Genome-wide Identification and Expression Pattern Analysis of the Medicago sativa 4-Coumarate: CoA ligase (4CL) Gene Family

Genome-wide Identification and Expression Pattern Analysis of the Medicago sativa 4-Coumarate: CoA ligase (4CL) Gene Family | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide Identification and Expression Pattern Analysis of the Medicago sativa 4-Coumarate: CoA ligase (4CL) Gene Family Haiyue Lei, Shuyan Liu, Yuqi Zhang, Xinyue Ma, Li Zhao, Fei He, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9384279/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract 4-Coumarate: CoA ligase (4CL) is a pivotal enzyme in the phenylpropanoid metabolic pathway and plays a crucial role in plant growth, development, and stress responses. In this study, a genome-wide analysis was conducted to identify a total of 43 Ms4CL genes ( Ms4CLs ) in 'Xinjiangdaye' ( Medicago sativa ). The fundamental structure, phylogenetic relationships, cis -acting element analysis, and expression patterns of these genes were systematically analysed. The results indicate that Ms4CL genes can be classified into four subfamilies, and subcellular localization predictions suggest that most genes are localized to the plasma membrane. The number of exons ranges from four to thirteen, consistent with the characteristics of most 4CL gene families. The promoter regions are rich in stress response elements associated with MeJA, ABA, and drought, implying that the expression of these genes may be regulated by stress-induced hormonal signals. Collinearity analyses revealed that segmental duplications are the primary mechanisms driving the expansion and functional diversification of this family. Analysis of tissue-specific expression revealed that the four genes Ms4CL8 , Ms4CL22 , Ms4CL37 , and Ms4CL42 exhibit significant tissue-specific expression patterns. An analysis of responses to abiotic stresses indicates that Ms4CL5 and Ms4CL13 are stress-induced genes in response to salt and drought stresses, whereas Ms4CL3 is a stress-repressed gene in response to salt and drought stresses. These three genes exhibit distinct functional differentiation in stress responses. Cis -acting element analysis further revealed promoter enrichment in defense/stress-responsive and phytohormone-related elements, consistent with their roles in environmental adaptation. This study provides a theoretical foundation for elucidating the mechanisms of salt and drought tolerance in alfalfa and supports its genetic improvement. Medicago sativa L. Ms4CL abiotic stress expression analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Abiotic stress refers to the adverse effects of non-living factors on organisms[ 1 , 2 ]. Plants must survive under constantly changing environmental conditions, especially by fluctuations in various abiotic stress factors, such as salinity[ 3 , 4 ] and drought[ 5 – 7 ]. Soil salinization is considered to be one of the primary abiotic stress factors that limit agricultural production in many regions worldwide[ 8 ]. Various ions, including sodium, potassium, calcium, magnesium, and chloride ions, can lead to soil salinization[ 9 ], among these ions, sodium chloride is the most abundant salt in soil. Specifically, when the electrical conductivity (EC) of the saturated extract exceeds 4 dS·m⁻¹ under conditions of 25°C and 15% exchangeable sodium, the soil is classified as saline-alkali soil[ 10 ]. Soil salinization hinders plants’ ability to absorb water and nutrients from the soil, thereby causing plant damage[ 11 ] such as failed seed germination, reduced plant height, stunted root systems, and impaired flowering and fruiting, which in turn leads to decreased yield and quality[ 12 ]. Among various abiotic stresses, soil salinization has become a serious problem facing many regions worldwide, particularly in arid and semi-arid areas[ 13 , 14 ]. According to statistics, the global land area affected by salinization exceeds 800 million hectares, accounting for approximately 6% of the world’s total land area[ 15 – 17 ]. Projections indicate that by 2050, over 50% of the land will be affected by soil salinization[ 17 , 18 ]. Modern molecular genetic studies have confirmed that the primary cause of this phenomenon is that, under salt stress, normal physiological and metabolic functions in plants are impaired by ionic stress, osmotic stress, and oxidative stress[ 19 ]. Soil salinization has a significant impact on global agricultural yields, as it hinders plant establishment, development, and growth, ultimately leading to reduced crop yields[ 20 – 22 ]. Drought stress is also one of the most severe abiotic stresses facing plants worldwide[ 23 ]. With global warming and the impact of human activities, soil drought is becoming increasingly severe and has become a global issue that constrains ecological conservation, plant distribution, and productivity. Under natural conditions, water content in plant tissues accounts for 75%–90% of fresh tissue weight and plays a crucial role in various physiological processes such as growth, development, and metabolism. However, drought restricts water absorption and utilization capacity, thereby limiting crop growth and yield[ 24 – 26 ]. The effects of drought on crops are multifaceted, disrupting various cellular physiological processes, including signal perception, photosynthesis, and all molecular and biochemical functions of cells[ 27 , 28 ]. It does so by inhibiting processes such as cell differentiation, division, and elongation[ 29 ]. Disruption of these physiological processes can lead to delayed growth and development or even plant death, severely impacting agricultural production and hindering local economic development[ 30 – 32 ]. Medicago sativa L. is a perennial leguminous forage crop with a well-developed root system, offering natural advantages in improving the ecological environment and conserving soil and water. Due to its strong drought tolerance, cold tolerance, salt and alkali tolerance, dense foliage, palatability, and high nutritional value, alfalfa is not only used for genetic improvement but also as a high-quality forage to provide nutrition for livestock[ 33 ]. It is an excellent forage widely cultivated in arid and semi-arid regions with promising prospects for industrial development. As one of the most widely distributed and extensively cultivated forage species globally, it is hailed as the “King of Forages” due to its high nutritional value and yield potential, and is among the world’s most economically valuable forage crops[ 34 – 36 ]. 'Xinjiangdaye' ( Medicago sativa ) is recognized as a high-yielding and high-quality alfalfa variety. This variety is characterized by a well-developed root system, an upright growth habit, hollow square-shaped stems, exceptionally large leaves, purple flowers, prolonged longevity, and resistance to senescence. Therefore, there is an urgent need to breed salt-tolerant and drought-resistant alfalfa varieties to ensure the sustainable production of alfalfa worldwide. 4-Coumarate: CoA ligase (4CL) is a key enzyme in the biosynthesis of secondary metabolites such as lignin and flavonoids, and is closely related to plant stress resistance. Flavonoids are very important polyphenolic secondary metabolites in plants, involved in regulating physiological activities such as plant growth, flower color formation, and responses to biotic or abiotic stresses[ 37 ], playing a key role in helping plants adapt to their ecological environment or resist external invasions during growth and development. At the same time, they also possess various pharmacological effects, such as antitumor, antioxidant, antihypertensive, and anti-inflammatory activities[ 38 , 39 ]. 