Identification of 4CL Gene Family in Arachis hypogaea L. and Expression Analysis in Response to Drought and Salt Stress

doi:10.13560/j.cnki.biotech.bull.1985.2024-1266

Abstract

Abstract:

Objective To analyze the basic characteristics of peanut 4CL (4-coumarate:CoA ligase) gene family members and their responses to drought and salt stress in order to provide important target genes for breeding peanut varieties tolerant to drought and salt. Method The members of peanut 4CL gene family were identified by HMM file, NCBI CDD, and Pfam databases at the whole genome level. ExPASy-ProtParam was used to analyze the physicochemical properties of proteins. MEGA7 and itol tools were used for phylogenetic analysis. The conserved motifs and conserved domains of proteins were analyzed by MEME and CD-search tools in NCBI, respectively. The cis-acting elements were analyzed and visualized by PlantCARE and TBtools, respectively. The transcriptional changes of peanut 4CLs were analyzed by RNA-seq data and RT-qPCR. Result Based on the reference data of peanut Tifrunner genome, 56 peanut 4CL genes were identified, with amino acid length ranging from 239 to 1 208, pI ranging from 5.5 to 9.22, and aliphatic index ranging from 80.2 to 103.13. The instability index (II) was 25.51-48.79, and GRAVY was -0.367-0.139. The Ah4CLs were unevenly distributed on 20 chromosomes of peanut A and B genome, and the Ah4CLs gene density was the highest on chromosomes 5 and 15. In the same clustering branch, Ah4CLs had similar conserved motif composition and intron-exon distribution structure, with exon number ranging from 1 to 18. Ah4CLs promoter regions were rich in light, abiotic stress, hormone and growth response elements. The expression of Ah4CLs was tissue-specific and higher in the root, flower and seed. Under ABA, salt and drought stress, the transcription levels of some Ah4CLs significantly increased, especially Ah4CL28 was significantly up-regulated under ABA, drought, and salt stress. These genes may play an important role in peanut response to abiotic stress. Conclusion The 56 identified peanut 4CL gene family members have different structures and characteristics, and some motifs and domains are conserved. Ah4CLs not only affects plant growth and development but also participates in abiotic stress response.

Key words: peanut, 4CL genes, gene family, phylogenetic analysis, synteny analysis, promoter analysis, drought stress, salt stress

ZHANG Ze, YANG Xiu-li, NING Dong-xian. Identification of 4CL Gene Family in Arachis hypogaea L. and Expression Analysis in Response to Drought and Salt Stress[J]. Biotechnology Bulletin, 2025, 41(7): 117-127.

Figures/Tables 10

Table 1 Genetic primers for RT-qPCR

基因名称 Gene name	正向引物 Forward primer （5′‒3′）	反向引物 Reverse primer （5′‒3′）
Ahactin7	TTGGAATGGGTCAGAAGGATGC	AGTGGTGCCTCAGTAAGAAGC
Ah4CL21	GAGAGAGCGAGGATGAAGGC	AGCTCCTCTGACCACGATCT
Ah4CL22	TCTTGGACAGAGCGGGGATA	GTGTCCTCTTCCAATGCCGA
Ah4CL28	GCTACACTTGGACCCTTGCT	GGCAGAGGAAGGATGGTGTC
Ah4CL37	AGCCCTTGTGCCTTTCTCTC	CATGGCCCTCAACTGAACCT
Ah4CL49	CGAGAATTTGAGCAGCGTGG	ACTCCACCATCTCCTCCTCC
Ah4CL53	CTCTGCACTCCCTCAACCTG	GCCTGGTTTACGCTCTCCTT

Fig. 1 Phylogenetic analysis of 4CL protein in peanut (Arachis hypogaea L.) and Arabidopsis thalianaDifferent regional colors indicate different classification subgroup branches, including Clade 4CL and Clade A‒F

Fig. 2 Analysis of chromosome distribution of 4CL gene in peanut

Fig. 3 Syntenic relationship of peanut 4CL gene family membersThe pink box indicates the peanut chromosomes, while the gray lines denote the positions of the 4CL genes in peanuts. The blue lines illustrate the collinearity observed between the Ah4CL genes

Fig. 4 Phylogenetic relationship (A), conserved motif (B), conserved domain (C), and gene structure (D) analysis of peanut 4CL family members

Table 2 Conserved motif sequences of peanut 4CL family proteins

基序 Motif	序列 Sequence	E值 E_value
Motif 1	GWLHTGDLGYIDEDGYJFIVDRLKELIKYKGEQVAPAELEAVLYSHP	3.8e-1 534
Motif 2	LLYSSGTTGLPKGVVLTHRGL	1.8e-653
Motif 3	GEICIRGPTIMKGYLKBPEAT	2.1e-581
Motif 4	DAAVVPRPDEEAGEVPCAFVV	4.8e-540
Motif 5	VVFIDSJPKTSTGKILRKDLR	2.8e-488
Motif 6	KSEDVYLWTLPMFHVNGLCFP	2.8e-448
Motif 7	SPAFYELHLAVPMAGAVLTTANP	7.2e-402
Motif 8	AVGGTNVCMRKFDAKAILEAIEKHKVT	5.4e-457
Motif 9	PGAIVSQGYGMTETG	6.9e-322
Motif 10	ITEEEIIEFCAKQVAPYKRPK	3.5e-402

