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作物学报 ›› 2022, Vol. 48 ›› Issue (8): 1996-2006.doi: 10.3724/SP.J.1006.2022.13047

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

人工老化玉米种胚ROS产生及ATP合成酶亚基mRNA完整性研究

王天波(), 赫文学, 张峻铭, 吕伟增, 梁雨欢, 卢洋, 王雨露, 谷丰序, 宋词, 陈军营*()   

  1. 河南农业大学农学院, 河南郑州 450046
  • 收稿日期:2021-07-09 接受日期:2021-11-29 出版日期:2022-08-12 网络出版日期:2021-12-24
  • 通讯作者: 陈军营
  • 作者简介:E-mail: 15093455085@163.com
  • 基金资助:
    国家自然科学基金项目(31971998);国家自然科学基金项目(31571761)

ROS production and ATP synthase subunit mRNAs integrity in artificially aged maize embryos

WANG Tian-Bo(), HE Wen-Xue, ZHANG Jun-Ming, LYU Wei-Zeng, LIANG Yu-Huan, LU Yang, WANG Yu-Lu, GU Feng-Xu, SONG Ci, CHEN Jun-Ying*()   

  1. College of Agronomy, Henan Agricultural University, Zhengzhou 450046, Henan, China
  • Received:2021-07-09 Accepted:2021-11-29 Published:2022-08-12 Published online:2021-12-24
  • Contact: CHEN Jun-Ying
  • Supported by:
    National Natural Science Foundation of China(31971998);National Natural Science Foundation of China(31571761)

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摘要：

为了揭示玉米种子衰老机理, 本研究以“郑单958”玉米杂交种为材料, 研究了人工老化处理(温度45℃﹑相对湿度100%)后种子活力﹑种胚O2-产生和积累﹑种胚ATP含量和ATP合成酶活性, 并用反转录阻断-双引物扩增法分析种胚及不同器官(盾片﹑胚根和胚芽) ATP合成酶亚基mRNA的完整性。结果表明, 人工老化处理后, 玉米种子的萌发率和胚根生长速率下降; 种胚生活力降低; 种子呼吸强度减弱。种胚O2-产生速率在老化3 d时达到高峰, 随后下降; 胚根和胚芽O2-产生速率和含量显著增加; 种胚ATP含量和ATP合成酶活性降低。ATP合成酶ε亚基和γ亚基的mRNA完整性先升高后降低; δ亚基mRNA的完整性没有明显变化; α亚基和β亚基mRNA的完整性则持续降低; 胚根和胚芽ATP合成酶亚基mRNA的损伤程度大于盾片。说明在老化过程中胚根和胚芽ROS产生和积累, 导致ATP合成酶亚基mRNA不同程度的损伤, ATP合成酶活性降低, ATP含量减少, 造成萌发过程种胚供能不足, 这可能是导致玉米种子衰老的重要原因之一。

关键词: 玉米种胚, 人工老化, ROS产生, ATP合成酶, mRNA完整性

Abstract:

In this study, hybrid maize (Zea mays L.) cultivar ‘Zhengdan 958' seeds were used as materials to reveal the mechanism of seed aging. After artificial aging treatment (45℃, 100% relative humidity), the seed vigor, O2- production, ATP content, and ATP synthase activity of seed embryo were investigated, and the integrity of ATP synthase subunit mRNAs in seed embryo and different organs (scutellum, radicle, and plumule) was analyzed by the reverse transcription blocking and double-primer amplification method. The results showed that the germination rate and radicle growth rate of maize seeds decreased, the viability of embryo decreased and the respiration intensity of seeds decreased after artificial aging treatment. The O2- production rate in embryo reached the peak on the 3rd day of aging treatment, and then decreased, the O2- production rate and content in radicle and plumule increased significantly. The ATP content and ATP synthase activity in embryo decreased. The mRNA integrity of ATP synthase ε-subunit and γ-subunit increased first and then decreased. The mRNA integrity of δ-subunit did not change significantly. The mRNA integrity of α-subunit and β-subunit decreased continuously in artificial aging treatment. The damage degree of mRNA integrity of ATP synthase subunits in radicle and plumule was much greater than that in scutellum. These results revealed that the production and accumulation of ROS in radicle and plumule could lead to different degrees of damage to ATP synthase subunit mRNAs and the activity of ATP synthase and the content of ATP in embryo decreased, which resulted in the lack of energy supply during germination, and this maybe one of the major factors causing maize seed aging.