4-Coumarate: CoA ligase (4CL) is a key enzyme linking the phenylpropanoid metabolic pathway with the lignin synthesis pathway. Located at the terminal end of the phenylalanine metabolic pathway, it catalyzes the conversion of cinnamic acid and its hydroxy or methoxy derivatives into corresponding coenzyme A esters. Intermediate products derived from different 4CL reactions are subsequently distributed to metabolic branches for the synthesis of phenylpropanoid derivatives such as flavonoids or lignans. Furthermore, they play a crucial regulatory role in the metabolic pathways of phenylpropanoids, including flavonoids, lignans, and coumarins. Based on the functions of the proteins encoded by 4CL genes, 4CLs can be classified into three categories: Class I primarily regulates the biosynthesis of plant lignan compounds; Class II primarily regulates the formation of flavonoid compounds; and Class III consists of 4CL-like proteins, whose specific functions remain unclear[ 40 ]. Taking Arabidopsis thaliana as an example, studies have shown that At4CL1 , At4CL2 , and At4CL4 are involved in lignin formation in Arabidopsis[ 41 , 42 ], while At4CL3 is involved in the biosynthesis of flavonoids[ 43 , 44 ]. Since 4CL genes are widely regulated during plant stress responses, research into their regulatory mechanisms and expression levels has become increasingly extensive[ 45 ]. 4CL genes have been extensively identified across plant species. In 1981, the first 4CL gene was cloned and identified in Petroselinumsativum . Subsequently, 13 At4CL genes were identified in Arabidopsis[ 46 ], and 14 Os4CL genes were identified in rice[ 46 ], 10 4CL genes were identified in the potato genome[ 47 ], and 50 4CL genes were documented in cassava[ 48 ], as well as in soybean[ 49 ] and other plant species. Similar studies in Arabidopsis, rice, and other species have shown that 4CL genes are closely associated with lignin synthesis, flavonoid biosynthesis, and stress responses[ 50 , 51 ]. In this study, 43 members of the 4CL gene family were identified in 'Xinjiangdaye' ( Medicago sativa ). The characteristics of the alfalfa 4CL gene family were systematically analyzed, and comprehensive analyses were conducted on gene structure, physicochemical properties, phylogenetic relationships, and cis -acting elements. To elucidate its functional role in stress responses, this study investigated the expression profiles of the 4CL gene under two major abiotic stress conditions (drought and salt stress). The transcriptomic results were validated through RT-qPCR experiments, provided theoretical support for future research into the functions of the 4CL gene family and its role in alfalfa’s abiotic stress response. Materials and Methods Identification of members of the alfalfa Ms4CL gene family The complete genomic resources for alfalfa ( Medicago sativa L. ), including protein sequences, gene annotation files (GFF format), and genomic DNA sequences, were obtained from the publicly available whole-genome sequencing project ( https://fgshare.com/projects/whole_genome_sequencing_and_assembly_of_Medicago_sativa/66380 )[ 52 ]. Protein sequences of members of the 4CL gene family from Arabidopsis thaliana L. were downloaded from TAIR as reference sequences, including At4CL1 (At1g51680), At4CL2 (At3g21240), At4CL3 (At1g65060), At4CL4(At3g21230), AtACS1(At1g20480), AtACS2(At1g20490), AtACS3(At1g20500), AtOPCL1(At1g20510), AtACS5(At1g62940), AtACS6(At4g05160), AtACS7(At4g19010), AtACS8(At5g38120), AtACS9 (At5g63380) and 13 other At4CL proteins. Initially, BLAST homology searches were performed on protein sequences from 'Xinjiangdaye' ( Medicago sativa ) using 13 protein sequences, including At4CL1 and AtACS9 from Arabidopsis thaliana , as query sequences, with a strict E-value cutoff of 1E − 5 . Concurrently, the hidden Markov model (HMM) of the 4CL family conserved domain (PF00501)[ 53 ] was retrieved from the Pfam database ( http://pfam.xfam.org/ )[ 54 ]. Finally, by integrating the screening results from BLAST and HMMER, 43 members of the Ms4CL gene family were identified. Subsequently, the Protein Parameter Calc plugin in TBtools software was used to predict the physicochemical properties of the identified Ms4CL proteins, including the number of amino acids, molecular weight (MW), isoelectric point (pI), and instability index. Finally, subcellular localization predictions for Ms4CL family members were conducted using the WoLF PSORT online prediction system ( https://wolfpsort.hgc.jp/ ). Chromosomal localization analysis of the Ms4CL gene in alfalfa Based on the genome annotation file (GFF format) of 'Xinjiangdaye' ( Medicago sativa ), this study determined the physical locations of Ms4CL genes on chromosomes and analyzed the distribution of Ms4CL gene family members across these chromosomal locations using TBtools software. Phylogenetic tree analysis of the Ms4CL gene in alfalfa Multiple sequence alignments of the 4CL protein sequences from Arabidopsis thaliana , ‌Glycine max and Medicago sativa were performed using the MEGA11.0[ 55 , 56 ] software. Based on the alignment results, a phylogenetic tree was constructed using the Maximum likelihood (ML) estimation, and branch support was evaluated through 1,000 bootstrap repetitions. The evolutionary tree was then visualized using the online platform Evolview ( https://evolgenius.info//evolview-v2/ ). Collinearity analysis of the Ms4CL gene in alfalfa Using MCScanX[ 56 ] (E-value threshold: 1E − 10 ), we analyzed the colinearity of the alfalfa 4CL gene family between Arabidopsis and soybean. The colinearity results were visualized using TBtools software. The gene structure of Ms4CL was analyzed based on the genome annotation file (GFF format), and colinearity analysis between alfalfa and alfalfa was performed using MCScanX[ 56 ] (E-value cutoff: 1E-10). The results were visualized using Advanced Circos. Genetic structure and conserved motif analysis of the 4CL Genes in Medicago sativa The 4CL gene sequence of alfalfa was obtained from the genome annotation file, and the structure and conserved motifs of the alfalfa 4CL gene were analyzed using the TBtools software and the MEME website. To investigate protein characteristics, the MEME suite[ 57 ] (Multi-Functional Motif Extractor) was used with default parameters to identify conserved motifs in the Ms4CL protein, predicting a total of 10 conserved motifs. TBtools[ 58 ] was used to visualize the gene structure and motif distribution of the Ms4CL protein. Cis -acting element analysis of the alfalfa Ms4CL genes To investigate potential regulatory elements in the promoter region of the Ms4CL gene, the TBtools software was used to retrieve the 2000 bp upstream sequences of alfalfa 4CL gene family members from the alfalfa genome database, which were designated as the putative promoter region. Subsequently, these sequences were submitted to the PlantCARE database ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) for comprehensive cis-regulatory element prediction, followed by further screening. Finally, the cis-regulatory elements were visualized using TBtools software to illustrate the cis-regulatory elements in the promoter region. Expression profiling of alfalfa Ms4CL genes in different tissues To characterize the expression patterns of the Ms4CL gene across different tissues, this study retrieved RNA-seq datasets for six tissue types (elongated stems, flowers, leaves, nodules, pre-elongated stems, and roots) from the NCBI database. In addition, transcriptomic data from alfalfa subjected to drought stress and salt stress were analyzed, and the results were visualized using