Fig. 5 Analysis of cis-acting elements in the promoter regions of peanut 4CL genes

Fig. 6 Expression analysis of peanut 4CL genes in different tissues (A) and after drought, salt and ABA treatment (B)

Fig. 7 Ah4CL28 and Ah4CL37 expression alterations after simulated drought treatmentSignificance analysis is conducted by Student's t-test, with * indicating P<0.05 and ** indicating P<0.01. The same below

Fig. 8 Ah4CL21, Ah4CL22, Ah4CL28, Ah4CL49, and Ah4CL53 expression alterations after salt treatment

References 36

[1]	刘福星, 汪可欣, 张璐, 等. 国内油料作物市场整合关系研究——以油菜籽、花生和芝麻为例 [J]. 中国油料作物学报, 2022, 44(5): 957-965.
	Liu FX, Wang KX, Zhang L, et al. Study on domestic market integration of oil crops—Taking rapeseed, peanut and sesame for examples [J]. Chin J Oil Crop Sci, 2022, 44(5): 957-965.
[2]	刘海东, 陈庆政, 林秀芳, 等. 干旱胁迫对花生生理特性与产质量的影响[J]. 贵州农业科学, 2022, 50: 25-34.
	Liu HD, Chen QZ, Lin XF, et al. Effects of drought stress on physiological characteristics, yield and quality of peanut[J]. Guizhou Agricultural Sciences, 2022, 50: 25-34.
[3]	朱统国, 高华援, 周玉萍, 等. 花生耐盐性鉴定研究进展 [J]. 中国农学通报, 2014, 30(21): 19-23.
	Zhu TG, Gao HY, Zhou YP, et al. Research advances on salt tolerance identification of peanut [J]. Chin Agric Sci Bull, 2014, 30(21): 19-23.
[4]	徐扬, 丁红, 张冠初, 等. 盐胁迫下花生种子萌发期代谢组学分析 [J]. 生物技术通报, 2023, 39(1): 199-213.
	Xu Y, Ding H, Zhang GC, et al. Metabolomics analysis of germinating peanut seed under salt stress [J]. Biotechnol Bull, 2023, 39(1): 199-213.
[5]	徐扬, 张瑞英, 戴良香, 等. 盐胁迫下氮素对花生种子萌发和种子际细菌菌群结构的调控 [J]. 生物技术通报, 2024, 40(2): 253-265.
	Xu Y, Zhang RY, Dai LX, et al. Regulation of nitrogen application on peanut seed germination and spermosphere bacterial community structure under salt stress [J]. Biotechnol Bull, 2024, 40(2): 253-265.
[6]	Dong NQ, Lin HX. Contribution of phenylpropanoid metabolism to plant development and plant-environment interactions [J]. J Integr Plant Biol, 2021, 63(1): 180-209.
[7]	Schneider K, Hövel K, Witzel K, et al. The substrate specificity-determining amino acid code of 4-coumarate: CoA ligase [J]. Proc Natl Acad Sci USA, 2003, 100(14): 8601-8606.
[8]	Hahlbrock K, Scheel D. Physiology and molecular biology of phenylpropanoid metabolism [J]. Annu Rev Plant Physiol Plant Mol Biol, 1989, 40: 347-369.
[9]	Fulda M, Heinz E, Wolter FP. The fadD gene of Escherichia coli K12 is located close to rnd at 39.6 min of the chromosomal map and is a new member of the AMP-binding protein family [J]. Mol Gen Genet, 1994, 242(3): 241-249.
[10]	Stuible H, Büttner D, Ehlting J, et al. Mutational analysis of 4-coumarate: CoA ligase identifies functionally important amino acids and verifies its close relationship to other adenylate-forming enzymes [J]. FEBS Lett, 2000, 467(1): 117-122.
[11]	De Azevedo Souza C, Barbazuk B, Ralph SG, et al. Genome-wide analysis of a land plant-specific acyl: coenzyme A synthetase (ACS) gene family in Arabidopsis, poplar, rice and Physcomitrella [J]. New Phytol, 2008, 179(4): 987-1003.
[12]	Costa MA, Eric Collins R, Anterola AM, et al. An in silico assessment of gene function and organization of the phenylpropanoid pathway metabolic networks in Arabidopsis thaliana and limitations thereof [J]. Phytochemistry, 2003, 64(6): 1097-1112.
[13]	Shockey JM, Fulda MS, Browse J. Arabidopsis contains a large superfamily of acyl-activating enzymes. Phylogenetic and biochemical analysis reveals a new class of acyl-coenzyme a synthetases [J]. Plant Physiol, 2003, 132(2): 1065-1076.
[14]	Lavhale SG, Kalunke RM, Giri AP. Structural, functional and evolutionary diversity of 4-coumarate-CoA ligase in plants [J]. Planta, 2018, 248(5): 1063-1078.
[15]	Chowdhury MEK, Choi B, Cho BK, et al. Regulation of 4CL, encoding 4-coumarate: coenzyme a ligase, expression in kenaf under diverse stress conditions [J]. Plant OMICS, 2013, 6(4): 254-262.
