紫花苜蓿老化种子吸胀过程中胚根线粒体抗氧化生理及激素响应规律

doi:10.3864/j.issn.0578-1752.2025.19.016

摘要/Abstract

摘要：

【目的】种子在贮藏过程中不可避免发生老化劣变，主要表现为线粒体功能障碍和活性氧（reactive oxygen species, ROS）累积。线粒体作为细胞能量代谢中心，其抗坏血酸—谷胱甘肽（AsA-GSH）循环是清除ROS的关键防线。探究紫花苜蓿（Medicago sativa L.）胚根线粒体AsA—GSH循环响应老化的抗氧化机制和种子吸胀期间胚根内源激素的变化规律，为揭示紫花苜蓿种子胚根细胞代谢与老化种子萌发过程之间的关系奠定基础。【方法】通过控制劣变处理获得中等活力紫花苜蓿种子，以未老化种子作为对照，研究了种子发芽特性、不同吸胀时间（6、12、24和36 h）胚根线粒体抗氧化酶活性、抗氧化物含量以及不同吸胀时间（12、24和36 h）胚根激素含量的变化规律。【结果】控制劣变处理抑制了紫花苜蓿种子的萌发和幼苗的生长，主要表现为降低种子的发芽势、发芽率和幼苗的根长。在吸胀36 h期间，老化种子胚根线粒体中过氧化氢酶（catalase，CAT）、抗坏血酸过氧化物酶（ascorbate peroxidase，APX）和单脱氢抗坏血酸还原酶（monodehydroascorbate reductase，MDHAR）活性以及抗坏血酸（ascorbic acid，AsA）和谷胱甘肽（glutathione，GSH）含量降低；从吸胀24 h开始，老化种子胚根线粒体中过氧化物酶（peroxidase，POD）和谷胱甘肽还原酶（glutathione reductase，GR）活性升高。随着吸胀的进行，老化和未老化种子AsA/氧化型抗坏血酸（oxidized ascorbic acid, DHA）比值差距渐小，GSH/氧化型谷胱甘肽（oxidized glutathione，GSSG）比值差距渐大。在吸胀过程中胚根脱落酸（abscisic acid，ABA）、赤霉素（gibberellin，GA₃）、生长素（indole acetic acid，IAA）和油菜素内酯（brassinolide，BR）含量的变化趋势基本一致。【结论】老化种子在吸胀过程中，胚根线粒体内的ROS积累，产生氧化损伤，造成种子萌发延迟。老化种子吸胀24 h后，胚根线粒体中POD和GR活性升高，保持一定抗氧化能力和萌发能力。在紫花苜蓿老化种子吸胀萌发过程中，POD和GR活性的增强对于种子活力水平的保持发挥重要作用，为深入探索AsA—GSH循环在抗氧化反应中的作用机制提供了依据，对优化牧草种质资源库的保存策略、延长种子寿命研究具有参考价值。

关键词: 紫花苜蓿, 种子吸胀, 线粒体, 抗氧化系统, AsA—GSH循环, 植物激素

Abstract:

【Objective】Aging deterioration of seeds is inevitable during storage, which is mainly manifested by mitochondrial dysfunction and reactive oxygen species (ROS) accumulation. Mitochondria, as the center of cellular energy metabolism, and its ascorbic acid-glutathione (AsA-GSH) cycle are the key defense to scavenging ROS. This experiment was designed to investigate the antioxidant mechanism of the mitochondrial AsA-GSH cycle and the rules of endogenous hormones changed in the radicle during imbibition of aged alfalfa (Medicago sativa L.) seeds, and to lay a foundation for revealing the relationship between the metabolism of the radicle cells and the imbibed process in the aged alfalfa seeds.【Method】In this experiment, seed samples were treated with controlled deterioration and obtained at a medium level of alfalfa seed vigor, and unaged seeds were used as the control. After the seed germination characteristics were tested, the changes in mitochondrial antioxidant enzyme activity and antioxidant content of the radicle during imbibition (6, 12, 24 and 36 h), and the changes in hormone content of the radicle during imbibition (12, 24 and 36 h).【Result】Controlled deterioration treatment inhibited the seed germination and seedling growth, which mainly presented the decrease in seed germination potential, germination percentage and root length of seedlings. The activities of catalase (CAT), ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR), as well as the contents of ascorbic acid (AsA) and glutathione (GSH) were decreased in the radicle mitochondria of aged seeds within 36 h imbibition; and the activities of peroxidase (POD) and glutathione reductase (GR) increased in the radicle mitochondria of aged seeds after 24 h of imbibition. With the progress of imbibition, the difference of AsA/ oxidized ascorbic acid (DHA) and GSH/ oxidized glutathione (GSSG) ratios between aged and unaged seeds was gradually reduced. The contents of abscisic acid (ABA), gibberellin (GA₃), growth hormone (IAA) and oleuropein lactone (BR) in the radicles presented the similar tendency during the imbibition.【Conclusion】During the process of imbibition, ROS accumulated in the mitochondria of the radicle, resulting in oxidative damage and delayed seed germination. After 24 h imbibition of aged seeds, POD and GR activities in radicle mitochondria increased to maintain a certain antioxidant capacity and germination ability. It could be suggested that the enhancement of POD and GR activities would play an important role in the maintenance of seed vigor level during the imbibition of aged alfalfa seeds, which provided a basis for an in-depth exploration of the mechanism of the role of the AsA-GSH cycle in antioxidant responses, and provided a reference for optimizing the conservation strategy of forage germplasm bank and prolonging seed life.

