菊花CmASMT的克隆及其在高温抗性中的作用

doi:10.3864/j.issn.0578-1752.2025.08.012

摘要/Abstract

摘要：

【背景】菊花是原产于我国的十大传统名花和世界四大切花之一，在世界各地广泛种植，适宜生长在气候温和凉爽的环境。夏季高温天气的持续出现，以及封闭或半封闭性设施环境栽培，严重影响了菊花的产量和品质。【目的】外源应用褪黑素有助于调节植物应对包括高温胁迫在内的各种逆境胁迫，N-乙酰-5-羟色胺甲基转移酶（ASMT）是褪黑素生物合成的限速酶。探究CmASMT的功能，研究CmASMT对高温胁迫下菊花生长的影响，探讨内源褪黑素的合成对菊花高温抗性的影响，为褪黑素调控植物高温抗性的分子机制及菊花分子育种奠定理论基础。【方法】以切花菊‘神马’为试验材料克隆得到褪黑素生物合成关键酶基因CmASMT，对其进行生物信息学分析、亚细胞定位和时空表达特性分析；利用病毒诱导的基因沉默（VIGS）技术构建CmASMT沉默植株，从光合作用、膜系统的稳定性和抗氧化系统等角度分析CmASMT对菊花耐热性的影响。【结果】CmASMT的ORF全长为1 059 bp，编码352个氨基酸，属于O-甲基转移酶家族；CmASMT在菊花营养生长时期的根、茎、叶和芽中均有表达，且在根中表达量最高；高温、低温、水涝、盐和干旱胁迫均诱导CmASMT表达，且CmASMT对高温胁迫响应最为突出；CmASMT蛋白在细胞膜、细胞质和细胞核中均有表达。菊花CmASMT沉默植株内源褪黑素含量显著降低。高温胁迫下，CmASMT沉默植株表现出光合作用的抑制、膜系统稳定性的降低、氧化胁迫的加重、抗氧化酶活性的减弱，以及变性蛋白质的增加。CmASMT可能通过调节菊花植株内源褪黑素的合成，协同提高抗氧化酶活性，清除过量活性氧，减轻膜结构损伤和光合色素的降解，进而提高菊花的光合效率，从而增强菊花的高温抗性。【结论】CmASMT通过调控内源褪黑素的合成，在菊花应答高温胁迫的过程中起重要作用。

关键词: 菊花, 高温胁迫, 褪黑素, CmASMT, 病毒诱导的基因沉默

Abstract:

【Background】 Chrysanthemum is one of the ten Chinese traditional flowers and four most important cut flowers in the world, originating from China and widely cultivated throughout the world. As chrysanthemum thrives in mild and cool climates, the summer high-temperature weather leads to continuous high temperature in protected cultivation environments, which seriously affects the yield and quality of chrysanthemum. 【Objective】Exogenous application of melatonin helps to regulate plant response to various abiotic stresses, including high-temperature stress. N-acetyl-5-hydroxytryptamine methyltransferase (ASMT) is the rate-limiting enzyme for melatonin biosynthesis. This study investigated the function of CmASMT, examined its effect on chrysanthemum growth under high-temperature stress, and explored how endogenous melatonin synthesis influences thermotolerance. This paper provides a theoretical basis for the molecular mechanism of melatonin-regulated thermotolerance in plants and the molecular breeding of chrysanthemum. 【Method】The CmASMT involved in melatonin biosynthesis was cloned from Chrysanthemum morifolium Jinba and analyzed for bioinformatics, subcellular localization, and spatiotemporal expression properties. CmASMT gene-silenced plants were generated by using virus-induced gene silencing (VIGS) technology to investigate the effects of CmASMT on heat tolerance of chrysanthemum through photosynthesis, the stability of the membrane system, and antioxidant system. 【Result】The ORF of CmASMT is 1 059 bp in length, encodes 352 amino acids, and belongs to the O-methyltransferases family. During the vegetative growth stage, CmASMT is expressed in roots, stems, leaves and buds of chrysanthemum, with the highest levels observe in the roots. Expression of CmASMT was induced by high temperature, low temperature, waterlogging, salt and drought stress, and CmASMT was most responsive to high-temperature stress. The CmASMT protein localized to the cell membranes, cytoplasm and nucleus. The content of endogenous melatonin in CmASMT-silenced plants of chrysanthemum was significantly reduced. Under high-temperature stress, CmASMT-silenced plants exhibited inhibition of photosynthesis, reduction of membrane system stability, aggravation of oxidative stress, weakening of antioxidant enzyme activity, and an increase of denatured proteins. CmASMT may enhance the photosynthetic efficiency of chrysanthemum by regulating the synthesis of endogenous melatonin, while improving the activity of antioxidant enzymes, scavenging excess reactive oxygen species, alleviating membrane structure damage and degradation of photosynthetic pigments, thereby improving chrysanthemum thermotolerance. 【Conclusion】CmASMT plays an important role in responding to high-temperature stress in chrysanthemum by regulating endogenous melatonin synthesis.