[16]	Nie TK, Sun XX, Wang SL, et al. Genome-wide identification and expression analysis of the 4-coumarate: CoA ligase gene family in Solanum tuberosum [J]. Int J Mol Sci, 2023, 24(2): 1642.
[17]	Sun SC, Xiong XP, Zhang XL, et al. Characterization of the Gh4CL gene family reveals a role of Gh4CL7 in drought tolerance [J]. BMC Plant Biol, 2020, 20(1): 125.
[18]	Zhong J, Qing J, Wang Q, et al. Genome-wide identification and expression analyses of the 4-coumarate: CoA ligase (4CL) gene family in Eucommia ulmoides [J]. Forests, 2022, 13(8): 1253.
[19]	Bertioli DJ, Jenkins J, Clevenger J, et al. The genome sequence of segmental allotetraploid peanut Arachis hypogaea [J]. Nat Genet, 2019, 51(5): 877-884.
[20]	Zhao XB, Li CJ, Wan SB, et al. Transcriptomic analysis and discovery of genes in the response of Arachis hypogaea to drought stress [J]. Mol Biol Rep, 2018, 45(2): 119-131.
[21]	Zhang H, Zhao XB, Sun QX, et al. Comparative transcriptome analysis reveals molecular defensive mechanism of Arachis hypogaea in response to salt stress [J]. Int J Genomics, 2020, 2020: 6524093.
[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.
[23]	Sahraeian SME, Mohiyuddin M, Sebra R, et al. Gaining comprehensive biological insight into the transcriptome by performing a broad-spectrum RNA-seq analysis [J]. Nat Commun, 2017, 8(1): 59.
[24]	Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR [J]. Nucleic Acids Res, 2001, 29(9): e45.
[25]	Douglas CJ. Phenylpropanoid metabolism and lignin biosynthesis: from weeds to trees [J]. Trends Plant Sci, 1996, 1(6): 171-178.
[26]	Vogt T. Phenylpropanoid biosynthesis [J]. Mol Plant, 2010, 3(1): 2-20.
[27]	Sun HY, Li Y, Feng SQ, et al. Analysis of five rice 4-coumarate: coenzyme A ligase enzyme activity and stress response for potential roles in lignin and flavonoid biosynthesis in rice [J]. Biochem Biophys Res Commun, 2013, 430(3): 1151-1156.
[28]	Cheng Y, Ahammed GJ, Yao ZP, et al. Comparative genomic analysis reveals extensive genetic variations of WRKYs in Solanaceae and functional variations of CaWRKYs in pepper [J]. Front Genet, 2019, 10: 492.
[29]	陈雷, 杨明达, 贺群岭, 等. 基于不同生育时期干旱对花生抗旱性的综合评定 [J]. 花生学报, 2024, 53(2): 31-38, 46.
	Chen L, Yang MD, He QL, et al. Comprehensive evaluation on drought resistance of peanuts at different growth stages [J]. J Peanut Sci, 2024, 53(2): 31-38, 46.
[30]	吴兰荣, 陈静, 许婷婷, 等. 花生全生育期耐盐鉴定研究 [J]. 花生学报, 2005, 34(1): 20-24.
	Wu LR, Chen J, Xu TT, et al. Identification of salt tolerance in peanut growth duration [J]. J Peanut Sci, 2005, 34(1): 20-24.
[31]	Chen XH, Wang HT, Li XY, et al. Molecular cloning and functional analysis of 4-coumarate: CoA ligase 4 (4CL-like 1) from Fraxinus mandshurica and its role in abiotic stress tolerance and cell wall synthesis [J]. BMC Plant Biol, 2019, 19(1): 231.
[32]	Wang CH, Yu J, Cai YX, et al. Characterization and functional analysis of 4-coumarate: CoA ligase genes in mul-berry [J]. PLoS One, 2016, 11(5): e0155814.
[33]	陈烨, 单思杰, 钟磊坚, 等. 甜橙过氧化氢酶基因CsCAT2启动子克隆及活性分析 [J]. 农业生物技术学报, 2024, 32(12): 2755-2763.
	Chen Y, Shan SJ, Zhong LJ, et al. Cloning and activity analysis of the CsCAT2 promoter in Citrus sinensis [J]. J Agric Biotechnol, 2024, 32(12): 2755-2763.
[34]	Danquah A, de Zelicourt A, Colcombet J, et al. The role of ABA and MAPK signaling pathways in plant abiotic stress responses [J]. Biotechnol Adv, 2014, 32(1): 40-52.
[35]	Liu QQ, Luo L, Zheng LQ. Lignins: biosynthesis and biological functions in plants [J]. Int J Mol Sci, 2018, 19(2): 335.
[36]	Shomali A, Das S, Arif N, et al. Diverse physiological roles of flavonoids in plant environmental stress responses and tolerance [J]. Plants, 2022, 11(22): 3158.