Overexpression of RcELIP1 from Racomitrium canescens Enhances the Tolerance to Drought and High Temperature in Transgenic Arabidopsisthaliana

doi:10.13560/j.cnki.biotech.bull.1985.2025-1065

Abstract

Abstract:

Objective This study investigated the function of the early light-induced protein (ELIP) gene RcELIP1 from the moss Racomitrium canescens in conferring tolerance to drought and high-temperature stress, and also preliminarily explored the underlying mechanisms, aiming to provide further theoretical insight into the role of bryophyte ELIPs in plant stress responses. Method The expression patterns of RcELIP1 in R. canescens during dehydration, rehydration, and 45 ℃ high-temperature stress were analyzed using quantitative real-time PCR (RT-qPCR), the characteristics of the encoded protein were predicted via bioinformatics tools, the coding sequence of RcELIP1 was cloned into an overexpression vector and transformed into Arabidopsis thaliana to generate transgenic lines, phenotypic differences between transgenic and wild-type plants under drought and high-temperature stress were compared, and physiological and biochemical indices were measured. Result RT-qPCR analysis confirmed that RcELIP1 expression was significantly altered in response to both dehydration and high-temperature stress. RcELIP1 comprises 216 amino acid residues, it was predicted to contain a chlorophyll a/b-binding domain, and was characterized as a hydrophilic, unstable transmembrane protein localized to chloroplasts. Under drought and high-temperature stress, transgenic RcELIP1-overexpressed A. thaliana showed significantly higher survival rates than wild-type plants, the transgenic lines also maintained higher chlorophyll and proline content, elevated antioxidant enzyme activities, and lower malondialdehyde levels. Conclusion The overexpression of RcELIP1 enhances plant tolerance to drought and high-temperature stress, likely through mechanisms involving chloroplast protection, increased osmotic regulation, and improved antioxidant capacity.

Key words: Racomitrium canescens, early light-induced protein gene, RcELIP1, tolerance to drought, tolerance to high-temperature

CAO Wan-di, BAO Wei, SUN Tian-guo, LIANG Shu-ting, QIN Xu-yang, ZHANG Mei-juan, SHA Wei, PENG Yi-fang, MA Tian-yi. Overexpression of RcELIP1 from Racomitrium canescens Enhances the Tolerance to Drought and High Temperature in Transgenic Arabidopsisthaliana[J]. Biotechnology Bulletin, 2026, 42(7): 246-256.

Figures/Tables 8

Fig. 1 Analysis of RcELIP1 expression changes in Racomitrium canescens during rehydration, dehydration, and high-temperature treatmentsA: Desiccated R. canescens under rehydration treatments. B: Dehydration treatments. C: 45 ℃ high-temperature treatments. The error bars indicate the standard deviation, the different letters indicate that the difference is significant (P<0.05), the same below

Fig. 2 Cloning of the coding sequence and the sequence information of RcELIP1A: PCR amplification results of RcELIP1 coding sequence (M: DL 2000 DNA marker; -: water control; 1,2: RcELIP1 coding sequence). B: The nucleotide sequence and the encoded amino acid sequence of RcELIP1

Fig. 3 Bioinformatics prediction analysis of RcELIP1A. Prediction of hydrophilicity and hydrophobicity. B. Transmembrane structure prediction. C. Protein secondary structure prediction; blue: alpha helix, green: beta turn, red: random coil, purple: extended strand. D. Protein tertiary structure prediction. E. Signal peptide prediction. F. Sequence alignment of RcELIP1 and homologous proteins

Fig. 4 Screening and identification of transgenic RcELIP1-overexpressed transgenic A.thaliana plantsA: Antibiotic screening of RcELIP1 overexpressed transgenic Arabidopsis thaliana plants, the red circles indicate the successfully screened RcELIP1 T1 generation plants; B: Expression analysis of RcELIP1 in T2 generation RcELIP1-overexpressedtransgenic A. thaliana plants

Fig. 5 Phenotypes performance of RcELIP1-overexpressed transgenic A.thaliana plants under drought stress

Fig. 6 Changes in physiological and biochemical indexes of wild-type and RcELIP1-overexpressed transgenic A. thaliana under drought stress

Fig. 7 Phenotypes of RcELIP1-overexpressed transgenic A. thaliana under 45 ℃ high-temperature treatments

