Screening Differentially Expressed Proteins in Response to Salt Stress of Tomato Leaves Based on TMT and PRM Techniques

doi:10.6048/j.issn.1001-4330.2022.06.013

Abstract

Abstract:

【Objective】 Salt stress is one of the key abiotic stresses that cause yield loss and quality decline of tomato.Therefore, it is of great significance to reveal the molecular mechanism of tomato salt tolerance. 【Methods】 In this study, tomato salt-tolerant introduction system IL-7-5-5 and tomato salt-sensitive M82 were used as test materials, and isotope relative labeling and absolute quantification (TMT) technology were combined with quantitative parallel reaction monitoring (PRM) technology.The proteomics of leaves of tomato seedlings under 200 mM salt stress for 12 h were studied to screen out potential target proteins with significant salt stress response.【Results】 (1)A total of 286 differentially expressed proteins (DEPs) were identified.Under salt stress, 191 DEPs were identified by IL-7-5-5, of which 119 were up-regulated and 72 down-regulated.157 DEPs were identified in M82, of which 84 were up-regulated and 73 down-regulated.Venn diagram analysis showed that 129 and 95 differential proteins were specific to IL-7-5-5 and M82, respectively.There were 62 differentially expressed proteins, among which 28 were up-regulated in IL-7-5-5 and M82, and 15 were down-regulated, showing consistent response to salt stress.Among the 19 differentially expressed proteins, 5 proteins were down-regulated in ST and up-regulated in SS, and 14 differentially expressed proteins were up-regulated in ST and down-regulated in SS; (2) The proteomic analysis showed that the GO enrichment tomato had strong salt reactive protein type induction ability, mainly related to metabolic process, single-tissue processes, and cellular processes and the cellular composition mainly involved cells, organelles, molecular compounds and membrane. And gene ontology revealed that these differential proteins were mainly involved in the regulation of catalytic activity, bound with molecular function,.(3) PRM validation results of 11 significantly different proteins showed the same trend as TMT quantitative results.Differential proteins included A0A3Q7E8T9, A0A3Q7EK65, A0A3Q7FY19, A0A3Q7G430, A0A3Q7ITH0, A0A3Q7J1Y7, P05116, Q43779, A0A3Q7F8W6,A0A3Q7GKU3 and A0A3Q7J0Z4, which might be potential target proteins for salt tolerance of tomato seedlings. 【Conclusion】 In this study, TMT combined with PRM technology was used to screen out differentially expressed proteins in tomato seedlings responding to salt stress, which could lay a foundation for further understanding of the molecular regulatory mechanism of tomato seedlings responding to salt stress.

Key words: tomato; salt stress; proteomics; TMT; PRM

摘要：

【目的】 盐胁迫是造成番茄产量损失和品质严重下降的关键非生物胁迫之一,研究番茄耐盐的分子机制,为认识番茄幼苗应答盐胁迫分子调控机制奠定基础。【方法】 研究利用番茄耐盐渐渗系IL-7-5-5和番茄盐敏感M82为试材,采用同位素相对标记与绝对定量(TMT)技术结合定量蛋白质组平行反应监测(PRM)技术,对200 mM盐胁迫12 h的番茄幼苗叶片进行蛋白质组学研究,筛选出盐胁迫响应显著的潜在靶标蛋白。【结果】 (1)共鉴定出286个差异显著表达蛋白(DEPs)。盐胁迫下IL-7-5-5鉴定到191个DEPs,其中119个表达上调,72个表达下调。在M82中鉴定出157个DEPs,其中84个表达上调,73个表达下调。维恩图分析显示,有129和95个差异蛋白分别特异于IL-7-5-5和M82。有62个显著差异蛋白共表达,其中 28 个在IL-7-5-5和M82中均上调,15 个均为下调,表现出对盐胁迫的一致响应。19个显著差异蛋白在表达相反,有5个蛋白质在ST中下调,在SS中上调,14个差异蛋白在ST中上调,在SS中下调;(2)番茄盐反应蛋白种类诱导能力强,主要与代谢过程、单组织过程以及细胞过程有关,细胞组成主要涉及细胞、细胞器、分子复合物和膜,基因本体分子功能显示,这些差异性蛋白主要参与催化活性、绑定和分子功能调控。(3)选择11个显著差异蛋白PRM验证结果与TMT 定量表现出相同的趋势。差异蛋白包括 A0A3Q7E8T9、A0A3Q7EK65、A0A3Q7FY19、A0A3Q7G430、A0A3Q7ITH0、A0A3Q7J1Y7、P05116、Q43779、A0A3Q7F8W6、A0A3Q7GKU3、A0A3Q7J0Z4,可能是番茄幼苗耐盐的潜在靶标蛋白。【结论】 研究采用 TMT 结合 PRM 技术,筛选出番茄幼苗响应盐胁迫差异表达蛋白。