Key words: alfalfa, seed imbibition, mitochondria, antioxidant system, AsA-GSH cycle, phytohormone

李若鸿, 马馼, 赵士强, 毛培胜. 紫花苜蓿老化种子吸胀过程中胚根线粒体抗氧化生理及激素响应规律[J]. 中国农业科学, 2025, 58(19): 4014-4025.

LI RuoHong, MA Wen, ZHAO ShiQiang, MAO PeiSheng. Antioxidant Physiology and Hormonal Response Pattern of Embryonic Root Mitochondria During Imbibition of Aging Seeds of Alfalfa[J]. Scientia Agricultura Sinica, 2025, 58(19): 4014-4025.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2025.19.016

https://www.chinaagrisci.com/CN/Y2025/V58/I19/4014

图/表 6

图1

图2

图3

图4

图5

图6

参考文献 40

[1]	SOLBERG S Ø, YNDGAARD F, ANDREASEN C, VON BOTHMER R, LOSKUTOV I G, ASDAL Å. Long-term storage and longevity of orthodox seeds: A systematic review. Frontiers in Plant Science, 2020, 11: 1007. doi: 10.3389/fpls.2020.01007 pmid: 32719707
[2]	ZHANG K L, ZHANG Y, SUN J, MENG J S, TAO J. Deterioration of orthodox seeds during ageing: Influencing factors, physiological alterations and the role of reactive oxygen species. Plant Physiology and Biochemistry, 2021, 158: 475-485. doi: 10.1016/j.plaphy.2020.11.031 pmid: 33250322
[3]	吕巨智, 贺囡囡, 石达金, 唐国荣, 李发桥, 谢小东, 邹成林, 程伟东, 张述宽. 人工老化过程中玉米种子活力生理指标的变化. 江苏农业科学, 2023, 51(11): 68-73.
	LÜ J Z, HE N N, SHI D J, TANG G R, LI F Q, XIE X D, ZOU C L, CHENG W D, ZHANG S K. Changes of physiological indices of maize seed vitality during artificial aging. Jiangsu Agricultural Sciences, 2023, 51(11): 68-73. (in Chinese)
[4]	吴旭鹏, 张小燕. 人工老化对鹰嘴豆种子发芽和膜透性的影响. 青海畜牧兽医杂志, 2023, 53(2): 19-23.
	WU X P, ZHANG X Y. Effect of artificially ageing treatments on thegermination and membrane permeability of Cicer arietinum l. seed. Chinese Qinghai Journal of Animal and Veterinary Sciences, 2023, 53(2): 19-23. (in Chinese)
[5]	宋丽媛. 高温高湿对水稻种子萌发及生理生化特性的影响. 北方水稻, 2023, 53(2): 19-23.
	SONG L Y. Effects of high temperature and humidity on seed germination and physiological and biochemical characteristics of rice. North Rice, 2023, 53(2): 19-23. (in Chinese)
[6]	牛震, 姜汇, 张瑞, 徐礼莹, 吕广超, 张志新, 田迅. 少花蒺藜草异型性种子人工老化过程中抗氧化生理响应差异. 中国草地学报, 2023, 45(1): 115-120.
	NIU Z, JIANG H, ZHANG R, XU L Y, LÜ G C, ZHANG Z X, TIAN X. Differences in antioxidant physiological responses of heteromorphic Cenchrus pauciflorus seeds during artificial aging. Chinese Journal of Grassland, 2023, 45(1): 115-120. (in Chinese)
[7]	刘延安. 人工老化处理对藜麦种子活力及生理特性的影响. 乡村科技, 2023, 14(13): 71-73. doi:10.19345/j.cnki.1674-7909.2023.13.040.
	LIU Y A. Effects of artificial aging treatment on vigor and physiological characteristics of Chenopodium quinoa Willd. seeds. Rural Science and Technology, 2023, 14(13): 71-73. doi:10.19345/j.cnki.1674-7909.2023.13.040. (in Chinese)
[8]	刘娟, 归静, 高伟, 马俊峰, 王佺珍. 种子老化的生理生化与分子机理研究进展. 生态学报, 2016, 36(16): 4997-5006.
	LIU J, GUI J, GAO W, MA J F, WANG Q Z. Review of the physiological and biochemical reactions and molecular mechanisms of seed aging. Acta Ecologica Sinica, 2016, 36(16): 4997-5006. (in Chinese)