Fig. 8 Changes in physiological and biochemical indexes of wild-type and RcELIP1-overexpressed transgenic A. thaliana under 45 ℃ high-temperature treatments

References 39

[1]	刘丹. 植物对环境胁迫的生理响应研究进展 [J]. 农家参谋, 2020(20): 97.
	Liu D. Research progress on physiological response of plants to environmental stress [J]. Farmers Consult, 2020(20): 97.
[2]	Arora NK. Impact of climate change on agriculture production and its sustainable solutions [J]. Environ Sustain, 2019, 2(2): 95-96.
[3]	史勇, 郑兰杰, 王晨, 等. 植物耐高温机制的研究进展 [J]. 河南农业大学学报, 2023, 57(5): 713-725.
	Shi Y, Zheng LJ, Wang C, et al. Research progress on mechanisms of high temperature tolerance in plants [J]. J Henan Agric Univ, 2023, 57(5): 713-725.
[4]	齐学礼, 赵明忠, 尹文生, 等. 小麦耐高温特性的生理机制与基因挖掘研究进展 [J]. 南方农业学报, 2022, 53(10): 2885-2893.
	Qi XL, Zhao MZ, Yin WS, et al. Physiological mechanism and gene discovery for high temperature tolerance of wheat: a review [J]. J South Agric, 2022, 53(10): 2885-2893.
[5]	刘琴, 孙辉, 何道文. 干旱和高温对植物胁迫效应的研究进展 [J]. 西华师范大学学报: 自然科学版, 2005, 26(4): 364-368.
	Liu Q, Sun H, He DW. Plant responses to the high temperature and moisture stress [J]. J China West Norm Univ Nat Sci, 2005, 26(4): 364-368.
[6]	吴玉环, 黄国宏, 高谦, 等. 苔藓植物对环境变化的响应及适应性研究进展 [J]. 应用生态学报, 2001, 12(6): 943-946.
	Wu YH, Huang GH, Gao Q, et al. Research advance in response and adaptation of bryophytes to environmental change [J]. Chin J Appl Ecol, 2001, 12(6): 943-946.
[7]	沙伟, 王欢, 师帅. 旱后复水对东亚砂藓生理生化指标的影响 [J]. 武汉植物学研究, 2010(2): 246-249.
	Sha W, Wang H, Shi S. Effects of rewatering on the physio-biochemical indexes of Racomitrium japonicum in long-time drought [J]. J Wuhan Bot Res, 2010(2): 246-249.
[8]	王晶晶, 白玉, 蔡璨, 等. 砂藓属植物抗逆特性及相关功能基因研究进展 [J]. 高师理科学刊, 2024, 44(5): 87-92.
	Wang JJ, Bai Y, Cai C, et al. Advances in studies on stress resistance characteristics and the related functional genes of Racomitrium plants [J]. J Sci Teach Univ, 2024, 44(5): 87-92.
[9]	沙伟, 王鑫, 张梅娟, 等. 砂藓抗高温相关基因RcDAPDC的克隆及表达分析 [J]. 分子植物育种, 2020, 18(24): 8092-8098.
	Sha W, Wang X, Zhang MJ, et al. Cloning and expression analysis of high-temperature resistance related gene RcDAPDC in Racomitrium canescens [J]. Mol Plant Breed, 2020, 18(24): 8092-8098.
[10]	周雅, 张道远. 植物早期光诱导蛋白的结构、功能及表达模式研究进展 [J]. 基因组学与应用生物学, 2015, 34(6): 1339-1346.
	Zhou Y, Zhang DY. The research progress of early light induced protein in plant [J]. Genom Appl Biol, 2015, 34(6): 1339-1346.
[11]	Pinto F, Berti M, Olivares D, et al. Leaf development, temperature and light stress control of the expression of early light-inducible proteins (ELIPs) in Vitis vinifera L. [J]. Environ Exp Bot, 2011, 72(2): 278-283.
[12]	Heddad M, Adamska I. Light stress-regulated two-helix proteins in Arabidopsis thaliana related to the chlorophyll a/b-binding gene family [J]. Proc Natl Acad Sci U S A, 2000, 97(7): 3741-3746.