关键词: 番茄, 盐胁迫, 蛋白质组学, TMT, PRM

CLC Number:

S188
S641.2

WANG Qiang, LIU Huifang, HAN Hongwei, ZHUANG Hongmei, WANG Baike, WANG Juan, YANG Tao, WANG Hao, QIN Yong. Screening Differentially Expressed Proteins in Response to Salt Stress of Tomato Leaves Based on TMT and PRM Techniques[J]. Xinjiang Agricultural Sciences, 2022, 59(6): 1418-1428.

王强, 刘会芳, 韩宏伟, 庄红梅, 王柏柯, 王娟, 杨涛, 王浩, 秦勇. 基于 TMT和PRM 技术筛选番茄响应盐胁迫差异表达蛋白[J]. 新疆农业科学, 2022, 59(6): 1418-1428.

Figures/Tables 5

Fig.1 Comparative analysis of differentially expressed proteins in two tomato varieties. Note: (A) Number of up-regulated and down-regulated differentially expressed proteins (DEPs). (B) Venn diagram of tomato DEPs in two maize varieties.

Fig.2 Gene ontology enrichment analysis of differentially expressed protein Note:A and D represent the biological processes of SS12 vs SS0 and ST12 vs ST0 respectively.Figure2- B and E represent SS12 vs SS0 and ST12 vs ST0 cell components respectively.Figure 2-C and F represent the molecular functions of SS12 vs SS0 and ST12 vs ST0 respectively

Fig.3 KOG functional classification of ST12vsST0 differential proteins in response to salt stress

Fig.4 KOG functional classification of SS12vsSS0 differential proteins in response to salt stress

Table 1 Comparison of PRM and TMT quantification result

蛋白号 Protein Accession	蛋白质的描述 Protein description	SS12/SS0 TMT PRM		ST12/ST0 TMT PRM
A0A3Q7E8T9	过氧化物酶^*	/	/	1.56	2.72
A0A3Q7EK65	精氨酸酶 2^*	/	/	1.97	3.98
A0A3Q7FY19	可能乙酰辅酶a乙酰转移酶^*	/	/	1.64	2.51
A0A3Q7G430	黄氧素脱氢酶	0.64	0.54	3.37	3.25
A0A3Q7ITH0	过氧化物酶	1.82	1.24	1.64	1.89
A0A3Q7J1Y7	可能多胺氧化酶 2	0.81	0.61	2.26	6.47
P05116	1-氨基环丙烷-1-羧酸氧化酶1	3.41	4.96	1.67	2.67
Q43779	超氧化物歧化酶^*	1.53	1.8	/	/
A0A3Q7F8W6	微管蛋白α链	0.6	0.48	1.57	1.63
A0A3Q7GKU3	40S核糖体蛋白S13样^*	/	/	1.62	1.44
A0A3Q7J0Z4	延长因子1α	0.57	0.3	2.22	3.48