Key words: chrysanthemum, high temperature stress, melatonin, CmASMT, virus induced gene silencing

孟慧, 罗丙玉, 卢正宇, 王鹏, 康冬茹, 郑成淑, 王文莉. 菊花CmASMT的克隆及其在高温抗性中的作用[J]. 中国农业科学, 2025, 58(8): 1617-1626.

MENG Hui, LUO BingYu, LU ZhengYu, WANG Peng, KANG DongRu, ZHENG ChengShu, WANG WenLi. Cloning of CmASMT and Its Role in Thermotolerance of Chrysanthemum[J]. Scientia Agricultura Sinica, 2025, 58(8): 1617-1626.

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.0578-1752.2025.08.012

https://www.chinaagrisci.com/CN/Y2025/V58/I8/1617

图/表 7

图1

图2

图3

图4

图5

图6

图7

参考文献 34

[1]	SETH P, SEBASTIAN J. Plants and global warming: Challenges and strategies for a warming world. Plant Cell Reports, 2024, 43(1): 27. doi: 10.1007/s00299-023-03083-w pmid: 38163826
[2]	RAY D K, WEST P C, CLARK M, GERBER J S, PRISHCHEPOV A V, CHATTERJEE S. Climate change has likely already affected global food production. PLoS ONE, 2019, 14(5): e0217148.
[3]	MATHUR S, AGRAWAL D, JAJOO A. Photosynthesis: Response to high temperature stress. Journal of Photochemistry and Photobiology B: Biology, 2014, 137: 116-126.
[4]	SALVUCCI M E, CRAFTS-BRANDNER S J. Relationship between the heat tolerance of photosynthesis and the thermal stability of rubisco activase in plants from contrasting thermal environments. Plant Physiology, 2004, 134(4): 1460-1470. doi: 10.1104/pp.103.038323 pmid: 15084731
[5]	FAN J B, XIE Y, ZHANG Z C, CHEN L. Melatonin: A multifunctional factor in plants. International Journal of Molecular Sciences, 2018, 19(5): 1528.
[6]	AFREEN F, ZOBAYED S M A, KOZAI T. Melatonin in Glycyrrhiza uralensis: Response of plant roots to spectral quality of light and UV-B radiation. Journal of Pineal Research, 2006, 41(2): 108-115.
[7]	ARNAO M B, HERNÁNDEZ-RUIZ J. Functions of melatonin in plants: A review. Journal of Pineal Research, 2015, 59(2): 133-150. doi: 10.1111/jpi.12253 pmid: 26094813
[8]	DEY S, BISWAS A, DENG Y, BIRHANIE Z M, CHEN W T, LI D F. Exogenous melatonin enhances low-temperature stress of jute seedlings through modulation of photosynthesis and antioxidant potential. Heliyon, 2023, 9(8): e19125.
[9]	KUPPUSAMY A, ALAGARSWAMY S, KARUPPUSAMI K M, MADURAIMUTHU D, NATESAN S, RAMALINGAM K, MUNIYAPPAN U, SUBRAMANIAN M, KANAGARAJAN S. Melatonin enhances the photosynthesis and antioxidant enzyme activities of mung bean under drought and high-temperature stress conditions. Plants, 2023, 12(13): 2535.
[10]	JAHAN M S, GUO S R, SUN J, SHU S, WANG Y, ABOU EL- YAZIED A, ALABDALLAH N M, HIKAL M, MOHAMED M H M, IBRAHIM M F M, HASAN M M. Melatonin-mediated photosynthetic performance of tomato seedlings under high-temperature stress. Plant Physiology and Biochemistry, 2021, 167: 309-320. doi: 10.1016/j.plaphy.2021.08.002 pmid: 34392044
