Melatonin Contents in Eutrema salsugineum and Arabidopsis thaliana Under Salt Stress, and Expression Pattern Analysis of Synthesis Related Genes

doi:10.13560/j.cnki.biotech.bull.1985.2022-0975

Abstract

Abstract:

The halophyte Eutrema salsugineum has strong salt tolerance and the characteristics of model plants. It is an ideal material for studying the mechanism of plant coping with adversity. As a multifunctional hormone, the role of melatonin in the salt tolerance of E. salsugineum remains unclear. In this study, we performed the enzyme-linked immunosorbent assay of melatonin and real-time fluorescence quantitative PCR analysis. The accumulations of melatonin in different tissues, the synthesis of melatonin, the expression patterns of related genes and the effect of exogenous melatonin on the phenotype response to salt stress were analyzed in E. salsugineum and Arabidopsis. The results showed the melatonin synthesis in both plants were the highest in the young leaves, the endogenous melatonin of E. salsugineum was significantly higher than in Arabidopsis. The melatonin level was induced by salt stress in both plants, but the melatonin synthesis in Arabidopsis obviously decreased after salt stress for 7 d, which was different from the accumulation pattern in E. salsugineum. It was found that the genes related to melatonin synthesis in Arabidopsis and E. salsugineum were closely among different species of plants through sequence alignment analysis. Salt-responsive expression analysis showed that the expression abundance of SNAT1, ASMT and COMT were significantly up-regulated after salt stress for 3 d in E. salsugineum, which also induced by salt treatment for 1 d and 3 d in Arabidopsis, and then turned to down regulation under 7 d treatment; while there was no change in E. salsugineum, revealing that the expression changes of related genes upon salt condition in E. salsugineum and Arabidopsis was distinct. Furthermore, exogenous melatonin treatment significantly alleviated the stress phenotype of both plants under salt stress. Taken together, melatonin effectively contributed to the salt resistance of E. salsugineum and is involved in the regulation of salt tolerance of E. salsugineum and Arabidopsis, while the induction pattern of melatonin synthesis for them was different.

Key words: melatonin, Eutrema salsugineum, Arabidopsis thaliana, salt tolerance, melatonin synthesis gene, gene expression, exogenous treatment

LI Zhi-qi, YUAN Yue, MIAO Rong-qing, PANG Qiu-ying, ZHANG Ai-qin. Melatonin Contents in Eutrema salsugineum and Arabidopsis thaliana Under Salt Stress, and Expression Pattern Analysis of Synthesis Related Genes[J]. Biotechnology Bulletin, 2023, 39(5): 142-151.

Figures/Tables 7

Table 1 Primer sequences used for RT-qPCR analysis

基因名称 Gene name	引物方向 Primer direction	引物序列 Primer sequence（5'-3'）
AtACTIN	Forward	CATCAGGAAGGACTTGTACGG
AtACTIN	Reverse	GATGGACCTGACTCGTCATAC
AtSNAT1	Forward	CGTCTATTAGCCCTCCTCC
AtSNAT1	Reverse	GCCATCCCACCTTATCACA
AtSNAT2	Forward	GGCACAATCTCCACTCCTC
AtSNAT2	Reverse	TCAACCACAACATCCCATA
AtASMT	Forward	GGTCAACGGTTCAACTCCA
AtASMT	Reverse	ATCGCCCTCAACATTCTCA
AtCOMT	Forward	ATGCTCCTTCTCATCCTGG
AtCOMT	Reverse	CACGCAATGTTCGTCACTC
EsACTIN	Forward	GCACAATCCAAAAGAGGTATTCTCACCT
EsACTIN	Reverse	GGAGCCTCGGTAAGAAGAACAGGG
EsSNAT1	Forward	AAGAATAACAATACGACCCA
EsSNAT1	Reverse	ACATCAATCTCACCACCAG
EsASMT	Forward	ATGTTACCGAAGTTGCG
EsASMT	Reverse	CGTTCTTTGCCTGTGCT
EsCOMT	Forward	CGGCTTACTCCGTCCTCAC
EsCOMT	Reverse	TAGCACCAATGCCACCACC

Fig. 1 Melatonin contents in different tissues of E. salsug-ineum and A. thaliana A: Vegetative growth stage. B: Reproductive growth stage. Different letters indicate significant difference（P < 0.05）. The same below

Fig. 2 Melatonin contents in E. salsugineum (A) and A. thaliana (B) leaves under salt stress