References 56

[1]	Zhai Y, Yang Q, Hou M. The Effects of Saline Water Drip Irrigation on Tomato Yield, Quality, and Blossom-End Rot Incidence-A 3a Case Study in the South of China[J]. PLoS ONE, 2015, 10:e0142204. DOI URL
[2]	Munns R, Tester M. Mechanisms of salinity tolerance[J]. Annual Review of Plant Biology, 2008, 59(0):651-681. DOI URL
[3]	Pérez-Alfocea F, Balibrea M E, Cruz A.S, et al. Agronomical and physiological characterization of salinity tolerance in a commercial tomato hybrid[J]. Plant and Soil, 1996, 180(2):251-257. DOI URL
[4]	Zhu J K. Salt and drought stress signal transduction in plants[J]. Annual Review of Plant Biology, 2002, 53(1):247-273. DOI URL
[5]	Zhu J K. Abiotic stress signaling and responses in plants[J]. Cell, 2016, 167(2):313-324. DOI URL
[6]	Ishikawa T, Shabala S. Control of xylem Na(+) loading and transport to the shoot in rice and barley as a determinant of differential salinity stress tolerance[J]. Physiologia Plantarum, 2018, 165(3):619-631. DOI URL
[7]	Zhang Z, Mao C Y, Shi Z, et al. The amino acid metabolic and carbohydrate metabolic pathway play important roles during salt-stress response in tomato[J]. Frontiers in Plant Science, 2017, 8:1231. DOI PMID
[8]	Manaa A, Faurobert M, Valot B, et al. Effect of salinity and calcium on tomato fruit proteome[J]. Omics A Journal of Integrative Biology, 2013, 17(6):338-352. DOI URL
[9]	Keutgen A J, Pawelzik E. Contribution of amino acids to strawberry fruit quality and their relevance as stress indicators under NaCl salinity[J]. Food Chemistry, 2008, 111(3):642-647. DOI URL
[10]	Galli v, Messias R D S, Perin E C, et al. Mild salt stress improves strawberry fruit quality[J]. LWT-Food Science and Technology, 2016, 73:693-699. DOI URL
[11]	Lu S W, Li T L, Jing J. Effects of tomato fruit under Na⁺-salt and Cl-salt stresses on sucrose metabolism[J]. African Journal of Agricultural Research, 2010, 5(16):2227-2231.
[12]	Chen C, Plant A. Salt-induced protein synthesis in tomato roots: the role of ABA[J]. Journal of Experimental Botany, 1999, 50(334):677-687. DOI URL
[13]	Amini F, Ehsanpour A, Hoang Q, et al. Protein pattern changes in tomato under in vitro salt stress[J]. Russian Journal of Plant Physiology, 2007, 54(4):464-471. DOI URL
[14]	Chen S, Gollop N, Heuer B. Proteomic analysis of salt-stressed tomato (Solanum lycopersicum) seedlings: effect of genotype and exogenous application of glycinebetaine[J]. Journal of Experimental Botany, 2009, 60(7):2005-2019. DOI URL
[15]	Ibrahimova U F, Mammadov A C, Feyziyev Y M. The effect of NaCl on some physiological and biochemical parameters in Triticum aestivum L. genotypes[J]. Plant Physiol Report, 2019, 24(3):370-375. DOI URL
[16]	Pailles Y, Awlia M, Julkowska M M, et al. Diverse Traits Contribute to Salinity Tolerance of Wild Tomato Seedlings from the Galapagos Islands[J]. Plant Physiology, 2020, 182(1):534-546. DOI URL
[17]	Pang Q S, Chen S, Dai Y, et al. Comparative proteomics of salt tolerance in Arabidopsis thaliana and Thellungiella halophila[J]. Proteome Research, 2010, 9(5):2584-2599. DOI URL
[18]	Zhou J, Palmer J, Zhou S, et al. Differential root proteome expression tomato genotypes with contrasting drought tolerance exposed to dehydration[J]. Journal of the American Society for Horticultural Science, 2013, 138(2):131-141. DOI URL
[19]	Peterson A C, Russell J D, Bailey D J. Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics[J]. Molecular & Cellular Proteomics: MCP, 2012, 11(11):1475-1488. DOI URL
[20]	Wang Z X, Shang P, Li Q G, et al. iTRAQ-based proteomic analysis reveals key proteins affecting muscle growth and lipid deposition in pigs[J]. Scientific reports, 2017, 7(1):46717. DOI URL