[11]	周永海. 甜瓜种质资源耐热性评价及外源物质对热胁迫的缓解效应[D]. 杨凌: 西北农林科技大学, 2021.
	ZHOU Y H. Evaluation of heat tolerance of melon germplasm resources and alleviating effects of exogenous substances on heat stress[D]. Yangling: Northwest A & F University, 2021. (in Chinese)
[12]	LI Z G, XU Y, BAI L K, ZHANG S Y, WANG Y. Melatonin enhances thermotolerance of maize seedlings (Zea mays L.) by modulating antioxidant defense, methylglyoxal detoxification, and osmoregulation systems. Protoplasma, 2019, 256(2): 471-490.
[13]	XU W, CAI S Y, ZHANG Y, WANG Y, AHAMMED G J, XIA X J, SHI K, ZHOU Y H, YU J Q, REITER R J, ZHOU J. Melatonin enhances thermotolerance by promoting cellular protein protection in tomato plants. Journal of Pineal Research, 2016, 61(4): 457-469. doi: 10.1111/jpi.12359 pmid: 27484733
[14]	齐晓媛, 王文莉, 胡少卿, 刘梦雨, 郑成淑, 孙宪芝. 外源褪黑素对高温胁迫下菊花光合和生理特性的影响. 应用生态学报, 2021, 32(7): 2496-2504. doi: 10.13287/j.1001-9332.202107.025
	QI X Y, WANG W L, HU S Q, LIU M Y, ZHENG C S, SUN X Z. Effects of exogenous melatonin on photosynthesis and physiological characteristics of Chrysanthemum seedlings under high temperature stress. Chinese Journal of Applied Ecology, 2021, 32(7): 2496-2504. (in Chinese)
[15]	PARK S, BYEON Y, BACK K. Functional analyses of three ASMT gene family members in rice plants. Journal of Pineal Research, 2013, 55(4): 409-415. doi: 10.1111/jpi.12088 pmid: 24033370
[16]	ZUO B X, ZHENG X D, HE P L, WANG L, LEI Q, FENG C, ZHOU J Z, LI Q T, HAN Z H, KONG J. Overexpression of MzASMT improves melatonin production and enhances drought tolerance in transgenic Arabidopsis thaliana plants. Journal of Pineal Research, 2014, 57(4): 408-417.
[17]	ZHOU K, LI Y, HU L Y, ZHANG J Y, YUE H, YANG S L, LIU Y, GONG X Q, MA F W. Overexpression of MdASMT9, an N- acetylserotonin methyltransferase gene, increases melatonin biosynthesis and improves water-use efficiency in transgenic apple. Tree Physiology, 2022, 42(5): 1114-1126.
[18]	赵世杰. 植物生理学实验指导. 北京: 中国农业科学技术出版社, 2002.
	ZHAO S J. Techniques of Plant Physiological Experiment. Beijing: China Agricultural Science and Technology Press, 2002. (in Chinese)
[19]	SHAH K, KUMAR R G, VERMA S, DUBEY R S. Effect of cadmium on lipid peroxidation, superoxide anion generation and activities of antioxidant enzymes in growing rice seedlings. Plant Science, 2001, 161(6): 1135-1144.
[20]	李合生. 植物生理生化实验原理和技术. 北京: 高等教育出版社, 2000.
	LI H S. Principles and Techniques of Plant Physiological Biochemical Experiment. Beijing: Higher Education Press, 2000. (in Chinese)
[21]	曹建康, 姜微波, 赵玉梅. 果蔬采后生理生化实验指导. 北京: 中国轻工业出版社, 2007.
	CAO J K, JIANG W B, ZHAO Y M. Guidance on Postharvest Physiological and Biochemical Experiments of Fruits and Vegetables. Beijing: China Light Industry Press, 2007. (in Chinese)
