994. Gene expression during leaf senescence. New Phytologist, 126 (3):419-448. doi: 10.1111/j.1469-8137.1994.tb04243.x pmid: 33874468
[29]	Song L, Tan Z, Zhang W, Li Q, Jiang Z, Shen S, Luo S, Chen X. 2022. Exogenous melatonin improves the chilling tolerance and preharvest fruit shelf life in eggplant by affecting ROS- and senescence-related processes. Horticultural Plant Journal, 9 (3):523-540.
[30]	Sun Hu. 2016. Effects of nitrogen fertilizer on tomato aging control and production. Northern Horticulture,(24):35-37. (in Chinese)
	孙虎. 2016. 氮肥对番茄衰老调控及产量的影响. 北方园艺,(24):35-37.
[31]	Tan X L, Zhao Y T, Shan W, Kuang J F, Lu W J, Su X G, Tao N G, Lakshmanan P, Chen J Y. 2020. Melatonin delays leaf senescence of postharvest Chinese flowering cabbage through ROS homeostasis. Food Research International, 138:109790.
[32]	Tsunoda Y, Hano S, Imoto N, Shibuya T, Ikeda H, Amagaya K, Kato K, Shirakawa H, Aso H, Kanayama Y. 2021. Physiological roles of tryptophan decarboxylase revealed by overexpression of SlTDC1in tomato. Scientia Horticulturae, 275:109672.
[33]	Wada S, Ishida H, Izumi M, Yoshimoto K, Ohsumi Y, Mae T, Makino A. 2009. Autophagy plays a role in chloroplast degradation during senescence in individually darkened leaves. Plant Physiology, 149 (2):885-893. doi: 10.1104/pp.108.130013 pmid: 19074627
[34]	Wang Aiguo, Luo Guanghua. 1990. Quantitative relationship between hydroxylamine reaction and free radical of superoxide in plants. Plant Physiology Bulletin,(6):55-57. (in Chinese)
	王爱国, 罗广华. 1990. 植物的超氧物自由基与羟胺反应的定量关系. 植物生理学通讯,(6):55-57.
[35]	Wang P, Sun X, Li C, Wei Z W, Liang D, Ma F W. 2013. Long-term exogenous application of melatonin delays drought-induced leaf senescence in apple. Journal of Pineal Research, 54 (3):292-302. doi: 10.1111/jpi.12017 pmid: 23106234
[36]	Wang P, Yin L H, Liang D, Li C, Ma F W, Yue Z Y. 2012. Delayed senescence of apple leaves by exogenous melatonin treatment:toward regulating the ascorbate-glutathione cycle. Journal of Pineal Research, 53:11-20.
[37]	Wang Rong, Cheng Menglin, Liu Huina, Zhao Daqiu, Tao Jun. 2018. Effects of melatonin on flavonoids content and related gene expression levels of gardenia under dark conditions. Bulletin of Botanical Research, 38 (4):559-567. (in Chinese)
	王荣, 成梦琳, 刘慧娜, 赵大球, 陶俊. 2018. 黑暗条件下褪黑素对栀子叶片类黄酮含量及相关基因表达水平的影响. 植物研究, 38 (4):559-567. doi: 10.7525/j.issn.1673-5102.2018.04.010
[38]	Wang Rong. 2019. Effects of melatonin on senescence process of gardenia leaves under darkness[M. D. Dissertation]. Jiangsu: Yangzhou University. (in Chinese)
	王荣. 2019. 褪黑素对黑暗胁迫下栀子叶片衰老进程的影响[硕士论文]. 江苏: 扬州大学.
[39]	Wang Yuhang, Li Dou, Wang Chunheng, Jin Xin, Cheng Yajuan, Dai Zibo, Feng Lidan, Yang Jiangshan. 2024. The effect of melatonin on the subcellular reactive oxygen species metabolism during development and senescence in grape leaves. Acta Horticulturae Sinica, 51 (1):103-120. (in Chinese) doi: 10.16420/j.issn.0513-353x.2022-1214
	王宇航, 李斗, 王春恒, 金鑫, 陈亚娟, 戴子博, 冯丽丹, 杨江山. 2024. 褪黑素对葡萄叶片发育衰老过程中亚细胞活性氧代谢的影响. 园艺学报, 51 (1):103-120. doi: 10.16420/j.issn.0513-353x.2022-1214
[40]	Weaver L M, Amasino R M. 2001. Senescence is induced in individually darkened Arabidopsis leaves,but inhibited in whole darkened plants. Plant Physiology, 127:876-886. pmid: 11706170
[41]	Woo H R, Kim H J, Lim P O, Nam H G. 2019. Leaf senescence:systems and dynamics aspects. Annual Review of Plant Biology, 70:347-376.
[42]	Woolhouse H W. 1967. The nature of senescence in plants. Symposia of the Society for Experimental Biology, 21:179-213. pmid: 4860954
[43]	Yu J C, Lu J Z, Cui X Y, Guo L, Wang Z J, Liu Y D, Wang F, Qi M F, Liu Y F, Li T L. 2022. Melatonin mediates reactive oxygen species homeostasis via SlCV to regulate leaf senescence in tomato plants. Journal of Pineal Research, 73 (2):e12810.
[44]	Zhang H H, Xu Z S, Huo Y Z, Guo K W, Wang Y, He G Q, Sun H W, Li M B, Li X, Xu N, Sun G Y. 2020. Overexpression of Trx CDSP32 gene promotes chlorophyll synthesis and photosynthetic electron transfer and alleviates cadmium-induced photoinhibition of PSⅡ and PSⅠ in tobacco leaves. Journal of Hazardous Materials, 398:122899.
[45]	Zhang T, Wang Y, Ma X J, Ouyang Z P, Deng L, Shen S S, Dong X X, Du N S, Dong H, Guo Z X, Meng G, Piao F Z, Sun K L. 2022. Melatonin alleviates copper toxicity via improving ROS metabolism and antioxidant defense response in tomato seedlings. Antioxidants, 11 (4):758.
[46]	Zhao Y, Tan D X, Lei Q, Chen H, Wang L, Li Q T, Gao Y A, Kong J. 2013. Melatonin and its potential biological functions in the fruits of sweet cherry. Journal of Pineal Research, 55 (1):79-88. doi: 10.1111/jpi.12044 pmid: 23480341
[47]	Zheng Sha,Zhu Yingxue,Zhu Baozhong,Zhang Hong,Xiang Zhonghuai,Zhao Aichun. 2021. Transgenic overexpression of MnTDC gene from mulberry in tobacco enhances its salt tolerance. Science of Sericulture, 47 (5):401-408. (in Chinese)
	郑莎, 朱映雪, 朱宝中, 张红, 向仲怀, 赵爱春. 2021. 过表达桑树色氨酸脱羧酶基因(MnTDC)转基因烟草提高其耐盐性. 蚕业科学, 47 (5):401-408.