HeatMap. RT-qPCR analysis of the Ms4CL Gene Under Drought and Salt Stress The experiment utilized Zhongmu No. 4 alfalfa cultivar obtained from the Institute of Animal Science, Chinese Academy of Agricultural Sciences. Seeds were treated at 4°C for 3 days, followed by cultivation in a greenhouse for 2 weeks (16/8-hour photoperiod, 70–80% relative humidity, day/night temperatures of 24°C/20°C). Two-week-old seedlings were subjected to two types of abiotic stress: salt stress and drought stress. Salt stress using 250 mM NaCl solution with leaf samples collected at 0, 0.5, 1, 3, 6, 12, and 24 h post-treatment, with 0 h serving as the control. Drought stress using 400 mM mannitol with leaf samples collected at 0, 1, 3, 6, 12, and 24 h, with 0 h serving as the control. Each stress treatment was set up in triplicate, with each replicate consisting of five seedlings; the untreated control plants were grown under normal conditions. Total RNA was extracted from leaf tissue using the Eastep® Super Total RNA Extraction Kit according to the manufacturer’s instructions, and the corresponding cDNA was synthesized using the TransScript ® Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal). Gene-specific primers for members of the Ms4CL gene family were designed using Primer-BLAST on the NCBI website ( https://www.ncbi.nlm.nih.gov/ ) (Table S7 ). Real-time quantitative PCR (qPCR) assays were performed on a Bio-Rad CFX96 real-time quantitative PCR system. Each sample included three technical replicates. The alfalfa actin gene served as the internal control, and relative gene expression levels were calculated using 2^(−ΔΔCT) method[ 59 ]. Results Genome-Wide Identification and Physicochemical Properties of 4CL Family Members in the Medicago sativa Genome. This study analyzed the basic physicochemical properties of 43 members of the Ms4CL gene family, the final prediction results are shown in Table 1 . The number of amino acids ranges from 433 to 1120 AA, and the molecular weight (MW) of the proteins ranges from 47.59 kDa to 122.12 kDa. The average isoelectric point (pI) is 6.92, ranging from 5.1 to 9.06. Among the 43 Ms4CL proteins, 25 are acidic, 17 are basic, and 1 is neutral, with a roughly equal distribution of charge types. Approximately 67% of the genes encoding these proteins exhibit an instability index below 40, indicating that most are relatively stable. Predictions of subcellular location indicate that 29 Ms4CL genes are localized to the plasma membrane. Others are localized to the endoplasmic reticulum, cytoplasm, peroxisomes, and chloroplasts. Table 1 Physicochemical properties and predicted subcellular location of the Ms4CL gene family Sequence ID Gene Name Number of Amino Acid Mw (kDa) Theoretical pI Instability Index Chr Location Subcellular Location MS.gene98031.t1 Ms4CL1 511 55.49 8.08 38.96 109656917-11054 plasma membrane MS.gene27109.t1 Ms4CL2 553 60.28 8.42 39.64 chr1.1135253-138331 plasma membrane MS.gene051439.t1 Ms4CL3 513 55.81 6.48 40.57 chr1.147712126-47715162 plasma membrane MS.gene051440.t1 Ms4CL4 521 56.55 5.1 40.11 chr1.147741135-47745007 plasma membrane MS.gene004713.t1 Ms4CL5 534 57.83 9.06 37.92 chr1.155144807-55150997 chloroplast MS.gene032245.t1 Ms4CL6 570 61.93 5.57 42.8 chr1.166251979-66254285 plasma membrane MS.gene032243.t1 Ms4CL7 570 61.93 5.57 42.8 chr1.166266001-66268307 plasma membrane MS.gene029771.t1 Ms4CL8 540 58.89 9.05 39.59 chr1.248165949-48173152 plasma membrane MS.gene029775.t1 Ms4CL9 545 59.20 6.63 43.58 chr1.248218079-48222552 plasma membrane MS.gene029773.t1 Ms4CL10 555 60.65 6.37 42.33 chr1.248260290-48264767 Endoplasmic Reticulum MS.gene029776.t1 Ms4CL11 556 60.50 7 43.32 chr1.248290193-48294705 Endoplasmic Reticulum MS.gene32098.t1 Ms4CL12 547 59.40 8.97 37.01 chr1.255821500-55826837 Cytoplasm MS.gene24088.t1 Ms4CL13 553 60.31 8.42 39.64 chr1.260938-64016 plasma membrane MS.gene68419.t1 Ms4CL14 571 61.95 5.39 42.55 chr1.268019361-68021669 plasma membrane MS.gene035028.t1 Ms4CL15 1120 122.12 7.24 41.75 chr1.345587979-45602628 plasma membrane MS.gene95463.t1 Ms4CL16 547 59.39 8.87 37.73 chr1.352629427-52635128 Cytoplasm MS.gene88935.t1 Ms4CL17 555 60.47 8.61 40.43 chr1.354865-57948 plasma membrane MS.gene68421.t1 Ms4CL18 570 61.93 5.57 42.8 chr1.363534484-63536790 plasma membrane MS.gene005769.t1 Ms4CL19 570 62.00 5.62 41.74 chr1.363574277-63576583 plasma membrane MS.gene88987.t1 Ms4CL20 553 60.31 8.42 39.64 chr1.4462664-465742 plasma membrane MS.gene40027.t1 Ms4CL21 528 57.06 5.87 36.2 chr1.459775198-59782133 peroxisome MS.gene21415.t1 Ms4CL22 570 62.01 5.62 42.08 chr1.471967508-71969814 plasma membrane MS.gene028660.t1 Ms4CL23 541 59.48 8.21 29.29 chr2.17999944-8004267 plasma membrane MS.gene43857.t1 Ms4CL24 539 59.14 8.21 30.76 chr2.26260300-6264969 plasma membrane MS.gene43858.t1 Ms4CL25 505 55.27 5.7 35.15 chr2.26275274-6280133 plasma membrane MS.gene31229.t1 Ms4CL26 539 59.12 8.21 30.76 chr2.47118015-7122682 plasma membrane Table 1 (continued) Sequence ID Gene Name Number of Amino Acid Mw (kDa) Theoretical pI Instability Index Chr Location Subcellular Location MS.gene31230.t1 Ms4CL27 507 55.43 5.74 33.92 chr2.47133513-7137610 plasma membrane MS.gene98283.t1 Ms4CL28 562 61.03 6.53 39.79 chr3.161512018-61517425 peroxisome MS.gene022444.t1 Ms4CL29 562 60.94 6.42 38.92 chr3.267600791-67607624 peroxisome MS.gene29781.t1 Ms4CL30 562 60.98 6.42 39.72 chr3.364732306-64741053 peroxisome MS.gene35477.t1 Ms4CL31 433 47.59 8.23 36.67 chr3.473563592-73574480 peroxisome MS.gene35476.t1 Ms4CL32 562 60.94 6.42 38.92 chr3.473577439-73583135 peroxisome MS.gene09098.t1 Ms4CL33 479 52.02 5.89 34.43 chr4.140440362-40452390 plasma membrane MS.gene09104.t1 Ms4CL34 542 58.92 6.29 40.32 chr4.140575186-40578911 plasma membrane MS.gene09107.t1 Ms4CL35 581 63.24 5.26 38.46 chr4.140668323-40673618 plasma membrane MS.gene049799.t1 Ms4CL36 451 49.23 6.17 35.2 chr4.245978451-45982896 plasma membrane MS.gene049798.t1 Ms4CL37 542 59.01 6.63 38.1 chr4.246006941-46011662 Endoplasmic Reticulum MS.gene09153.t1 Ms4CL38 542 59.08 6.23 37.28 chr4.343371506-43374762 Endoplasmic Reticulum MS.gene007715.t1 Ms4CL39 542 59.08 6.23 37.28 chr4.445661522-45664778 Endoplasmic Reticulum MS.gene016573.t1 Ms4CL40 564 61.64 7.14 35.07 chr8.164342047-64346426 plasma membrane MS.gene61077.t1 Ms4CL41 564 61.58 7.14 35.07 chr8.260542431-60546713 plasma membrane MS.gene88460.t1 Ms4CL42 551 60.17 6.61 36.31 chr8.357778400-57782757 plasma membrane MS.gene61755.t1 Ms4CL43 563 61.48 8.07 36.56 chr8.458607134-58611518 plasma membrane Chromosomal localization Analysis of Ms4CLs In this study, we performed chromosomal localization analysis on members of the Ms4CL gene family. Based on their sequence order and chromosomal positions, they were named Ms4CL1 to Ms4CL4 3. The results are shown in Fig. 1 . Of the 43 Ms4CL genes, 21 are located on chromosome 1, accounting for 48.84% of the total Ms4CL genes. Among these, the highest number of genes is found on chr1.2, with 7 Ms4CL genes located there. Only 4 Ms4CL genes are located on chromosome 8, representing the smallest proportion. Additionally, the Ms4CL1 gene has not been anchored. Phylogenetic and collinearity analyses of Ms4CLs To investigate the phylogenetic relationships of 4CL proteins in Arabidopsis thaliana , ‌Glycine max and Medicago sativa , a phylogenetic tree was constructed in this study. The results showed that among the 60 4CL proteins analyzed, 13 were from Arabidopsis thaliana , 4 were from ‌Glycine max , and 43 were from Medicago sativa . The phylogenetic analysis classified the 60 4CL protein sequences from these three species into four distinct subfamilies (I, II, III, and IV). Specifically, Group I contains six Ms4CL members, Group