Key words: maize embryo, artificial aging, ROS production, ATP synthesis, integrity of mRNAs

图1

反转录阻断法和双引物扩增原理图 圆点为氧化损伤位点。"

图2

老化玉米种子吸胀过程中的萌发率(a)、胚根生长长度(b)及吸胀60 h时的生长状况(c)"

图3

人工老化处理对种子生活力的影响 (a) 玉米种胚TTC染色情况, S为盾片, R为胚根, P为胚芽, 标尺为2.0 mm; (b) 老化处理对玉米种胚脱氢酶活性的影响, 误差线上不同小写英文字母表示在0.05水平上的差异显著。"

图4

吸胀过程中老化玉米种子呼吸速率的变化 误差线上小写英文字母表示同一吸胀时间不同老化处理间的差异。"

图5

老化处理后玉米种胚的NBT染色情况 S: 盾片; R: 胚根; P: 胚芽。"

图6

人工老化对种胚(a)及不同器官(b) O2-产生速率的影响 Embryo: 种胚; Scutellum: 盾片; Radicle: 胚根; Plumule: 胚芽。误差线上小写英文字母表示同一器官不同老化处理间的差异。"

图7

吸胀过程中老化玉米种子ATP含量的变化 误差线上小写英文字母表示同一吸胀时间不同老化处理间的差异。"

图8

老化处理后玉米种胚(a)及不同器官(b) ATP合成酶活性的变化 误差线上小写英文字母表示同一器官不同老化处理间的差异。"

表1

ATP合成酶亚基基因及其引物序列"

基因ID
Gene ID		 引物方向
Primer direction		 引物序列
Primer sequence (5′-3′)		 位置
Position		 退火温度
Tm (℃)		 产物长度
Product length (bp)
Zm00001d040880-5' (ATP-α) F ACAGCCTTCTCTTCTCAT 314 59.0 123
R CTTAGTTCAGAGTTGCCTC 436 59.1
Zm00001d040880-3' (ATP-α) F GCGACTTAGGCATTACTC 1784 59.0 139
R CTAGTGGCATTCGATCAC 1922 59.0
Zm00001d038929-5' (ATP-β) F TGGACGCATCATAAATGTTA 494 59.0 88
R GGCTTCACGATGAATAGG 581 59.0
Zm00001d038929-3' (ATP-β) F GCAGTCATTCTACATGGTT 1589 59.0 88
R GAAGCCTCCTTATGAAGC 1676 58.9
Zm00001d028207-5' (ATP-γ) F CTCAAGTTGTGAGGAACC 305 59.0 188
R AATCACATTCTTCTTGACGT 492 59.1
Zm00001d028207-3' (ATP-γ) F CTCACGCTTACTTACAACA 1054 59.0 99
R TCTCTTCACCTATCCTTCAA 1152 59.1
Zm00001d006439-5' (ATP-δ) F AAGGAGGTTGACATGGTA 384 58.9 87
R CTTGAGCTCTGCAATTGT 470 59.4
Zm00001d006439-3' (ATP-δ) F TGATATTGTGGCTGTTGAG 559 59.0 149
R CACTGTGAACATCGACTC 707 59.2
Zm00001d044441-5' (ATP-ε) F GAAGGTCCATTTCTCCATC 229 59.1 77
R ATTCATCGGATTCTGTGC 305 58.9
Zm00001d044441-3' (ATP-ε) F AGTCATGTGAACATCATGG 479 59.0 84
R TAAGGACCAGACATAGAATCT 562 59.2
Zm00001d010159 (Actin1) F GAACTGCCATGATAAGGTTA — 59.0 194
R GGTGACGAATATACGAGTG 59.0