[13]	Berti M, Pinto M. Expression of early light induced protein in grapevine and pea, under different conditions and its relation with photoinhibition [J]. Chilean J Agric Res, 2012, 72(3): 371-378.
[14]	Shimosaka E, Sasanuma T, Handa H. A wheat cold-regulated cDNA encoding an early light-inducible protein (ELIP): its structure, expression and chromosomal location [J]. Plant Cell Physiol, 1999, 40(3): 319-325.
[15]	Peng YF, Ma TY, Wang X, et al. Proteomic and transcriptomic responses of the desiccation-tolerant moss Racomitrium canescens in the rapid rehydration processes [J]. Genes, 2023, 14(2): 390.
[16]	鲍薇, 王晶晶, 沙伟, 等. 砂藓SMPD基因的克隆及对干旱和高温胁迫响应分析 [J]. 广西植物, 2025, 45(8): 1530-1542.
	Bao W, Wang JJ, Sha W, et al. Cloning of SMPD in Racomitrium canescens and analysis of its responses to drought and high-temperature stresses [J]. Guihaia, 2025, 45(8): 1530-1542.
[17]	马天意, 许家佳, 路文婧, 等. ‘金小童’大白菜BrcGASA3基因在盐碱胁迫下的表达分析及抗性鉴定 [J]. 生物技术通报, 2025, 41(2): 127-138.
	Ma TY, Xu JJ, Lu WJ, et al. Expression analysis and resistance identification of BrcGASA3 in Chinese cabbage ‘Jinxiaotong’ Cultivar under saline-alkali stress [J]. Biotechnol Bull, 2025, 41(2): 127-138.
[18]	刘博. 毛尖紫萼藓抗旱相关基因的克隆与功能分析 [D]. 齐齐哈尔: 齐齐哈尔大学, 2015.
	Liu B. Cloning and function analysis of three articles of drought resistance related genes from Grimmia pilifera [D]. Qiqihar: Qiqihar University, 2015.
[19]	信雨萌, 沙伟, 张梅娟, 等. 砂藓抗旱相关转录因子基因RcbZIP1的克隆与表达分析 [J]. 基因组学与应用生物学, 2018, 37(7): 2949-2957.
	Xin YM, Sha W, Zhang MJ, et al. Cloning and expression analysis of drought-resistant related transcription factor gene RcbZIP1 from Racomitrium canescens [J]. Genom Appl Biol, 2018, 37(7): 2949-2957.
[20]	马天意, 张时通, 朱巍巍, 等. 砂藓RcPLD基因的抗旱功能分析 [J]. 西北植物学报, 2021, 41(7): 1091-1100.
	Ma TY, Zhang ST, Zhu WW, et al. Analysis of drought-resistance function of RcPLD gene from Racomitrium canescens [J]. Acta Bot Boreali Occidentalia Sin, 2021, 41(7): 1091-1100.
[21]	杨帆, 苗灵凤, 胥晓, 等. 植物对干旱胁迫的响应研究进展 [J]. 应用与环境生物学报, 2007, 13(4): 586-591.
	Yang F, Miao LF, Xu X, et al. Progress in research of plant responses to drought stress [J]. Chin J Appl Environ Biol, 2007, 13(4): 586-591.
[22]	Zhuo CL, Cai JL, Guo ZF. Overexpression of early light-induced protein (ELIP) gene from Medicago sativa ssp. falcata increases tolerance to abiotic stresses [J]. Agron J, 2013, 105(5): 1433-1440.
[23]	刘秀瑾, 闫振, 张道远. 齿肋赤藓早期光诱导蛋白基因ScELIPs响应非生物胁迫的功能鉴定 [J]. 植物生理学报, 2021, 57(8): 1679-1689.
	Liu XJ, Yan Z, Zhang DY. Functional analysis of Syntrichia caninervis early light-induced protein genes ScELIPs in response to abiotic stresses [J]. Plant Physiol Commun, 2021, 57(8): 1679-1689.
[24]	Hutin C, Nussaume L, Moise N, et al. Early light-induced proteins protect Arabidopsis from photooxidative stress [J]. Proc Natl Acad Sci U S A, 2003, 100(8): 4921-4926.
[25]	Kufryk G, Kieselbach T, Vermaas W, et al. ELIP/CAB-type proteins associated with photosystem Ⅱ during normal growth of Cyanobacterium synechocystis sp. PCC 6803 [M]//Photosynthesis. Energy from the Sun. DordrechtSpringer Netherlands 2008: 723-727.