II contains twenty Ms4CL members, Group III contains nine Ms4CL members, Group IV contains eight Ms4CL members. The shortest branch of the phylogenetic tree consists of ten 4CL proteins, containing only one AtACS9 gene, with the remaining members being Ms4CL proteins. Additionally, all four Gm4CL proteins are classified in Group I (Fig. 2 ). Phylogenetic analysis indicates that Medicago sativa 4CL genes are more closely related to ‌Glycine max genes than to Arabidopsis thaliana genes. To further validate this finding, we analyzed the colinearity relationships among Medicago sativa , Arabidopsis thaliana , and ‌Glycine max . The Medicago sativa Ms4CL gene exhibits homological synteny with both Arabidopsis thaliana and ‌Glycine max . There are 4 syntenic pairs between Ms4CL and the Arabidopsis thaliana ( At4CL ) gene, whereas there are 32 syntenic pairs between Ms4CL and the ‌Glycine max ( Gm4CL ) gene, indicating a closer evolutionary relationship between the Medicago sativa 4CL gene and ‌Glycine max , a plant of the same family (Fig. 3 ). Gene structure and conserved motifs of Ms4CL gene family To elucidate the structure-function relationship of Ms4CL proteins, this study systematically analyzed their gene architectures and conserved motifs. Using the MEME suite analysis, ten distinct motifs were identified among 43 Ms4CL proteins (Fig. 4 ). Among these, Motifs 1, 2, 3, 5, 6, and 8 were present in all 43 Ms4CL genes, indicating that these motifs exhibit a group-specific pattern of conservation. Phylogenetic tree-based clustering classified these motifs into four major groups (Fig. 5 ), with motifs within each group exhibiting significant sequence homology. Variations in gene structure may influence functional differences. Therefore, this study characterized the gene structures of all identified Ms4CLs (Fig. 5 ). The results of the gene structure analysis indicate that Ms4CLs contain between four and thirteen exons. Specifically, there are six Ms4CL genes containing four exons, ten Ms4CL genes containing five exons, fourteen Ms4CL genes containing six exons, eleven Ms4CL genes containing seven exons, one Ms4CL gene containing eight exons, and one Ms4CL gene containing thirteen exons. Ms4CL15 has the highest number of exons, with thirteen. It is worth noting that some 4CL genes contain longer introns, particularly Ms4CL15 . Gene duplication events and collinearity analysis of Ms4CL genes in Medicago sativa To investigate the role of gene duplication events in the evolution of the Ms4CL gene family, this study identified tandem and segmental duplication events across chromosomes. A total of five tandem duplication events were identified (Table S1 ), involving the gene pairs Ms4CL3 / Ms4CL4 , Ms4CL24 / Ms4CL25 , Ms4CL26 / Ms4CL27 , Ms4CL31 / Ms4CL32 , Ms4CL36 / Ms4CL37 . These tandem duplication events are primarily located on five chromosomes: chr1.1, chr2.2, chr2.4, chr3.4, and chr4.2 (Fig. 1 ). In the Ms4CL gene family, we identified 32 segmental duplication events (Table S2 ) unevenly distributed across chromosomes. Data analysis revealed that the chr1 region contains the largest number of genes and exhibits the highest density, the Ms4CL gene clustered on chr1 shows stronger homology and colinearity with itself and other chromosomes. No repetitive events were detected on chr2.3, chr5.1-chr5.4, chr6.1-chr6.4, chr7.1-chr7.4, indicating an uneven distribution of genes and selective collinearity (Fig. 6 ). Prediction of cis-elements in the promoter sequences of Ms4CL genes To elucidate the regulatory potential of the Ms4CL gene, this study analyzed cis -acting elements within the 2000 bp promoter region of alfalfa ( Medicago sativa) Ms4CL gene family members (Table S3 ). A total of eight elements were identified, including plant growth regulators, MeJA responsive, light responsive, salicylic acid responsive, anaerobic induction, low temperature responsive, drought responsive, involvement in defense and stress response. Furthermore, members of the alfalfa 4CL gene family are likely to respond to these stimuli. Jasmonic acid plays a crucial role by regulating numerous key processes in plant growth and development. As a signaling molecule, jasmonic acid responds to abiotic stresses (such as salt stress) to regulate the expression of a large number of genes and promote the initiation of specific defense mechanisms[ 60 ]. Among the 43 genes, 35 contain MeJA responsive elements, indicating that the Ms4CL gene family may participate in the salt stress response mediated by jasmonic acid. Additionally, 21 genes contain drought responsive, suggesting that the Ms4CL gene family may respond to drought stress. Furthermore, all 43 members of the Ms4CL gene family possess light responsive elements and plant growth regulators elements (including auxin, GA, and ABA). The Ms4CL gene family occupies a central hub position in the plant hormone regulatory network and plays a key regulatory role in balancing plant growth and development with stress responses. Among them, Ms4CL23 contains the fewest response elements (only 13), while Ms4CL35 contains the most (up to 33) (Fig. 7 ). Response elements associated with low temperature, drought, defense, and salicylic acid are widely distributed across the Ms4CL gene family, indicating its significant role in stress response regulatory networks. The types and numbers of stress-related elements vary among different Ms4CL gene members, suggesting functional differentiation among family members in response to different stresses. Tissue-Specific Gene Expression of Ms4CLs To elucidate the biological functions of Ms4CL genes in alfalfa ( Medicago sativa ), this study examined their in elongated stems, flowers, leaves, nodules, pre-elongated stems, and roots (Fig. 8 and Table S4 ) across six different tissues. Among the 43 Ms4CL genes, only 30 showed detectable expression levels. Among these, 13 genes showed no detectable expression in any of the analyzed tissues. Specifically, Ms4CL8 and Ms4CL42 were expressed only in roots, Ms4CL22 were expressed only in flowers, and Ms4CL37 was elongated stems-specific. These four genes exhibit tissue-specific expression patterns, while the remaining 26 Ms4CL genes show a multi-tissue expression profile. Among the six different tissues, the highest number of highly expressed genes was found in leaves tissue. In elongated stems, flowers, and pre-elongated stems, the expression levels of most Ms4CL genes were relatively low, with localized high expression observed only at a few gene loci. Expression analysis of 4CL genes Medicaogo sativa leaves under drought and salt stress To elucidate the functional divergence of Ms4CL genes under abiotic stress (Tables S5-S6), this study analyzed the expression profiles of alfalfa ( Medicago sativa ) Ms4CL genes under drought and salt stress using RNA-seq data. As demonstrated in Fig. 9 , the Ms4CL genes exhibited differential responses to both drought stress and salt stress. Among these, the Ms4CL34 gene was statistically significant ( P < 0.05 ) upregulation only under salt stress. The eleven genes Ms4CL1 , Ms4CL3-5 , Ms4CL12-13 , Ms4CL15-17 , and Ms4CL20-21 all exhibited statistically significant ( P < 0.05 ) differences under both drought stress and salt stress. Notably, Ms4CL13 was specifically activated only under salt and drought stress, its expression was undetected in the non-stressed control group but was significantly induced under both salt and drought conditions ( P 0.05 ). Expression of the Ms4CL gene in alfalfa under abiotic stress based on RT-qPCR Based on differential gene expression analysis, three representative genes ( Ms4CL3 , Ms4CL5 , and Ms4CL13 ) that responded to