[22]	CHE R M, LIU Y R, YAN S Q, YANG C, SUN Y B, LIU C H, MA F W. Elongation factor MdEF-Tu coordinates with heat shock protein MdHsp70 to enhance apple thermotolerance. The Plant Journal, 2024, 117(4): 1250-1263. doi: 10.1111/tpj.16561 pmid: 37991990
[23]	LERNER A B, CASE J D, TAKAHASHI Y. Isolation of melatonin and 5-methoxyindole-3-acetic acid from bovine pineal glands. Journal of Biological Chemistry, 1960, 235: 1992-1997. pmid: 14415935
[24]	KOLÁR J, MACHÁCKOVÁ I. Melatonin in higher plants: Occurrence and possible functions. Journal of Pineal Research, 2005, 39(4): 333-341. doi: 10.1111/j.1600-079X.2005.00276.x pmid: 16207287
[25]	BYEON Y, BACK K. Melatonin synthesis in rice seedlings in vivo is enhanced at high temperatures and under dark conditions due to increased serotonin N-acetyltransferase and N-acetylserotonin methyltransferase activities. Journal of Pineal Research, 2014, 56(2): 189-195.
[26]	杨永娟. 梅花品种耐旱性差异分析及褪黑素合成基因功能研究[D]. 北京: 北京林业大学, 2021.
	YANG Y J. Analysis on the difference of drought tolerance of plum blossom varieties and study on the function of melatonin synthesis gene[D]. Beijing: Beijing Forestry University, 2021. (in Chinese)
[27]	HASANUZZAMAN M, NAHAR K, ALAM M M, ROYCHOWDHURY R, FUJITA M. Physiological, biochemical, and molecular mechanisms of heat stress tolerance in plants. International Journal of Molecular Sciences, 2013, 14(5): 9643-9684. doi: 10.3390/ijms14059643 pmid: 23644891
[28]	SHARKEY T D. Effects of moderate heat stress on photosynthesis: Importance of thylakoid reactions, rubisco deactivation, reactive oxygen species, and thermotolerance provided by isoprene. Plant, Cell & Environment, 2005, 28(3): 269-277.
[29]	RAJA V, QADIR S U, ALYEMENI M N, AHMAD P. Impact of drought and heat stress individually and in combination on physio- biochemical parameters, antioxidant responses, and gene expression in Solanum lycopersicum. 3 Biotech, 2020, 10(5): 208.
[30]	CHAKI M, BEGARA-MORALES J C, BARROSO J B. Oxidative stress in plants. Antioxidants, 2020, 9(6): 481.
[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]	须文. 褪黑素在调控番茄耐热性中的作用及机制[D]. 杭州: 浙江大学, 2016.
	XU W. Role and mechanism of melatonin in regulating heat tolerance of tomato[D]. Hangzhou: Zhejiang University, 2016. (in Chinese)
[33]	JIA C H, YU X J, ZHANG M, LIU Z G, ZOU P, MA J, XU Y C. Application of melatonin-enhanced tolerance to high-temperature stress in cherry radish (Raphanus sativus L. var. radculus pers). Journal of Plant Growth Regulation, 2020, 39(2): 631-640.
[34]	高腾腾. 苹果MdASMT9介导的褪黑素合成对温度和低氮胁迫的调控机制研究[D]. 杨凌: 西北农林科技大学, 2022.
	GAO T T. Regulation mechanism of melatonin synthesis mediated by MdASMT9 on temperature and low nitrogen stress in apple[D]. Yangling: Northwest A & F University, 2022. (in Chinese)