Identification and Interaction Analysis of SMAS Gene Family in Tea Plant（Camellia sinensis）

doi:10.13560/j.cnki.biotech.bull.1985.2022-1045

Abstract

Abstract:

S-adenosylmethionine synthase（SAMS）is the only enzyme that catalyzes the synthesis of S-adenosylmethionine（SAM）from methionine and ATP. Research shows that SAMS participates in lignin biosynthesis. In this study, we analyzed the expression patterns and protein interaction networks of the SAMS gene family identified from ‘Huangdan’ tea plant（Camellia sinensis）to explore candidate genes of CsSAMS that may be involved in lignin synthesis. Then we identified the members of CsSAMS gene family by bioinformatics based on the genome of ‘Huangdan’, and further analyzed their protein physicochemical properties, phylogenetic tree, chromosome location, gene structure, protein structure and expression pattern. We also studied the protein interaction network by yeast two-hybrid technology（Y2H）. Finally, we determined the lignin contents of the first bud and the two leaves of ‘Haungdan’, ‘Tieguanyin’, ‘Jinguanyin’ and ‘Fuding Dahao’ by UV spectrophotometer. The results of bioinformatics analysis showed that 4 CsSAMS gene family members were identified in ‘Huangdan’. The number of encoded amino acids was 345-519, and the isoelectric point ranged from 6.12 to 6.47. The prediction results of subcellular localization revealed that CsSAMS1 was located in the chloroplast, CsSAMS2 and CsSAMS3 were located in the cytoplasm, and CsSAMS4 was located in the cytoskeleton. Expression of CsSAMS and lignin content detection of different tea varieties indicated that CsSAMS2, CsSAMS3 and CsSAMS4 might potentially regulate lignin content. In addition, Y2H results showed that CsSAMS4 formed homodimers with itself. In this study, the physicochemical properties of four CsSAMS members are identified and analyzed, and their functions are predicted. The expression patterns of CsSAMS genes in different tissue sites, under nitrogen and fluoride treatments, as well as the potential involvement of CsSAMS in lignin synthesis process, are clarified.

Key words: Camellia sinensis, SAMS, bioinformatics analysis, lignin, expression analysis, interacting proteins

WANG Yi-qing, WANG Tao, WEI Chao-ling, DAI Hao-min, CAO Shi-xian, SUN Wei-jiang, ZENG Wen. Identification and Interaction Analysis of SMAS Gene Family in Tea Plant（Camellia sinensis）[J]. Biotechnology Bulletin, 2023, 39(4): 246-258.

Figures/Tables 15

Table 1 Primer sequences

基因名称Gene name	引物序列Primer sequence（5'-3'）	备注Note
SAMS1	F CATGCCCAAGTGTAACAATAG R AGAGACGGAGAGAGAGAAGA	荧光定量 Quantitative real-time PCR
SAMS2	F CCACTGATGAAACACCTGAAC R GTATGAACTCTAATGGGGACC
SAMS3	F CACGGGGTTCTTTCTCTCTGTCT R GGTGTCCCTCATTCACTGATTCA
SAMS4	F AGCCCGTCATCCCTTCCCAATA R CTCCACCGTGAGCACCCCAACC
BD- SAMS2	F CATGGAGGCCGAATTCATGGATAGTTTCCTC R GCAGGTCGACGGATCCTCAAGCTTTGGGCT	酵母双杂交 Yeast two-hybrid
BD- SAMS4	F CATGGAGGCCGAATTCATGGAAACCTTCCTCT R GCAGGTCGACGGATCCTCAAGCTTTGGGCT
AD- SAMS2	F CATCGATACGGGATCATGGATAGTTTCCTCTT R ACGATTCATCTGCAGCTCAAGCTTTGGGCTTAA
AD- SAMS4	F CATCGATACGGGATCATGGAAACCTTCCTCT R ACGATTCATCTGCAGCTCAAGCTTTGGGCTTG