Fig. 3 Multiple alignment of amino acid sequences of melatonin synthases in different plants Black boxes indicate conserved functional sites or domains in proteases, and * indicates the binding sites of phenolic substrate. At: Arabidopsis thaliana; Es: Eutrema salsugineum; Vv: Vitis vinifera L.; Sl: Solanum lycopersicum L.; Os: Oryza sativa L.; Zm: Zea mays L. The same below

Fig. 4 Evolutionary analysis of melatonin synthesis genes A：SNAT1；B：SNAT2；C：ASMT；D：COMT

Fig. 5 Expression pattern of melatonin synthesis genes in response to salt stress A: E. salsugineum. B: A. thaliana. * indicates significant difference between untreated and salt treated plants at different time point（P < 0.05）

Fig. 6 Salt-responsive phenotypes of E. salsugineum and A. thaliana treated with exogenous melatonin A: E. salsugineum. B: A. thaliana. C: Control. MT: Melatonin treatment. S: Salt treatment. S+MT: Salt and melatonin treatment

References 30

[1]	Morton MJL, Awlia M, Al-Tamimi N, et al. Salt stress under the scalpel - dissecting the genetics of salt tolerance[J]. Plant J, 2019, 97(1): 148-163. doi: 10.1111/tpj.2019.97.issue-1 URL
[2]	Gong ZZ, Xiong LM, Shi HZ, et al. Plant abiotic stress response and nutrient use efficiency[J]. Sci China Life Sci, 2020, 63(5): 635-674. doi: 10.1007/s11427-020-1683-x pmid: 32246404
[3]	Parihar P, Singh S, Singh R, et al. Effect of salinity stress on plants and its tolerance strategies: a review[J]. Environ Sci Pollut Res Int, 2015, 22(6): 4056-4075. doi: 10.1007/s11356-014-3739-1 URL
[4]	Apse MP, Blumwald E. Engineering salt tolerance in plants[J]. Curr Opin Biotechnol, 2002, 13(2): 146-150. doi: 10.1016/S0958-1669(02)00298-7 URL
[5]	Zhu JK. Abiotic stress signaling and responses in plants[J]. Cell, 2016, 167(2): 313-324. doi: 10.1016/j.cell.2016.08.029 URL
[6]	Park HJ, Kim WY, Yun DJ. A new insight of salt stress signaling in plant[J]. Mol Cells, 2016, 39(6): 447-459. doi: 10.14348/molcells.2016.0083 pmid: 27239814
[7]	Kanwar MK, Yu JQ, Zhou J. Phytomelatonin: Recent advances and future prospects[J]. J Pineal Res, 2018, 65(4): e12526. doi: 10.1111/jpi.2018.65.issue-4 URL
[8]	Lerner AB, Case JD, Takahashi Y, et al. Isolation of melatonin, the pineal gland factor that lightens melanocytes[J]. J Am Chem Soc, 1958, 80(10): 2587.
[9]	Dubbels R, Reiter RJ, Klenke E, et al. Melatonin in edible plants identified by radioimmunoassay and by high performance liquid chromatography-mass spectrometry[J]. J Pineal Res, 1995, 18(1): 28-31. doi: 10.1111/j.1600-079x.1995.tb00136.x pmid: 7776176
[10]	Hattori A, Migitaka H, Iigo M, et al. Identification of melatonin in plants and its effects on plasma melatonin levels and binding to melatonin receptors in vertebrates[J]. Biochem Mol Biol Int, 1995, 35(3): 627-634. pmid: 7773197
[11]	Lee HY, Byeon Y, Lee K, et al. Cloning of Arabidopsis serotonin N-acetyltransferase and its role with caffeic acid O-methyltransferase in the biosynthesis of melatonin in vitro despite their different subcellular localizations[J]. J Pineal Res, 2014, 57(4): 418-426. doi: 10.1111/jpi.12181 URL
[12]	宋雪飞. 褪黑素调控番茄幼苗耐盐性的浓度效应及其生理机制的研究[D]. 扬州: 扬州大学, 2018.
	Song XF. Study on the concentration effect of melatonin on salt tolerance of tomato seedlings and its physiological mechanism[D]. Yangzhou: Yangzhou University, 2018.
[13]	乔沛, 殷菲胧, 王雨萱, 等. 外源褪黑素处理对采后荔枝褐变及活性氧代谢的影响[J]. 食品工业科技, 2021, 42(6): 282-287.
	Qiao P, Yin FL, Wang YX, et al. Effects of exogenous melatonin on browning and active oxygen metabolism of postharvest Litchi[J]. Sci Technol Food Ind, 2021, 42(6): 282-287.