[9]	张立勇, 阮燕晔, 张春宇, 张立军. 种子吸胀萌发期抗氧化酶活性的研究进展. 安徽农业科学, 2005, 33(7): 1274-1275.
	ZHANG L Y, RUAN Y Y, ZHANG C Y, ZHANG L J. Research progress on antioxidant enzyme activity during imbibition germination of seeds. Journal of Anhui Agricultural Sciences, 2005, 33(7): 1274-1275. (in Chinese)
[10]	顾炳朝, 岳绪国, 杨军, 唐泽庆. 油菜种子老化处理后的生理生化变化. 福建农业学报, 2013, 28(4): 339-342.
	GU B C, YUE X G, YANG J, TANG Z Q. Physiological and biochemical characteristics of artificially aged rapeseeds. Fujian Journal of Agricultural Sciences, 2013, 28(4): 339-342. (in Chinese)
[11]	廖乐, 齐军仓, 华淼源, 林立昊, 惠宏杉, 吕小兰, 刘馨月, 王丹丹, 余泽新. 人工老化对大麦种子萌发过程中发芽特性及部分酶活性的影响. 麦类作物学报, 2015, 35(1): 71-79.
	LIAO L, QI J C, HUA M Y, LIN L H, HUI H S, LÜ X L, LIU X Y, WANG D D, YU Z X. Effects of artificial aging on germination characteristics and some enzymes' activities during imbibition of barley seeds. Journal of Triticeae Crops, 2015, 35(1): 71-79. (in Chinese)
[12]	韩亚琼. 人工老化条件下老芒麦生理生化特性及差异基因表达研究[D]. 呼和浩特: 内蒙古农业大学, 2018.
	HAN Y Q. Research on physiological and biochemical characteristics and differential expression gene during artificial aging in Elymus sibiricus L[D]. Hohhot: Inner Mongolia Agricultural University, 2018. (in Chinese)
[13]	刘备, 宋玉梅, 孙铭, 毛培胜. 燕麦劣变种子吸胀过程中线粒体AsA-GSH循环的生理响应. 草地学报, 2021, 29(2): 211-219. doi: 10.11733/j.issn.1007-0435.2021.02.001
	LIU B, SONG Y M, SUN M, MAO P S. Physiological responses of mitochondrial AsA-GSH cycle to imbibition of deteriated oat seeds. Acta Agrestia Sinica, 2021, 29(2): 211-219. (in Chinese) doi: 10.11733/j.issn.1007-0435.2021.02.001
[14]	蔡春菊, 范少辉, 刘凤, 曹帮华. 毛竹种子人工老化过程中生理生化变化. 林业科学, 2013, 49(8): 29-34.
	CAI C J, FAN S H, LIU F, CAO B H. Physiological and biochemical changes of moso bamboo (Phyllostachys edulis) seeds in artificial aging. Scientia Silvae Sinicae, 2013, 49(8): 29-34. (in Chinese)
[15]	王凤, 齐军仓, 林立昊, 郑许光, 郭亚南, 龚磊, 王少玉, 陈阿龙, 李忠豪, 宋瑞娇. 人工老化处理对大麦种子萌发早期胚乳内源激素的影响. 作物杂志, 2016(6): 160-167.
	WANG F, QI J C, LIN L H, ZHENG X G, GUO Y N, GONG L, WANG S Y, CHEN A L, LI Z H, SONG R J. Effects of artificial aging on endogenous hormones in the endosperm of barley seeds during early germination. Crops, 2016(6): 160-167. (in Chinese)
[16]	MÜLLER K, HESS B, LEUBNER-METZGER G. A role for reactive oxygen species in endosperm weakening. Seeds:Biology, development and ecology. Proceedings of the Eighth International Workshop on Seeds, Brisbane, Australia, May 2005. UK: CABI, 2007: 287-295.
[17]	GOGOI K, GOGOI H, BORGOHAIN M, SAIKIA R, CHIKKAPUTTAIAH C, HIREMATH S, BASU U. The molecular dynamics between reactive oxygen species (ROS), reactive nitrogen species (RNS) and phytohormones in plant’s response to biotic stress. Plant Cell Reports, 2024, 43(11): 263.
[18]	RAZA A, SALEHI H, RAHMAN M A, ZAHID Z, MADADKAR HAGHJOU M, NAJAFI-KAKAVAND S, CHARAGH S, OSMAN H S, ALBAQAMI M, ZHUANG Y H, et al. Plant hormones and neurotransmitter interactions mediate antioxidant defenses under induced oxidative stress in plants. Frontiers in Plant Science, 2022, 13: 961872.