Fig. 1 Chromosomal distribution of CsSAMS family genes

Table 2 Basic physicochemical properties of CsSAMS proteins

基因 Gene	序列号 Gene ID	蛋白长度 Amino acid/aa	分子量 Molecular weight/kD	等电点 pI	不稳定系数 Instability constant	亲水性 GRAVY	亚细胞定位 Subcellular localization	跨膜结构 Transmembrane domain	信号肽 Signal peptide
CsSAMS1	HD.05G0021870	519	56.98	6.47	28.69	-0.245	叶绿体Chloroplast	无 No	无 No
CsSAMS2	HD.06G0008900	390	42.75	6.24	25.34	-0.331	细胞质Cytoplasm	无 No	无 No
CsSAMS3	HD.08G0000160	345	37.79	6.42	18.7	-0.281	细胞质Cytoplasm	无 No	无 No
CsSAMS4	HD.11G0014340	390	42.68	6.12	27.59	-0.294	细胞骨架Cytoskeleton	无 No	无 No

Fig. 2 Phylogenetic tree（A）, conserved motifs（B）, intron and exon structure distribution（C）of CsSAMS

Fig. 3 Phylogene tree of CsSAMS CsSAM1-4：茶树 Camellia sinensis；AtMAT1（At1g02500）、AtMAT2（At4g01850）、AtMAT3（At2g36880）AtMAT4（At3g17390）：拟南芥 Arabidopsis thaliana；SlSAMS1（NP.001234425）、SlSAMS2（NP.001296305）、SlSAMS3（NP.001234004）、SlSAMS4（XP.010312254）：番茄 Solanum lycopersicum；ZmSAMS1（NP.001130734）、ZmSAMS2（NP.001146249）、ZmSAMS3（NP.001148708）、ZmSAMS4（NP.001132867）：玉米 Zea mays；MtSAMS1（XP.003626035）、MtSAMS2（XP.003609861）、MtSAMS3a（XP.003625682）、MtSAMS3b（XP.013468134）、MtSAMS4（XP.013463713）：蒺藜苜蓿 Medicago truncatula；TuSAMS（XP.020178978）、TuSAMS1（EMS55466）、TuSAMS4（EMS52834）：乌拉尔图小麦 Triticum urartu；OsSAMS1（NP.001389410）、OsSAMS2（NP.001393223）、OsSAMS3（XP.015614349）：水稻 Oryza sativa；DcSAMS（AAA33274）：康乃馨 Dianthus caryophyllus；LcSAMS（AAP13994）：荔枝 Litchi chinensis；PcSAMS（AAG17036）：扭叶松 Pinus contorta；RpSAMS（AIT39705）：刺槐 Robinia pseudoacacia；LrSAMS（AFC88125）：石蒜 Lycoris radiata；BjSAMS（AAK71234）：芥菜 Brassica juncea；CcSAMS（KYP71740）：木豆 Cajanus cajan

Fig. 4 Intraspecies collinearity analysis of CsSAMS

Table 3 Ka/Ks analysis of CsSAMS homologous genes

基因对名称 Gene pair name	非同义突变频率Nonsynonymous mutation Ka	同义突变频率Synonymous mutation Ks	Ka/Ks
CsSAMS1/CsSAMS3	0.033 115 095	1.036 155 399	0.031 959 584
CsSAMS2/CsSAMS4	0.022 137 640	0.526 911 233	0.042 013 983

Fig. 5 Collinearity analysis of CsSAMS within species of HD and TGY

Fig. 6 Analysis of cis-elements in the upstream region of CsSAMS family genes in tea plants I: Phytohormone response. Ⅱ: Abiotic stress response. Ⅲ: Light response. Ⅳ: TF recongnition and binding site. Ⅴ: Tissue specificity. Ⅵ: Plant growth. Ⅶ: Core

Fig. 7 Secondary and tertiary structure prediction of CsSAMS

Fig. 8 Expression patterns of CsSAMS A: Expression pattern of CsSAMS in different tissue. B: Expression pattern of CsSAMS under low nitrogen treatment. C: Expression pattern of CsSAMS under high nitrogen treatment. D: Expression pattern of CsSAMS under fluorine treatment

Fig. 9 Lignin content of different tea varieties Capital letters indicate significance at the P<0.01 level

Fig. 10 Expression pattern of CsSAMS gene in different tea varieties

Fig. 11 Correlation analysis between the lignin content and CsSAMS genes expression in different tea plant varieties * indicates significant correlation at the 0.05 level, and ** indicates significant correlation at the 0.01 level