[21]	Ira G W. The function of ribosomal protein outside the ribosome[J]. Life of Chemistry, 1997, 17(1):23-25.
[22]	Tadepalli A, Roney O L. Ribosomal protein S25 mRNA partners with MTF-1 and La to provide a p53-mediated mechanism for survival or death[J]. The Journal of Biological Chemistry, 2002, 277(6):4147. DOI URL
[23]	孙伟, 李燕, 赵彦修, 等. 盐地碱蓬延伸因子(SsEF-1α)的克隆与表达分析[J]. 西北植物学报, 2004, 24(9):1657-1661.
	SUN Wei, LI Yan, ZHAO Yanxiu, et al. Isolation and characterizing of a cDNA clone encoding an elongation factorEF-1α from halophyte Suaeda salsa[J]. Acta Botanica Boreali-Occidentalia Sinica, 2004, 24(9):1657-1661.
[24]	Padaria J C, Yadav R, Tarafdar A, et al. Molecular cloning and characterization of drought stress responsive abscisic acid-stress-ripening (Asr 1) gene from wild jujube, Ziziphus nummularia (Burm.f.) Wight & Arn[J]. Molecular biology reports, 2016, 43(8):849-859. DOI URL
[25]	Feng Z J, Xu Z S, Sun J, et al. Investigation of the ASR family in foxtail millet and the role of ASR1 in drought oxidative stress tolerance[J]. Plant Cell Reports, 2016, 35(1):115-128. DOI URL
[26]	Goldgur Y, Rom R, Ghirlando D, et al. Desiccation and zinc binding induce transition of tomato abscisic acid stress ripening 1, a water stress- and salt stress-regulated plant-specific protein, from unfolded to folded state[J]. Plant Physiology, 2007, 143(2):617-628. PMID
[27]	Cakir B, Agasse A, Gaillard C, et al. A grape ASR protein involved in sugar and abscisic acid signaling[J]. The Plant Cell, 2003, 15(9):2165-2180. DOI URL
[28]	Kalifa Y A, Gilad Z, Konrad M, et al. The water- and salt-stress-regulated Asr1(abscisic acid stress ripening) gene encodes a zinc-dependent DNA-binding protein[J]. The Biochemical journal, 2004, 381(Pt2):373-378. DOI URL
[29]	Nie H S, Wang Y L, Wei C C, et al. Embryogenic Calli Induction and Salt Stress Response Revealed by RNA-Seq in Diploid Wild Species Gossypium sturtianum and Gossypium Raimondi[J]. Frontiers in Plant Science, 2021, 12 : 715041-715041. DOI URL
[30]	Weng Q Y, Zhao Y M, Zhao Y N, et al. Identification of Salt Stress-Responsive Proteins in Maize (Zea may) Seedlings Using iTRAQ-Based Proteomic Technique[J]. Iranian Journal of Biotechnology, 2021, 19(1) : e2512-e2512.
[31]	陈娜, 胡冬青, 潘丽娟, 等. 花生中胁迫相关基因AhDHNI的克隆及非生物胁迫下表达分析[J]. 核农学报, 2014, 28(12):2159-2166. DOI
	CHEN Na, HU Dongqin, PAN Lijuan, et al. Cloning of a Dehydrin Gene AhDHN1 and Its Expression Analysis during Abiotic Stresses in Peanut[J]. Journal of Nuclear Agricultural Sciences, 2014, 28(12):2159-2166.
[32]	Chourey K, Ramani S, Apte S K. Accumulation of LEA proteins in salt (NaCl) stressed young seedlings of rice (Oryza sativa L.) cultivar Bura Rata and their degradation during recovery from salinity stress[J]. Journal of Plant Physiology, 2003, 160(10):1165-1174. PMID
[33]	Jia F J, Qi S D, Li H, et al. Overexpression of late embryogenesis abundant 14 enhances Arabidopsis salt stress tolerance[J]. Biochemical and Biophysical Research Communications, 2014, 454(4):505-511. DOI URL
[34]	Wang M Z, Li P, Li C, et al. SiLEA14, a novel atypical LEA protein, confers abiotic stress resistance in foxtail millet[J]. BMC Plant Biology, 2014, 14(1):290. DOI URL
[35]	Park S C, Kim Y H, Jeong J C, et al. Sweetpotato late embryogenesis abundant 14 (IbLEA14) gene influences lignifications and increases osmotic and salt stress-tolerance of transgenic calli[J]. Planta, 2011, 233(3):621-634. DOI URL
[36]	Yu H T, Wang T. Proteomic dissection of endosperm starch granule associated proteins reveals a network coordinating starch biosynthesis and amino acid metabolism and glycolysis in rice endosperms[J]. Frontiers in Plant Science, 2016, 7:707.