图9

老化过程中ATP合成酶亚基mRNA的完整性检测 误差线上不同小写英文字母表示在0.05水平上的差异显著。"

图10

种胚不同器官ATP合成酶亚基mRNA的完整性检测 误差线上不同小写字母表示同一器官不同老化处理间的差异。"

图11

玉米种子衰老可能的机理"

[1] Walters C, Ballesteros D, Vertucci V A. Structural mechanics of seed deterioration: standing the test of time. Plant Sci, 2010, 179: 565-573.
doi: 10.1016/j.plantsci.2010.06.016
[2] Ballesteros D, Walters C. Detailed characterization of mechanical properties and molecular mobility within dry seed glasses: relevance to the physiology of dry biological systems. Plant J, 2011, 68: 607-619.
doi: 10.1111/j.1365-313X.2011.04711.x
[3] Harman D. Aging: a theory based on free radical and radiation chemistry. Biol Sci Med Sci, 1956, 11: 298-300.
[4] Pukacka S, Ratajczak E. Antioxidative response of ascorbate-glutathione pathway enzymes and metabolites to desiccation of recalcitrant Acer saccharinum seeds. J Plant Physiol, 2006, 163: 1259-1266.
doi: 10.1016/j.jplph.2005.10.003
[5] Roach T, Beckett R P, Minibayeva F V, Colville L, Whitaker C, Chen H Y, Bailly C, Kranner I. Extracellular superoxide production, viability and redox poise in response to desiccation in recalcitrant Castanea sativa seeds. Plant Cell Environ, 2010, 33: 59-75.
[6] Ratajczak E, Maecka A, Ciereszko I, Staszak A. Mitochondria are important determinants of the aging of seeds. Int J Mol Sci, 2019, 20: 1568.
doi: 10.3390/ijms20071568
[7] Rhoads D M, Umbach A L, Subbaiah C C, Siedow J N. Mitochondrial reactive oxygen species. Contribution to oxidative stress and interorganellar signaling. Plant Physiol, 2006, 141: 357-366.
pmid: 16760488
[8] Xin X, Lin X H, Zhou Y C, Chen X L, Liu X, Lu X X. Proteome analysis of maize seeds: the effect of artificial ageing. Physiol Plant, 2011, 143: 126-138.
doi: 10.1016/S0176-1617(11)82112-7
[9] Li Y, Wang Y, Xue H, Pritchard H W, Wang X. Changes in the mitochondrial protein profile due to ROS eruption during ageing of elm (Ulmus pumila L.) seeds. Plant Physiol Biochem, 2017, 114: 72-87.
doi: 10.1016/j.plaphy.2017.02.023
[10] Beatriz F M, Ilse K, San S M, Unai A, Manuel L J, Luis V J, Pritchard H W, Jayanthi N, Fátima M, María B J. Evidence for the absence of enzymatic reactions in the glassy state: a case study of xanthophyll cycle pigments in the desiccation-tolerant moss Syntrichia ruralis. J Exp Bot, 2013: 3033-3043.
[11] Chen H, Osuna D, Colville L, Lorenzo O, Graeber K, Küster H, Kranner I. Transcriptome-wide mapping of pea seed ageing reveals a pivotal role for genes related to oxidative stress and programmed cell death. PLoS One, 2013, 8: e78471.
doi: 10.1371/journal.pone.0078471
[12] Bazin J, Langlade N, Vincourt P, Arribat S, Balzergue S, El-Maarouf-Bouteau H, Bailly C. Targeted mRNA oxidation regulates sunflower seed dormancy alleviation during dry after- ripening. Plant Cell, 2011, 23: 2196-2208.
doi: 10.1105/tpc.111.086694
[13] 杨伟飞, 张景龙, 吕伟增, 曹广灿, 陈军营. 人工劣变处理对玉米种胚差异基因表达的影响. 中国农业科学, 2014, 10: 1878-1893.