[26]	Casazza AP, Rossini S, Rosso MG, et al. Mutational and expression analysis of ELIP1 and ELIP2 in Arabidopsis thaliana [J]. Plant Mol Biol, 2005, 58(1): 41-51.
[27]	Adamska I, Kruse E, Kloppstech K. Stable insertion of the early light-induced proteins into etioplast membranes requires chlorophyll a [J]. J Biol Chem, 2001, 276(11): 8582-8587.
[28]	Niu QQ, Zhao LY, Dang YX, et al. Bryophyte ELIPs as evolutionary guardians: engineering chloroplast resilience and ABA-enhanced drought tolerance in crops [J]. Plant Cell Environ, 2025, 48(11): 7948-7960.
[29]	Noman M, Jameel A, Qiang WD, et al. Overexpression of GmCAMTA12 enhanced drought tolerance in Arabidopsis and soybean [J]. Int J Mol Sci, 2019, 20(19): 4849.
[30]	Liu WP, Zhao BG, Chao Q, et al. The maize AP2/EREBP transcription factor ZmEREB160 enhances drought tolerance in Arabidopsis [J]. Trop Plant Biol, 2020, 13(3): 251-261.
[31]	Hachiya T, Inaba J, Wakazaki M, et al. Excessive ammonium assimilation by plastidic glutamine synthetase causes ammonium toxicity in Arabidopsis thaliana [J]. Nat Commun, 2021, 12: 4944.
[32]	Tao L, Zeba N, Ashrafuzzaman M, et al. Heavy metal stress-inducible early light-inducible gene CaELIP from hot pepper (Capsicum annuum) shows broad expression patterns under various abiotic stresses and circadian rhythmicity [J]. Environ Exp Bot, 2011, 72(2): 297-303.
[33]	李玉红, 林荣呈. 叶绿素合成调控研究进展 [J]. 生命科学, 2024, 36(9): 1141-1148.
	Li YH, Lin RC. Advances in the regulation mechanism of chlorophyll synthesis [J]. Chin Bull Life Sci, 2024, 36(9): 1141-1148.
[34]	钟思彤, 张亚真, 孔祥瑞, 等. 高等植物捕光叶绿素a/b结合蛋白的研究进展 [J]. 农业生物技术学报, 2024, 32(2): 444-457.
	Zhong ST, Zhang YZ, Kong XR, et al. Advances of light-harvesting chlorophyll a/b binding protein (CAB) in higher plants [J]. J Agric Biotechnol, 2024, 32(2): 444-457.
[35]	王娟, 李德全. 逆境条件下植物体内渗透调节物质的积累与活性氧代谢 [J]. 植物学通报, 2001, 18(4): 459-465.
	Wang J, Li DQ. The accumulation of plant osmoticum and activated oxygen metabolism under stress [J]. Chin Bull Bot, 2001, 18(4): 459-465.
[36]	Chen Z, An YY, Wang LJ. ALA reverses ABA-induced stomatal closure by modulating PP2AC and SnRK2.6 activity in apple leaves [J]. Hortic Res, 2023, 10(6): uhad067.
[37]	赵天宏, 孙加伟, 付宇. 逆境胁迫下植物活性氧代谢及外源调控机理的研究进展 [J]. 作物杂志, 2008(3): 10-13.
	Zhao TH, Sun JW, Fu Y. Advances of research on metabolism of plant reactive oxygen species and exogenous regulation under abiotic stresses [J]. Crops, 2008(3): 10-13.
[38]	周丽, 粱新乐, 励建荣. 类胡萝卜素抗氧化作用研究进展 [J]. 食品研究与开发, 2003, 24(2): 21-23.
	Zhou L, Liang XL, Li JR. Research progress on antioxidant effect of carotenoids [J]. Food Res Dev, 2003, 24(2): 21-23.
[39]	Sun TH, Zhou F, Huang XQ, et al. ORANGE represses chloroplast biogenesis in etiolated Arabidopsis cotyledons via interaction with TCP14 [J]. Plant Cell, 2019, 31(12): 2996-3014.