both drought and salt stress were selected for real-time quantitative PCR (qPCR) analysis to ensure the reliability and biological representativeness of the results. qPCR analysis (Fig. 10 ) revealed distinct stress response patterns, the expression levels of the Ms4CL5 and Ms4CL13 genes fluctuated ( P < 0.01 ), while the expression level of the Ms4CL3 gene remained consistently lower than that of the control group. Under the drought stress, the gene expression levels of Ms4CL3 , Ms4CL5 , and Ms4CL13 all peaked during the M2 and M3 periods, after which they began to decline, under salt stress, gene expression levels during the S3 and S4 periods were also significantly higher than during other periods ( P < 0.01 ). The peak expression levels of Ms4CL13 under salt and drought stress occurred relatively later compared to those of Ms4CL5 . For Ms4CL5 , gene expression reached a peak at 3 hours under both drought and salt stress, with a 15.19-fold (drought) and 21.33-fold (salt) increase compared to the control group. For Ms4CL13 , gene expression reached a peak at 6 hours under both drought and salt stress, with a 4.07-fold (drought) and 10.44-fold (salt). This is consistent with expression at the transcriptome level, validating the accuracy of the transcriptome data. Discussion Flavonoids are an important class of secondary metabolites produced in plants via the phenylpropanoid pathway. 4CL is one of the key enzyme genes in the phenylpropanoid metabolic pathway and represents the final step in phenylpropanoid biosynthesis[ 61 ]. Members of the 4CL gene family not only play a crucial role in the complex processes of plant growth and development, but their activity levels also significantly influence the accumulation of plant compounds such as flavonoids, lignans and lignin, playing an important role in plant growth and development as well as in responses to biotic and abiotic stresses[ 40 , 61 , 62 ]. In this study, through genome-wide analysis, 43 4CL gene members were identified in the genome of 'Xinjiangdaye' ( Medicago sativa ) and systematically named Ms4CL1–Ms4CL43 according to the Arabidopsis thaliana nomenclature standards. This study comprehensively identified 43 Ms4CL genes and systematically analyzed their basic characteristic. By examining the expression patterns of these genes in different tissues and under drought and salt stress, the aim was to screen for Ms4CL genes with stress resistance potential, thereby providing candidate targets for the breeding of salt stress and drought stress alfalfa varieties. Subcellular localization predictions indicate that most Ms4CLs are localized to the plasma membrane (Table 1 ), while Jr4CLs and Jm4CLs are predominantly distributed between the plasma membrane and chloroplasts, Gh4CLs exhibit widespread distribution, whereas Cit4CLs are confined to the cytoplasm, which may be related to differences in the functional sites of 4CLs[ 45 ]. Based on phylogenetic relationships (Fig. 2 ), the identified 4CL genes can be classified into four distinct subfamilies (I, II, III, and IV). Group II contains the highest number of Ms4CL genes, indicating that the alfalfa 4CL gene family underwent extensive duplication during evolution, likely to adapt to its unique environmental stresses and the demands of lignin or flavonoid synthesis. The overlapping distribution of genes across three species in Group I indicates that the core function of 4CLs is highly conserved in legumes and crucifers, suggesting involvement in fundamental phenylpropanoid pathways (such as lignin synthesis). The species-specific branches in Groups II and IV suggest that these genes may have evolved new, species-specific functions, such as stress resistance and the synthesis of specialized secondary metabolites in alfalfa. The number of 4CL genes varies significantly among different species[ 46 , 53 , 63 – 65 ], which may be related to gene duplication events experienced by different species during evolution. Collinearity analysis revealed genomic clustering, with approximately 74.4% of Ms4CL genes localized to nine chromosomal regions (chr1.1–chr1.4, chr2.2, chr2.4, chr3.4, chr4.1–chr4.2) (Fig. 1 ). These regions show a significant enrichment of tandem and segmental duplication events. From an evolutionary perspective, segmental duplication events are the core drivers of the expansion and functional diversification of this gene family. Tandem duplication events (such as Ms4CL3 and Ms4CL4 ) and numerous segmental duplication events (such as Ms4CL3 and Ms4CL13 ) have collectively shaped the genomic distribution of family members. Gene copies generated by duplication may undergo subfunctionalization or functionalization during evolution, resulting in some copies (such as Ms4CL3 , Ms4CL5 , and Ms4CL13 ) retain and enhance stress response capabilities, while others have acquired tissue-specific expression patterns. Chromosomal mapping (Fig. 1 ) and colinearity analysis (Fig. 3 ) further corroborate this finding. The genes are clustered on chromosomes or distributed across chromosomes, and exhibit conserved colinearity with Arabidopsis thaliana and ‌Glycine max , indicating that this family possesses conserved domains during evolution. Comparative synteny analysis further indicates that the Medicago sativa Ms4CL gene shares homologous synteny with both Arabidopsis thaliana and ‌Glycine max , but with more syntenic pairs in ‌Glycine max , suggesting a closer evolutionary relationship between the Arabidopsis thaliana 4CL gene and ‌Glycine max , a plant of the same family. Changes in gene structure and conserved domains affect the function of the gene[ 66 ]. The results show (Fig. 5 ) that Ms4CLs have 4–13 exons, a phenomenon also observed in most species of the 4CL gene family. For example, the number of conserved motifs in Md4CL proteins ranges from 0 to 18[ 53 ], while the number of exons in Ta4CL genes ranges from 1 to 18[ 61 ]. In plants, an increase in the number of introns within a gene may generally be advantageous. As non-coding regions, introns can protect genes from mutations, thereby better preserving gene function[ 67 , 68 ]. Transcription factors in the promoter region of genes are used to predict the biological processes in which they may be involved[ 69 ], and they also participate in the regulation of gene expression. The various transcription factors in gene promoters may be associated with different gene functions[ 70 ].In other species, 4CL promoters are similarly rich in various stress and hormone response elements. For example, the 4CL promoter in Juglans species contains multiple elements associated with plant hormones (such as MeJA) and abiotic stress[ 45 ]. In maize, the 4CL promoter contains cis -acting elements responsive to drought, low temperature, light, MeJA, auxin, gibberellin, and salicylic acid[ 71 ]. This study found that the Ms4CL promoter exhibits similar compositional characteristics. A total of eight response elements were identified in the cis -acting element analysis, with the promoter regions of most genes containing cis -acting elements responsive to hormones such as MeJA and ABA, as well as abiotic stresses induced by drought (Fig. 7 ). This structural feature suggests that the expression of Ms4CL genes may be co-regulated by hormonal signaling and environmental stress. The Ms4CL family not only participates in basic lignin synthesis but also responds extensively to various stimuli, including light, hormones, low temperature, drought, and defense, serving as a key node linking growth and development with environmental adaptation. Further analysis revealed that the Ms4CL3 and Ms4CL13 genes contain not only drought response elements but