Fig. 12 Results of Y2H for SAMS

References 39

[1]	Vanholme R, de Meester B, Ralph J, et al. Lignin biosynthesis and its integration into metabolism[J]. Curr Opin Biotechnol, 2019, 56: 230-239. doi: 10.1016/j.copbio.2019.02.018 URL
[2]	王永鑫. 茶树木质素代谢分子机制研究[D]. 南京: 南京农业大学, 2019.
	Wang YX. Study of molecular mechanism of lignin metabolism in tea plant(Camellia sinensis)[D]. Nanjing: Nanjing Agricultural University, 2019.
[3]	Wang YX, Teng RM, Wang WL, et al. Identification of genes reveal-ed differential expression profiles and lignin accumulation during leaf and stem development in tea plant(Camellia sinensis(L.) O. Kuntze)[J]. Protoplasma, 2019, 256(2): 359-370. doi: 10.1007/s00709-018-1299-9
[4]	李寿田, 周健民, 朱世东, 等. 萝卜贮藏期间木质素、纤维素和可溶性糖含量变化及其与糠心的关系[J]. 安徽农业大学学报, 2001, 28(3): 255-258.
	Li ST, Zhou JM, Zhu SD, et al. Content changes of lignin, cellulose and soluble sugar and their correlations with hollowness during storage in radishes[J]. J Anhui Agric Univ, 2001, 28(3): 255-258.
[5]	Bedon F, Legay S. Lignin synthesis, transcriptional regulation and potential for useful modification in plants[J]. CABI Rev, 2011, 2011: 1-28.
[6]	杨建坤. 木质素与茶树新梢嫩度关系的研究[D]. 杨凌: 西北农林科技大学, 2019.
	Yang JK. Study on relation between lignin and shoot tendness of Camellia sinensis[D]. Yangling: Northwest A & F University, 2019.
[7]	Dixon RA, Barros J. Lignin biosynthesis: old roads revisited and new roads explored[J]. Open Biol, 2019, 9(12): 190215. doi: 10.1098/rsob.190215 URL
[8]	Monné M, Marobbio CMT, Agrimi G, et al. Mitochondrial transport and metabolism of the major methyl donor and versatile cofactor S-adenosylmethionine, and related diseases: a review[J]. IUBMB Life, 2022, 74(7): 573-591. doi: 10.1002/iub.2658 pmid: 35730628
[9]	Hanson AD, Roje S. One-carbon metabolism in higher plants[J]. Annu Rev Plant Physiol Plant Mol Biol, 2001, 52: 119-137. doi: 10.1146/arplant.2001.52.issue-1 URL
[10]	Li YY, Ogita S, Keya CA, et al. Expression of caffeine biosynthesis genes in tea(Camellia sinensis)[J]. Z Naturforsch C J Biosci, 2008, 63(3/4): 267-270.
[11]	吕焕青, 王志敏, 汤青林, 等. 多胺生物合成途径中两个关键酶基因研究进展[J]. 生物技术通报, 2015, 31(2): 61-64. doi: 10.13560/j.cnki.biotech.bull.1985.2015.02.008
	Lü HQ, Wang ZM, Tang QL, et al. Polyamine biosynthesis enzyme research progress in two key genes[J]. Biotechnol Bull, 2015, 31(2): 61-64. doi: 10.13560/j.cnki.biotech.bull.1985.2015.02.008
[12]	Roje S. S-Adenosyl-L-methionine: beyond the universal methyl group donor[J]. Phytochemistry, 2006, 67(15): 1686-1698. doi: 10.1016/j.phytochem.2006.04.019 pmid: 16766004
[13]	Sekula B, Ruszkowski M, Dauter Z. S-adenosylmethionine synthases in plants: structural characterization of type I and II isoenzymes from Arabidopsis thaliana and Medicago truncatula[J]. Int J Biol Macromol, 2020, 151: 554-565. doi: 10.1016/j.ijbiomac.2020.02.100 URL
[14]	Shen B, Li CJ, Tarczynski MC. High free-methionine and decreased lignin content result from a mutation in the Arabidopsis S-adenosyl-L-methionine synthetase 3 gene[J]. Plant J, 2002, 29(3): 371-380. doi: 10.1046/j.1365-313x.2002.01221.x pmid: 11844113