[14]	Sun CL, Liu LJ, Wang LX, et al. Melatonin: a master regulator of plant development and stress responses[J]. J Integr Plant Biol, 2021, 63(1): 126-145. doi: 10.1111/jipb.12993
[15]	Zhan HS, Nie XJ, Zhang T, et al. Melatonin: a small molecule but important for salt stress tolerance in plants[J]. Int J Mol Sci, 2019, 20(3): 709. doi: 10.3390/ijms20030709 URL
[16]	Li JP, Liu J, Zhu TT, et al. The role of melatonin in salt stress responses[J]. Int J Mol Sci, 2019, 20(7): 1735. doi: 10.3390/ijms20071735 URL
[17]	Gong QQ, Li PH, Ma SS, et al. Salinity stress adaptation competence in the extremophile Thellungiella halophila in comparison with its relative Arabidopsis thaliana[J]. Plant J, 2005, 44(5): 826-839. doi: 10.1111/tpj.2005.44.issue-5 URL
[18]	Amtmann A. Learning from evolution: Thellungiella generates new knowledge on essential and critical components of abiotic stress tolerance in plants[J]. Mol Plant, 2009, 2(1): 3-12. doi: 10.1093/mp/ssn094 pmid: 19529830
[19]	Inan G, Zhang Q, Li PH, et al. Salt cress. A halophyte and cryophyte Arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles[J]. Plant Physiol, 2004, 135(3): 1718-1737. doi: 10.1104/pp.104.041723 URL
[20]	Pelagio-Flores R, Muñoz-Parra E, Ortiz-Castro R, et al. Melatonin regulates Arabidopsis root system architecture likely acting independently of auxin signaling[J]. J Pineal Res, 2012, 53(3): 279-288. doi: 10.1111/j.1600-079X.2012.00996.x pmid: 22507071
[21]	张悦新. 褪黑素对棉花耐盐性的影响及相关基因GhSNAT34的功能研究[D]. 郑州: 郑州大学, 2021.
	Zhang YX. Study on effect of melatonin on salt tolerance of cotton and function of GhSNAT34 gene[D]. Zhengzhou: Zhengzhou University, 2021.
[22]	Li JH, Yang YQ, Sun K, et al. Exogenous melatonin enhances cold, salt and drought stress tolerance by improving antioxidant defense in tea plant(Camellia sinensis(L.) O. Kuntze)[J]. Molecules, 2019, 24(9): 1826. doi: 10.3390/molecules24091826 URL
[23]	吴艳迪. 葡萄褪黑素含量变化及其合成基因SNAT原核表达分析[D]. 北京: 中国农业科学院, 2018.
	Wu YD. Changes of melatonin content and prokaryotic expression analysis of SNAT gene in grapevine[D]. Beijing: Chinese Academy of Agricultural Sciences, 2018.
[24]	Arnao MB, Hernández-Ruiz J. Chemical stress by different agents affects the melatonin content of barley roots[J]. J Pineal Res, 2009, 46(3): 295-299. doi: 10.1111/j.1600-079X.2008.00660.x pmid: 19196434
[25]	Byeon Y, Lee HY, Lee K, et al. Cellular localization and kinetics of the rice melatonin biosynthetic enzymes SNAT and ASMT[J]. J Pineal Res, 2014, 56(1): 107-114. doi: 10.1111/jpi.12103 pmid: 24134674
[26]	Liu DD, Sun XS, Liu L, et al. Overexpression of the melatonin synthesis-related gene SlCOMT1 improves the resistance of tomato to salt stress[J]. Molecules, 2019, 24(8): 1514. doi: 10.3390/molecules24081514 URL
[27]	Chen YE, Mao JJ, Sun LQ, et al. Exogenous melatonin enhances salt stress tolerance in maize seedlings by improving antioxidant and photosynthetic capacity[J]. Physiol Plant, 2018, 164(3): 349-363. doi: 10.1111/ppl.2018.164.issue-3 URL
[28]	Yan FY, Zhang JY, Li WW, et al. Exogenous melatonin alleviates salt stress by improving leaf photosynthesis in rice seedlings[J]. Plant Physiol Biochem, 2021, 163: 367-375. doi: 10.1016/j.plaphy.2021.03.058 URL
[29]	Zhang ZH, Liu LT, Li HY, et al. Exogenous melatonin promotes the salt tolerance by removing active oxygen and maintaining ion balance in wheat(Triticum aestivum L.)[J]. Front Plant Sci, 2022, 12: 787062. doi: 10.3389/fpls.2021.787062 URL
[30]	Xu LL, Xiang GQ, Sun QH, et al. Melatonin enhances salt tolerance by promoting MYB108A-mediated ethylene biosynthesis in grapevines[J]. Hortic Res, 2019, 6: 114. doi: 10.1038/s41438-019-0197-4