also MeJA response elements, whereas the Ms4CL5 gene contains only drought response elements. This suggests that Ms4CL3 and Ms4CL13 may be involved in both drought stress responses and MeJA-mediated salt stress responses, implying that they have acquired a broader regulatory network during evolution. In contrast, Ms4CL5 primarily participates in drought stress responses and may have undergone functional specialization, focusing specifically on drought stress responses and being less likely to be regulated by MeJA. Thus, within the alfalfa 4CL gene family, different members may exhibit functional differentiation in stress responses, with some possessing multiple regulatory pathways and others exhibiting more specialized functions. Stress response elements dominate the promoters of Ms4CL genes. Among them, 14 Ms4CL genes contain TC-rich repetitive sequences associated with stress resistance, while 35 Ms4CL genes contain MeJA-responsive cis -acting elements (CGTCA-motif and TGACG-motif), indicating that this gene family may be involved in adaptation to abiotic stress. Notably, all Ms4CL promoters contain elements associated with major plant hormone pathways, including gibberellin response elements (GARE motifs, P-box), abscisic acid response elements (ABRE), and auxin (TGA) response elements; additionally, 9 genes contain salicylic acid-related elements (TCA elements). As shown in Fig. 7 , the presence of a large number of growth regulator-related elements suggests that the 4CL gene family may indirectly enhance stress resistance by participating in growth regulation, rather than being limited to direct responses to stress. This indicates that the 4CL gene family plays an important role in alfalfa’s adaptation to abiotic stress and in basic growth processes. 4CL is a regulatory gene. Studies on its expression regulation indicate that 4CL gene expression is primarily regulated by the plant developmental stage[ 72 ], and expression levels vary across different developmental stages and tissues. Among them , At4CL1 and At4CL2 are most strongly expressed in seedling roots, while At4CL3 is highly expressed in flowers[ 45 ]. In rice, the Os4CL2 gene exhibits tissue-specific expression, with the highest expression levels observed in anthers[ 44 ]. In soybean, the Gm4CL3 and Gm4CL4 genes, which are associated with flavonoid biosynthesis, are highly expressed in roots and the hypocotyl[ 73 ]. A similar phenomenon has also been observed in alfalfa, where these stress-responsive genes exhibit distinct tissue specificity: the Ms4CL3 gene is highly expressed in roots, while the Ms4CL5 gene is highly expressed in nodules, with relatively low expression in tissues such as stems and flowers (Fig. 8 ). This suggests that their functions are not limited to stress responses but also involve the regulation of tissue differentiation during normal plant growth and development. Previous studies have shown that 4CL genes can respond to plant stress, but different members of the same 4CL gene family within a species may respond differently to stress[ 40 ]. In Eucommia ulmoides, all 35 Euc4CLs responded to salt stress, and the expression levels of most Euc4CLs significantly increased after salt treatment, with Euc4CL9 , Euc4CL17, and Euc4CL27 [ 64 ] showing the highest expression levels. In mulberry, all 4 Ma4CLs responded to salt stress. Under salt stress, all Ma4CL1–3 showed overall upregulation, while Ma4CL4 exhibited a trend of upregulation in the stem and downregulation in the roots following salt stress[ 74 ]. In the potato, St4CL6 and St4CL7 were upregulated under PEG stress following PEG-induced drought treatment simulation, whereas St4CL4 and St4CL5 expression was suppressed[ 47 ]. Numerous studies have shown that 4CL genes play a crucial role in responding to and regulating processes related to drought and salt stress[ 75 – 78 ]. However, systematic studies on the function of 4CL in alfalfa’s response to abiotic stresses are currently lacking. To elucidate the functional relevance of Ms4CL genes under abiotic stress conditions, this study systematically characterized the expression patterns of Ms4CL genes under drought and salt stress (Fig. 10 ). Three genes ( Ms4CL3 , Ms4CL5 , and Ms4CL13 ) were selected and analyzed using real-time quantitative PCR (qPCR). Ms4CL5 was upregulated under both drought and salt stress, indicating the importance of this gene in abiotic stress responses. The Ms4CL13 gene was not upregulated in all treatment groups under drought and salt stress, but rather reached its peak only during specific time points (such as M3, S4). The three target genes exhibited distinct functional differentiation under drought and salt stress, Ms4CL5 and Ms4CL13 were stress-induced, while Ms4CL3 was stress-repressed, possibly related to growth maintenance. Combined with real-time quantitative PCR (qPCR) results, it can be observed that the genes identified in this study exhibit largely consistent responses to abiotic stress. The qPCR results for Ms4CL3 , Ms4CL5 , and Ms4CL13 generally align with the expression patterns observed under salt and drought stress (Fig. 9 ). 4-Coumarate:CoA ligase (4CL) catalyze the production of various phenolic secondary metabolites, particularly lignin and flavonoids, which play a key role in the regulation of plant stress resistance[ 79 , 80 ]. Although this study has revealed the expression profiles of Ms4CLs , their specific functions in the metabolic pathways of lignin, flavonoids, and other metabolites require further validation. Conclusion This study identified a total of 43 Ms4CL genes in the 'Xinjiangdaye' ( Medicago sativa ) genome, classified into four subfamilies, with Group II having the largest number of members, indicating that this family has undergone extensive duplication and diversification during evolution. Genomic structure analysis revealed that Ms4CLs contain 4 to 13 exons. Cis -acting element analysis indicated that the promoters of most Ms4CL genes contain elements responsive to MeJA, ABA, and drought stress. Notably, Ms4CL3 and Ms4CL13 contain both drought and MeJA response elements, whereas Ms4CL 5 contains only drought response elements, suggesting functional differentiation among family members. Tissue-specific expression analysis revealed that Ms4CL3 is highly expressed in roots, while Ms4CL5 is highly expressed in root nodules. qPCR results indicated that Ms4CL5 was significantly upregulated under both drought and salt stress, Ms4CL13 reached its expression peak during a specific time window, and Ms4CL3 exhibited a repressed expression pattern, demonstrating distinct functional differentiation among the three genes. This study preliminarily reveals the functional characteristics of alfalfa 4CL genes, laying the foundation for subsequent functional studies of the 4CL gene family. Future experiments will investigate the 4CL genes and elucidate their mechanisms of action in plant abiotic stress resistance, further validating the specific functions of Ms4CLs in the synthesis of metabolites such as lignin and flavonoids, as well as in stress resistance regulation, thereby providing a theoretical basis and candidate targets for the molecular breeding of salt- and drought-tolerant alfalfa varieties. Abbreviations MW: Molecular weight pI: isoelectric points AA: amino acid ML: Maximum likelihood estimation HMM: The hidden Markov model MEME: Multiple em for motif elicitation Declarations No datasets were generated or analysed during the current study. Author Contributions Haiyue Lei and Shuyan Liu jointly conceived and designed the study and drafted the original manuscript. Yuqi Zhang, Xinyue Ma, and Li Zhao were responsible for the visualization and production of the figures and tables. Fei He and Ruicai Long handled data organization and manuscript revision. Tiejun Zhang and Qingchuan Yang conducted data analysis and verified data accuracy. Min Lu and Lin Chen supervised the entire research project and provided scientific guidance. All authors reviewed and approved the final manuscript prior to submission. Funding This work was supported by the National Natural Science Foundation of China (32371757,32441018), the major demonstration project “The Open Competition” for Seed Industry Science and Technology Innovation in Inner Mongolia (No. 2022JBGS0016). Ethics approval and consent to participate Field and laboratory studies were conducted by local legislation. This article does not contain any studies with human participants or animals and does not involve any endangered or protected species. The plant materials sampled and experiments performed in this research complied with institutional, national, and international guidelines and legislation. Consent for publication Not applicable. 