[15]	Meng JJ, Wang LS, Wang JY, et al. METHIONINE ADENOSYLTRANSFERASE4 mediates DNA and histone methylation[J]. Plant Physiol, 2018, 177(2): 652-670. doi: 10.1104/pp.18.00183 pmid: 29572390
[16]	Yang SX, Wu TT, Ding CH, et al. SAHH and SAMS form a methyl donor complex with CCoAOMT7 for methylation of phenolic compounds[J]. Biochem Biophys Res Commun, 2019, 520(1): 122-127. doi: 10.1016/j.bbrc.2019.09.101 URL
[17]	Li Y, Xiong WD, He F, et al. Down-regulation of PvSAMS impairs S-adenosyl-L-methionine and lignin biosynthesis, and improves cell wall digestibility in switchgrass[J]. J Exp Bot, 2022, 73(12): 4157-4169. doi: 10.1093/jxb/erac147 URL
[18]	Yang X, Yu Z, Zhang BB, et al. Effect of fluoride on the biosynthesis of catechins in tea[Camellia sinensis(L.) O. Kuntze]leaves[J]. Sci Hortic, 2015, 184: 78-84. doi: 10.1016/j.scienta.2014.12.031 URL
[19]	肖清铁, 王经源, 郑新宇, 等. 水稻根系响应镉胁迫的蛋白质差异表达[J]. 生态学报, 2015, 35(24): 8276-8283.
	Xiao QT, Wang JY, Zheng XY, et al. Analysis of the differently expressed proteins in rice roots in response to cadmium stress[J]. Acta Ecol Sin, 2015, 35(24): 8276-8283.
[20]	杨婉莹, 孙莎莎, 巩彪, 等. 超表达SlSAMS1对番茄镉胁迫的缓解效应及抗氧化系统的影响[J]. 核农学报, 2020, 34(3): 487-496. doi: 10.11869/j.issn.100-8551.2020.03.0487
	Yang WY, Sun SS, Gong B, et al. Effects of overexpressing SlSAMS1 on tomato tolerance to cadmium toxicity and antioxidant system[J]. J Nucl Agric Sci, 2020, 34(3): 487-496.
[21]	Chen J, Wang ZB, Liu SD, et al. Nitrogen stress inhibits root growth by regulating cell wall and hormone changes in cotton(Gossypium hirsutum L.)[J]. J Agron Crop Sci, 2021, 207(6): 1006-1023. doi: 10.1111/jac.v207.6 URL
[22]	Chen CJ, Chen H, Zhang Y, et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data[J]. Mol Plant, 2020, 13(8): 1194-1202. doi: S1674-2052(20)30187-8 pmid: 32585190
[23]	Boerjan W, Bauw G, van Montagu M, et al. Distinct phenotypes generated by overexpression and suppression of S-adenosyl-L-methionine synthetase reveal developmental patterns of gene silencing in tobacco[J]. Plant Cell, 1994, 6(10): 1401-1414. doi: 10.1105/tpc.6.10.1401 pmid: 7994174
[24]	Heidari P, Mazloomi F, Nussbaumer T, et al. Insights into the SAM synthetase gene family and its roles in tomato seedlings under abiotic stresses and hormone treatments[J]. Plants(Basel), 2020, 9(5): 586.
[25]	赖春旺, 周小娟, 陈燕, 等. 龙眼乙烯合成途径基因鉴定及响应ACC处理的分析[J]. 中国农业科学, 2022, 55(3): 558-574. doi: 10.3864/j.issn.0578-1752.2022.03.011
	Lai CW, Zhou XJ, Chen Y, et al. Identification of ethylene synthesis pathway genes in longan and its response to ACC treatment[J]. Sci Agric Sin, 2022, 55(3): 558-574. doi: 10.3864/j.issn.0578-1752.2022.03.011
[26]	Markham GD, Pajares MA. Structure-function relationships in methionine adenosyltransferases[J]. Cell Mol Life Sci, 2009, 66(4): 636-648. doi: 10.1007/s00018-008-8516-1 pmid: 18953685