[19]	HERNÁNDEZ-CARRANZA P, AVILA-SOSA R, VERA-LÓPEZ O, NAVARRO-CRUZ A R, RUÍZ-ESPINOSA H, RUIZ-LÓPEZ I I, OCHOA-VELASCO C E. Uncovering the role of hormones in enhancing antioxidant defense systems in stressed tomato (Solanum lycopersicum) plants. Plants, 2023, 12(20): 3648.
[20]	ZAYED M, EL-KAFAFI E S, EL HAFNAWY S, EL-ARABY H. Effect of auxin treatment on growth and physiological traits in two sunflower cultivars under saline conditions. Journal of Plant Production, 2017, 8(2): 335-345.
[21]	张盛楠, 黄益宗, 李颜, 保琼莉, 黄永春. Cd胁迫下不同外源植物激素对水稻幼苗抗氧化系统及Cd吸收积累的影响. 环境科学, 2021, 42(4): 2040-2046.
	ZHANG S N, HUANG Y Z, LI Y, BAO Q L, HUANG Y C. Effects of different exogenous plant hormones on the antioxidant system and Cd absorption and accumulation of rice seedlings under Cd stress. Environmental Science, 2021, 42(4): 2040-2046. (in Chinese)
[22]	ASSOCIATION I S T. International rules for seed testing[S]. Wallisellen: The International Seed Testing Association, 2023: 114.
[23]	LYU W H, SELINSKI J, LI L, DAY D A, MURCHA M W, WHELAN J, WANG Y. Isolation and respiratory measurements of mitochondria from Arabidopsis thaliana. Journal of Visualized Experiments, 2018(131): e56627.
[24]	米春娇, 洪流, 马馼, 毛培胜. 谷胱甘肽引发对老化燕麦种胚线粒体抗氧化特性的影响. 草业学报, 2024, 33(9): 51-59. doi: 10.11686/cyxb2023420
	MI C J, HONG L, MA W, MAO P S. Effects of glutathione priming on the mitochondrial antioxidant characteristics of aged oat seed embryos. Acta Prataculturae Sinica, 2024, 33(9): 51-59. (in Chinese)
[25]	YANG Y M, XU C N, WANG B M, JIA J Z. Effects of plant growth regulators on secondary wall thickening of cotton fibres. Plant Growth Regulation, 2001, 35(3): 233-237.
[26]	YANG J, ZHANG J, WANG Z, ZHU Q, WANG W. Hormonal changes in the grains of rice subjected to water stress during grain filling. Plant Physiology, 2001, 127(1): 315-323. doi: 10.1104/pp.127.1.315 pmid: 11553759
[27]	ZHAO J, LI G, YI G X, WANG B M, DENG A X, NAN T G, LI Z H, LI Q X. Comparison between conventional indirect competitive enzyme-linked immunosorbent assay (icELISA) and simplified icELISA for small molecules. Analytica Chimica Acta, 2006, 571(1): 79-85. pmid: 17723423
[28]	米春娇, 洪流, 马馼, 毛培胜. 谷胱甘肽引发对老化燕麦种子发芽与幼苗生长特性的影响. 草地学报, 2024, 32(3): 928-934. doi: 10.11733/j.issn.1007-0435.2024.03.029
	MI C J, HONG L, MA W, MAO P S. Effects of glutathione priming on germination and seedling growth characteristics of aged oat seeds. Acta Agrestia Sinica, 2024, 32(3): 928-934. (in Chinese)
[29]	张玲, 曹磊, 蔡诚, 颜欣怡, 项博才, 艾佳, 詹新杨, 宋有洪, 朱玉磊. 自然老化条件下冬小麦种子活力及生理指标变化. 中国农业科学, 2025, 58(5): 877-889. doi: 10.3864/j.issn.0578-1752.2025.05.005.