[37]	Wang N B, Zhao J, He X Y, et al. Comparative proteomic analysis of drought tolerance in the two contrasting Tibetan wild genotypes and cultivated genotype[J]. BMC Genomics, 2015, 16(1):432. DOI URL
[38]	Maeda H, Dudareva N. The shikimate pathway and aromatic amino acid biosynthesis in plants[J]. Annual Review of Plant Biology, 2012, 63(1):73-105. DOI URL
[39]	Jander G, Joshi V. Recent progress in deciphering the biosynthesis of aspartate derived amino acids in plants[J]. Molecular Plant, 2010, 3(1):54-65. DOI PMID
[40]	Rothman S. How is the balance between protein synthesis and degradation achieved?[J]. Theoretical Biology and Medical Modelling, 2010, 7(1):25. DOI URL
[41]	Christiane R, Stephen P, Steffen R. Singlet oxygen signaling links photosynthesis to translation and plant growth[J]. Trends in Plant Science, 2010, 15(9):499-506. DOI PMID
[42]	Houda M-D, Houhamed D, Laure J, et al. An Arabidopsis mutant disrupted in ASN2 encoding asparagine synthetase 2 exhibits low salt stress tolerance[J]. Plant Physiology and Biochemistry, 2011, 49(6):623-628. DOI URL
[43]	Wang H B, Liu D C, Sun J Z, et al. Asparagine synthetase gene TaASN1 from wheat is up-regulated by salt stress, osmotic stress and ABA[J]. Journal of Plant Physiology, 2005, 162(1):81-89. DOI URL
[44]	Conroy C, Ching J, Gao Y, et al. Knockout of AtMKK1 enhances salt tolerance and modifies metabolic activities in Arabidopsis[J]. Plant signaling & behavior, 2013, 8(5):e24206.
[45]	Bouche N, Fromm H. GABA in plants: Just a metabolite?[J]. Trends in Plant Science, 2004, 9(3):110-115. DOI URL
[46]	Michaeli S, Fromm H. Closing the loop on the GABA shunt in plants: are GABA metabolism and signaling entwined?[J]. Frontiers in Plant Science, 2015, 6:419. DOI PMID
[47]	周翔, 吴晓岚, 李云, 等. 盐胁迫下玉米幼苗ABA和GABA的积累及其相互关系[J]. 应用与环境生物学报, 2005, 11(4):412-415.
	ZHOU Xiang, WU Xiaolan, LI Yun, et al. Accumulations and Correlations of ABA And GABA in Maize Seedling under Salt Stress[J]. Chinese Journal of Applied & Environmental Biology, 2005, 11(4):412-415.
[48]	Achard P, Cheng H, Liesbeth D G, et al. Integration of plant responses to environmentally activated phytohormonal signals[J]. Science, 2006, 311(5757):91-94. DOI URL
[49]	Li C Z, Jiao J, Wang G X. The important roles of reactive oxygen species in the relationship between ethylene and polyamines in leaves of spring wheat seedlings under root osmotic stress[J]. Plant Science, 2003, 166(2):303-315. DOI URL
[50]	Gil-Amado J A, Gomez-Jimenez M C. Regulation of polyamine metabolism and biosynthetic gene expression during olive mature-fruit abscission[J]. Planta, 2012, 235(6):1221-1237. DOI PMID
[51]	Farooq M, Wahid A N, Kobayashi D. Plant drought stress: effects, mechanisms and management[J]. Agronomy for Sustainable Development, 2009, 29(1):185-212. DOI URL
[52]	Zenda T, Liu S T, Wang X, et al. Comparative proteomic and physiological analyses of two divergent maize inbred lines provide more insights into drought-stress tolerance mechanisms[J]. International Journal of Molecular Sciences, 2018, 19(10):3225. DOI URL
[53]	Rasoulnia A, Bihamta M R, Peyghambari S A, et al. Proteomic response of barley leaves to salinity[J]. Molecular Biology Reports, 2011, 38(8):5055-5063. DOI URL
[54]	Klaus K, Heribert H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction[J]. Annual Review of Plant Biology, 2004, 55(1):373-399. DOI URL
[55]	Westermann S, Weber K. Post-translational modifications regulate microtubule function.[J]. Nature Reviews Molecular Cell Biology, 2003, 4(12): 938. PMID
[56]	Gaertig J, Verhey F. The tubulin code[J]. Cell Cycle, 2007, 6 (17):2152-2160. DOI URL