Yang W F, Zhang J L, Lyu W Z, Cao G C, Chen J Y. Study on the differential genes expression in maize embryo treated by a controlled deterioration treatment. Sci Agric Sin, 2014, 10: 1878-1893. (in Chinese with English abstract)
[14] ISTA. 国际种子检验规程. 北京: 中国农业出版社, 1996. pp 187-188.
ISTA. International Rules for Seed Testing. Beijing: China Agriculture Press, 1996. pp 187-188. (in Chinese)
[15] 胡晋. 对种子活力测定方法--TTC定量法的改进. 种子, 1986, (增刊1): 73-74.
Hu J. Improvement of TTC quantitative method for determination of seed vigor. Seed, 1986, (S1): 73-74. (in Chinese)
[16] 张志良. 植物生理学实验指导. 北京: 高等教育出版社, 2003.
Zhang Z L. Experimental Guidance of Plant Physiology. Beijing: Higher Education Press, 2003. (in Chinese)
[17] 李忠光, 龚明. 植物中超氧阴离子自由基测定方法的改进. 云南植物研究, 2005, 27(2): 211-216.
Li Z G, Gong M. Improvement of determination method of superoxide anion radical in plants. Acta Bot Yunnan, 2005, 27(2): 211-216. (in Chinese with English abstract)
[18] 马春花, 铁轶, 付汉江, 刘斌, 邢瑞云, 朱捷, 丁勇, 郑晓飞. mRNA氧化损伤检测方法的优化及p53 mRNA氧化损伤分析. 军事医学, 2013, 37(3): 180-183.
Ma C H, Tie Y, Fu J H, Liu B, Xing R Y, Zhu J, Ding Y, Zheng X F. Optimization of method to detect mRNA oxidative damage and quantification of p53 mRNA oxidative damage. Milit Med Sci, 2013, 37(3): 180-183. (in Chinese with English abstract)
[19] Basak O, Demir I, Mavi K, Matthews S. Controlled deterioration test for predicting seedling emergence and longevity of pepper (Capsicum annuum L.) seed lot. Seed Sci Technol, 2006, 34: 701-712.
doi: 10.15258/sst.2006.34.3.16
[20] Wang Y, Li Y, Xue H, Pritchard H W, Wang X. Reactive oxygen species provoked mitochondria-dependent cell death during ageing of elm (Ulmus pumila L.) seeds. Plant J, 2015, 81: 438-452.
doi: 10.1111/tpj.12737
[21] Fleming M B, Richards C M, Christina W. Decline in RNA integrity of dry-stored soybean seeds correlates with loss of germination potential. J Exp Bot, 2017, 68: 2219-2230.
doi: 10.1093/jxb/erx100
[22] Bewley J D, Black M. Seeds:Physiology of Development and Germination. New York and London: Plenum Press, 1994.
[23] Bailly C, El-Maarouf-Bouteau H, Corbineau F. From intracellular signaling networks to cell death: the dual role of reactive oxygen species in seed physiology. Comp Rendus Biol, 2008, 331: 806-814.
[24] Rajjou L, Lovigny Y, Groot S, Belghazi M, Job J D. Proteome-wide characterization of seed aging in Arabidopsis: a comparison between artificial and natural aging protocols. Plant Physiol, 2008, 148: 620-641.
doi: 10.1104/pp.108.123141 pmid: 18599647
[25] Yin G K, Xin X, Song C, Chen X, Zhang J, Wu S, Li R, Liu X, Lu X. Activity levels and expression of antioxidant enzymes in the ascorbate-glutathione cycle in artificially aged rice seed. Plant Physiol Biochem, 2014, 80: 1-9.
doi: 10.1016/j.plaphy.2014.03.006