[27]	张寒冰, 张书发, 李毛, 等. 大麦S-腺苷甲硫氨酸合成酶基因HvSAMS2对非生物胁迫响应的表达分析[J]. 农业生物技术学报, 2021, 29(1): 35-46.
	Zhang HB, Zhang SF, Li M, et al. Expression analysis of S-adenosylmethionine synthetase gene HvSAMS2 from Hordeum vulgare in response to abiotic stress[J]. J Agric Biotechnol, 2021, 29(1): 35-46.
[28]	谭政委, 李磊, 余永亮, 等. 红花S-腺苷甲硫氨酸合成酶基因的克隆与表达分析[J]. 核农学报, 2021, 35(9): 1994-2001. doi: 10.11869/j.issn.100-8551.2021.09.1994
	Tan ZW, Li L, Yu YL, et al. Cloning and expression analysis of S-adenosylmethionine synthetase gene from Carthamus tinctorius L[J]. J Nucl Agric Sci, 2021, 35(9): 1994-2001.
[29]	He MW, Wang Y, Wu JQ, et al. Isolation and characterization of S-adenosylmethionine synthase gene from cucumber and responsive to abiotic stress[J]. Plant Physiol Biochem, 2019, 141: 431-445. doi: 10.1016/j.plaphy.2019.06.006 URL
[30]	何美文. 黄瓜S-腺苷甲硫氨酸合成酶基因鉴定及其在响应盐胁迫中的功能[D]. 南京: 南京农业大学, 2019.
	He MW. Identification of S-adenosylmethionine synthetase gene and its salt stress response function in cucumber[D]. Nanjing: Nanjing Agricultural University, 2019.
[31]	Thomas J, Bowman MJ, Vega A, et al. Comparative transcriptome analysis provides key insights into gene expression pattern during the formation of nodule-like structures in Brachypodium[J]. Funct Integr Genomics, 2018, 18(3): 315-326. doi: 10.1007/s10142-018-0594-z
[32]	Budak H, Zhang BH. microRNAs in model and complex organisms[J]. Funct Integr Genomics, 2017, 17(2/3): 121-124. doi: 10.1007/s10142-017-0544-1 URL
[33]	Peleman J, Boerjan W, Engler G, et al. Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase[J]. Plant Cell, 1989, 1(1): 81-93. doi: 10.1105/tpc.1.1.81 pmid: 2535470
[34]	Chen Y, Zou T, McCormick S. S-adenosylmethionine synthetase 3 is important for pollen tube growth[J]. Plant Physiol, 2016, 172(1): 244-253. doi: 10.1104/pp.16.00774 pmid: 27482079
[35]	Bai ZT, Qi TX, Liu YC, et al. Alteration of S-adenosylhomocysteine levels affects lignin biosynthesis in switchgrass[J]. Plant Biotechnol J, 2018, 16(12): 2016-2026. doi: 10.1111/pbi.12935 pmid: 29704888
[36]	Villalobos DP, Díaz-Moreno SM, Said ESS, et al. Reprogramming of gene expression during compression wood formation in pine: coordinated modulation of S-adenosylmethionine, lignin and lignan related genes[J]. BMC Plant Biol, 2012, 12: 100. doi: 10.1186/1471-2229-12-100 pmid: 22747794
[37]	Scully ED, Gries T, Palmer NA, et al. Overexpression of SbMYB60 in Sorghum bicolor impacts both primary and secondary metabolism[J]. New Phytol, 2018, 217(1): 82-104. doi: 10.1111/nph.14815 pmid: 28944535
[38]	Srivastava AC, Chen F, Ray T, et al. Loss of function of folylpolyglutamate synthetase 1 reduces lignin content and improves cell wall digestibility in Arabidopsis[J]. Biotechnol Biofuels, 2015, 8: 224. doi: 10.1186/s13068-015-0403-z pmid: 26697113
[39]	Murray B, Antonyuk SV, Marina A, et al. Structure and function study of the complex that synthesizes S-adenosylmethionine[J]. IUCrJ, 2014, 1(Pt 4): 240-249. doi: 10.1107/S2052252514012585 pmid: 25075345