	ZHANG L, CAO L, CAI C, YAN X Y, XIANG B C, AI J, ZHAN X Y, SONG Y H, ZHU Y L. Changes in seed vigor and physiological index of winter wheat under natural aging condition. Scientia Agricultura Sinica, 2025, 58(5): 877-889. doi: 10.3864/j.issn.0578-1752.2025.05.005. (in Chinese)
[30]	朱迎树. 引发对老化大麦种子生理生化变化影响的研究[D]. 石河子: 石河子大学, 2019.
	ZHU Y S. Effects of priming on physiological and biochemical changes of aging barley seeds[D]. Shihezi: Shihezi University, 2019. (in Chinese)
[31]	GILL S S, TUTEJA N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry, 2010, 48(12): 909-930. doi: 10.1016/j.plaphy.2010.08.016 pmid: 20870416
[32]	VAN BREUSEGEM F, VRANOVÁ E, DAT J F, INZÉ D. The role of active oxygen species in plant signal transduction. Plant Science, 2001, 161(3): 405-414.
[33]	许英, 陈建华, 朱爱国, 栾明宝, 王晓飞, 孙志民. 低温胁迫下植物响应机理的研究进展. 中国麻业科学, 2015, 37(1): 40-49.
	XU Y, CHEN J H, ZHU A G, LUAN M B, WANG X F, SUN Z M. Research progress on response mechanism of plant under low temperature stress. Plant Fiber Sciences in China, 2015, 37(1): 40-49. (in Chinese)
[34]	孙守江, 唐艺涵, 马馼, 李曼莉, 毛培胜. 紫花苜蓿种子吸胀期胚根线粒体AsA-GSH循环对低温胁迫的响应. 草业学报, 2023, 32(3): 152-162. doi: 10.11686/cyxb2022272
	SUN S J, TANG Y H, MA W, LI M L, MAO P S. Response of the mitochondrial AsA-GSH cycle during alfalfa seed germination under low temperature stress. Acta Prataculturae Sinica, 2023, 32(3): 152-162. (in Chinese)
[35]	廖乐. 人工老化对大麦种子生理生化特性和麦芽品质影响的研究[D]. 石河子: 石河子大学, 2015.
	LIAO L. Effects of artificial aging on physiological and biochemical characteristics and malt quality of barley seeds[D]. Shihezi: Shihezi University, 2015. (in Chinese)
[36]	薛爽, 饶丽莎, 左丹丹, 汪凤林, 林思祖. 植物低温胁迫响应机理的研究进展. 安徽农业科学, 2016, 44(33): 17-19, 48.
	XUE S, RAO L S, ZUO D D, WANG F L, ZUO D D, WANG F L, LIN S Z. Research progress on the response mechanism of plants to low temperature stress. Journal of Anhui Agricultural Sciences, 2016, 44(33): 17-19, 48. (in Chinese)
[37]	石金玉, 孙守江, 马馼, 毛培胜. 不同温度条件下老芒麦种子萌发特性和抗氧化生理研究. 草地学报, 2024, 32(10): 3151-3158. doi: 10.11733/j.issn.1007-0435.2024.10.015
	SHI J Y, SUN S J, MA W, MAO P S. Study on germination characteristics and antioxidant physiology of Siberian wildrye seeds under different temperature conditions. Acta Agrestia Sinica, 2024, 32(10): 3151-3158. (in Chinese)
[38]	马书燕, 李吉跃, 彭祚登. 人工老化过程中柔枝松种子内源激素含量变化的研究. 种子, 2010, 29(2): 4-8.
	MA S Y, LI J Y, PENG Z D. Study on the variation changes of endogenous hormone in the seeds of Pinus flexilis James during artificial aging course. Seed, 2010, 29(2): 4-8. (in Chinese)
[39]	王凤. 人工老化对大麦种子萌发期内源激素、淀粉代谢及麦芽品质的影响[D]. 石河子: 石河子大学, 2017.
	WANG F. Effects of artificial aging on endogenous hormones, starch metabolism and malt quality of barley seeds in germination[D]. Shihezi: Shihezi University, 2017. (in Chinese)
[40]	刘春华, 王鹏, 夏超, 董晓秋, 刘兵, 赵丹, 韦如俊, 兰海. 种子休眠与萌发相关激素突变体研究进展. 作物杂志, 2014(3): 139-142.
	LIU C H, WANG P, XIA C, DONG X Q, LIU B, ZHAO D, WEI R J, LAN H. Overview of hormone mutants related on seed dormancy and germination. Crops, 2014(3): 139-142. (in Chinese)