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Supplementary Files TableS1.xlsx TableS2.xlsx TableS3.xlsx TableS4.xlsx TableS5.xlsx TableS6.xlsx TableS7.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 18 May, 2026 Reviewers agreed at journal 14 May, 2026 Reviews received at journal 28 Apr, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers invited by journal 20 Apr, 2026 Editor invited by journal 16 Apr, 2026 Editor assigned by journal 15 Apr, 2026 Submission checks completed at journal 15 Apr, 2026 First submitted to journal 10 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9384279","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":630870284,"identity":"10adfc8c-26e9-4e8c-80f1-8afd9d77d17a","order_by":0,"name":"Haiyue Lei","email":"","orcid":"","institution":"Beijing University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Haiyue","middleName":"","lastName":"Lei","suffix":""},{"id":630870285,"identity":"dedd8a47-318f-4560-a671-36a9430e2c29","order_by":1,"name":"Shuyan Liu","email":"","orcid":"","institution":"Beijing University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Shuyan","middleName":"","lastName":"Liu","suffix":""},{"id":630870286,"identity":"bd01ff11-866f-49a9-bbae-b441f1118c63","order_by":2,"name":"Yuqi Zhang","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yuqi","middleName":"","lastName":"Zhang","suffix":""},{"id":630870287,"identity":"0adf9207-09c5-443e-bf04-f5c9ac8be14a","order_by":3,"name":"Xinyue Ma","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xinyue","middleName":"","lastName":"Ma","suffix":""},{"id":630870288,"identity":"5007d599-7f56-42f0-be35-b2eb32f6a12e","order_by":4,"name":"Li Zhao","email":"","orcid":"","institution":"Chinese Academy of Agricultural 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Sciences","correspondingAuthor":false,"prefix":"","firstName":"Tiejun","middleName":"","lastName":"Zhang","suffix":""},{"id":630870292,"identity":"880cbd29-6578-4140-abb0-8d2c914b0e4d","order_by":8,"name":"Qingchuan Yang","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Qingchuan","middleName":"","lastName":"Yang","suffix":""},{"id":630870293,"identity":"f5a732ab-5df3-4a31-b7cb-d0d2ca9d8862","order_by":9,"name":"Min Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYBACPiA+AEbsDSCSCMAG18JzgAQtEF0SCUQ6jE0iO/FwAcOdxP6Zzx8eLqhhkOcXI2AZm0TuhsMzGJ4lzridY3B4xjEGw5mzCVgH1sLDcDix4XYOw2EeNoYEg9vEapl/8/iDwzz/SNGy4QaDwWHeNmK08LwFaXlmvPEM0C+8fRKE/cLPnrv5Mw/DHdl5x48//szzzUaeX5qAFjBg/AdnShChfBSMglEwCkYBQQAAU/lGj/X5bw8AAAAASUVORK5CYII=","orcid":"","institution":"Beijing University of Agriculture","correspondingAuthor":true,"prefix":"","firstName":"Min","middleName":"","lastName":"Lu","suffix":""},{"id":630870294,"identity":"7298bd8e-87b4-4e0f-9793-9f820acfeb49","order_by":10,"name":"Lin Chen","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2026-04-11 03:53:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9384279/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9384279/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108183036,"identity":"12f35385-c3b3-46e4-86dc-96153f70d8e3","added_by":"auto","created_at":"2026-04-30 08:59:45","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":209708,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal distribution of \u003cem\u003e4CL\u003c/em\u003e genes in \u003cem\u003eMedicago sativa\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/c2254ab37adb7ef7f9580312.jpg"},{"id":108137565,"identity":"d94b4e73-9eb2-4b79-ba5f-051240a9e6a7","added_by":"auto","created_at":"2026-04-29 18:05:04","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":541520,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of the 4CL gene in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003e‌Glycine max\u003c/em\u003e and \u003cem\u003eMedicago sativa\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/8a4503c4b080a172ba61748e.jpg"},{"id":108182875,"identity":"85990fd7-4b46-4d9a-957f-2d5220917cf9","added_by":"auto","created_at":"2026-04-30 08:59:39","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":266956,"visible":true,"origin":"","legend":"\u003cp\u003eCollinearity analysis of \u003cem\u003eMs4CL\u003c/em\u003e genes in \u003cem\u003eMedicago sativa\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, and \u003cem\u003e‌Glycine max\u003c/em\u003e. The gray lines in the background represent collinear blocks within \u003cem\u003eMedicago sativa\u003c/em\u003e and other species, while the colored lines highlight the collinear 4CL gene pairs.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/e8f586614f8742c26379bc25.jpg"},{"id":108182797,"identity":"cf177a53-a091-41a3-b2e6-f062469a6826","added_by":"auto","created_at":"2026-04-30 08:59:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":479206,"visible":true,"origin":"","legend":"\u003cp\u003eBasic information on the\u003cem\u003e Ms4CL\u003c/em\u003e protein motifs in \u003cem\u003eMedicago sativa\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/655017ce3962a26f1c5be3fc.jpg"},{"id":108137570,"identity":"f6857d83-7405-43e0-b0f1-00455815f45d","added_by":"auto","created_at":"2026-04-29 18:05:04","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":484839,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the gene structure and motifs of the \u003cem\u003eMs4CL\u003c/em\u003e genes in \u003cem\u003eMedicago sativa\u003c/em\u003e. Green boxes indicate coding sequences (CDS). Black lines indicate introns. The length of the CDS can be inferred from the scale bar at the bottom.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/048dfbd310d907360932ce59.jpg"},{"id":108182882,"identity":"5809b78a-2f12-46df-9258-bf26e966f6eb","added_by":"auto","created_at":"2026-04-30 08:59:39","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":622319,"visible":true,"origin":"","legend":"\u003cp\u003eRegional colinearity of the \u003cem\u003eMs4CL\u003c/em\u003e gene in \u003cem\u003eMedicago sativa\u003c/em\u003e. The red lines represent \u003cem\u003eMs4CL\u003c/em\u003e gene duplication events and the chromosome numbers are labeled within gray rectangles.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/a240ea6aa996d0844d7dc1cc.jpg"},{"id":108182706,"identity":"dd985536-4047-4ce5-b860-7977e03ea997","added_by":"auto","created_at":"2026-04-30 08:59:30","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":556345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCis\u003c/em\u003e-acting elements of the \u003cem\u003eMs4CL\u003c/em\u003e gene promoters in \u003cem\u003eMedicago sativa\u003c/em\u003e. The gray-shaded areas indicate \u003cem\u003ecis\u003c/em\u003e-acting elements, with different colors representing different types of \u003cem\u003ecis\u003c/em\u003e-acting elements. The heatmap represents the frequency of occurrence. The stacked bar chart on the far right displays the number of occurrences across the eight major categories of \u003cem\u003ecis\u003c/em\u003e-acting elements.