[26] Ratajczak E, Maecka A, Bagniewska Z A, Kalemba E M. The production, localization and spreading of reactive oxygen species contributes to the low vitality of long-term stored common beech (Fagus sylvatica L.) seeds. J Plant Physiol, 2015, 174: 147-156.
doi: 10.1016/j.jplph.2014.08.021
[27] Wang X F, Jing X M, Lin J. Starch mobilization in ultradried seed of maize (Zea mays L.) during germination. J Integr Plant Biol, 2005, 47: 443-451.
doi: 10.1111/j.1744-7909.2005.00088.x
[28] Benamar A, Rolletschek H, Borisjuk L, Avelange-Macherel M H, Curien G, Mostefai H A. Nitrite-nitric oxide control of mitochondrial respiration at the frontier of anoxia. Biochim Biophys Acta, 2008, 1777: 1268-1275.
[29] Navarro A, Boveris A. The mitochondrial energy transduction system and the aging process. Am J Physiol, 2007, 292: 670-686.
[30] Werner K. Structure and function of mitochondrial membrane protein complexes. BMC Biol, 2015, 13: 89.
doi: 10.1186/s12915-015-0201-x
[31] Marcus A, Feeley J. Activation of protein synthesis in the imbibition phase of seed germination. Proc Natl Acad Sci USA, 1964, 51: 1075-1079.
doi: 10.1073/pnas.51.6.1075
[32] Rajjou L, Duval M, Gallardo K, Catusse J, Bally J, Job C, Job D. Seed germination and vigor. Annu Rev Plant Biol, 2012, 63: 507-533.
doi: 10.1146/annurev-arplant-042811-105550 pmid: 22136565
[33] Kong Q, Lin C L. Oxidative damage to RNA: mechanisms, consequences, and diseases. Cell Mol Life Sci, 2010, 67: 1817-1829.
doi: 10.1007/s00018-010-0277-y
[34] Shcherbik N, Pestov D G. The impact of oxidative stress on ribosomes: from injury to regulation. Cells, 2019, 8: 1379.
doi: 10.3390/cells8111379
[35] Schmidt M, Luff M, Mollwo A, Kaminski M, Mittag M, Kreimer G. Evidence for a specialized localization of the chloroplast ATP-synthase subunits alpha, beta, and gamma in the eyespot apparatus of Chlamydomonas reinhardtii (Chlorophyceae). J Phycol, 2011, 43: 284-294.
doi: 10.1111/j.1529-8817.2007.00331.x
[36] Wang C, Chen Z, Li S, Zhang Y, Jia S, Li J, Chi Y, Miao Y, Guan Y, Yang J. Hepatic overexpression of ATP synthase β subunit activates PI3K/Akt pathway to ameliorate hyperglycemia of diabetic mice. Diabetes, 2014, 63: 947-959.
doi: 10.2337/db13-1096
[37] Lee T, Paul R, Langlais N, Hoffman L, Roust C. Mitochondrial ATP synthase beta subunit production rate and ATP synthase specific activity are reduced in skeletal muscle of humans with obesity. Exp Physiol, 2019, 104: 126-135.
doi: 10.1113/EP087278
[38] Shirakihara Y, Shiratori A, Tanikawa H, Nakasako M, Suzuki T. Structure of a thermophilic F1-ATPase inhibited by an ε-subunit: deeper insight into the ε-inhibition mechanism. FEBS J, 2015, 282: 2895-2913.
doi: 10.1111/febs.13329 pmid: 26032434
[39] Sun J, Hu H, Li Y, Wang L, Zhou Q, Huang X. Effects and mechanism of acid rain on plant chloroplast ATP synthase. Environ Sci Pollut Res, 2016, 23: 18296-18306.
doi: 10.1007/s11356-016-7016-3
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