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/aaa147c3aa357fe4acab777b.jpg"},{"id":108182759,"identity":"91b2a436-ec93-4f5a-acd2-ad45396ee091","added_by":"auto","created_at":"2026-04-30 08:59:32","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":157529,"visible":true,"origin":"","legend":"\u003cp\u003eTissue-specific expression profiles of \u003cem\u003eMs4CL\u003c/em\u003e genes in \u003cem\u003eMedicago sativa\u003c/em\u003e. The left figure shows the expression of the \u003cem\u003eMs4CL\u003c/em\u003e gene in multiple tissues, while the right figure shows the expression of the \u003cem\u003eMs4CL\u003c/em\u003e gene in a single tissue.\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/f9e8cf669de18f00c1067e3b.jpg"},{"id":108137576,"identity":"4cf1473d-fd1f-4e57-88b9-a9406b1520b7","added_by":"auto","created_at":"2026-04-29 18:05:04","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":362610,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression patterns of \u003cem\u003eMs4CL\u003c/em\u003e gene family members under two stresses. (A) Expression of \u003cem\u003eMs4CL\u003c/em\u003e genes in \u003cem\u003eMedicago sativa\u003c/em\u003e under drought stress. (B) Expression of \u003cem\u003eMs4CL\u003c/em\u003e genes in \u003cem\u003eMedicago sativa\u003c/em\u003e under salt stress. M1–M5 represent expression levels at 1 h, 3 h, 6 h, 12 h, and 24 h under drought stress, respectively. S1–S6 represent expression levels at 0.5 h, 1 h, 3 h, 6 h, 12 h, and 24 h under salt stress, respectively. 0 h serves as the control (CK).\u003c/p\u003e","description":"","filename":"Picture9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/01261a87274d4dfae24a8b8a.jpg"},{"id":108182250,"identity":"7501f4e1-8bd2-4272-ac2e-266af8f832ca","added_by":"auto","created_at":"2026-04-30 08:59:17","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":228721,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiles of \u003cem\u003eMs4CL\u003c/em\u003e under drought and salt stress as detected by RT-qPCR. Time-course designations: Drought stress: M1–M5 (1 h, 3 h, 6 h, 12 h, 24 h); Salt stress: S1–S6 (0.5 h, 1 h, 3 h, 6 h, 12 h, 24 h). The 0 h time point serves as the control (CK). (** indicates \u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e)\u003c/p\u003e","description":"","filename":"Picture10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/7c30e9a702045b6b8499ba53.jpg"},{"id":108490900,"identity":"2cf21997-ebc2-41a1-9666-539be6504764","added_by":"auto","created_at":"2026-05-05 09:49:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4612527,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/09269f73-8399-4dff-90c1-90c3e4e466e1.pdf"},{"id":108137562,"identity":"e24d89a3-40ea-4609-a4d6-76e9822c9d7c","added_by":"auto","created_at":"2026-04-29 18:05:04","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":11666,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/2abb2d23b724a723e7480f2c.xlsx"},{"id":108182256,"identity":"ad123720-3ad9-4333-ad9b-6f72a51d4022","added_by":"auto","created_at":"2026-04-30 08:59:17","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11465,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/caef94b0b437252a69c9597e.xlsx"},{"id":108182224,"identity":"29aba290-6328-401f-bdfc-a674f6555d62","added_by":"auto","created_at":"2026-04-30 08:59:15","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":43329,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/fdb26f5beea8bd63c5d72ecf.xlsx"},{"id":108137568,"identity":"18cf9ba5-813b-4f9a-ae43-5de1a867e040","added_by":"auto","created_at":"2026-04-29 18:05:04","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14287,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/1eaac452b22d1cb7e13a3fb9.xlsx"},{"id":108183234,"identity":"0a6e3263-e85b-4f7b-a394-7d00bbb2b043","added_by":"auto","created_at":"2026-04-30 09:00:00","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":14284,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/d958300814cf320ee1986be5.xlsx"},{"id":108182388,"identity":"089a70c4-85a4-4367-8cfb-a2f48b6e892f","added_by":"auto","created_at":"2026-04-30 08:59:20","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14758,"visible":true,"origin":"","legend":"","description":"","filename":"TableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/b2bffd968cca83ef4185c9a7.xlsx"},{"id":108137574,"identity":"47af11af-0d54-49b1-90a2-6256d8afebd4","added_by":"auto","created_at":"2026-04-29 18:05:04","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":10746,"visible":true,"origin":"","legend":"","description":"","filename":"TableS7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9384279/v1/166d6e93c9bd4772d2dc46f5.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide Identification and Expression Pattern Analysis of the Medicago sativa 4-Coumarate: CoA ligase (4CL) Gene Family","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAbiotic stress refers to the adverse effects of non-living factors on organisms[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Plants must survive under constantly changing environmental conditions, especially by fluctuations in various abiotic stress factors, such as salinity[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and drought[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSoil salinization is considered to be one of the primary abiotic stress factors that limit agricultural production in many regions worldwide[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Various ions, including sodium, potassium, calcium, magnesium, and chloride ions, can lead to soil salinization[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], among these ions, sodium chloride is the most abundant salt in soil. Specifically, when the electrical conductivity (EC) of the saturated extract exceeds 4 dS\u0026middot;m⁻\u0026sup1; under conditions of 25\u0026deg;C and 15% exchangeable sodium, the soil is classified as saline-alkali soil[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Soil salinization hinders plants\u0026rsquo; ability to absorb water and nutrients from the soil, thereby causing plant damage[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] such as failed seed germination, reduced plant height, stunted root systems, and impaired flowering and fruiting, which in turn leads to decreased yield and quality[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Among various abiotic stresses, soil salinization has become a serious problem facing many regions worldwide, particularly in arid and semi-arid areas[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. According to statistics, the global land area affected by salinization exceeds 800\u0026nbsp;million hectares, accounting for approximately 6% of the world\u0026rsquo;s total land area[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Projections indicate that by 2050, over 50% of the land will be affected by soil salinization[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Modern molecular genetic studies have confirmed that the primary cause of this phenomenon is that, under salt stress, normal physiological and metabolic functions in plants are impaired by ionic stress, osmotic stress, and oxidative stress[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Soil salinization has a significant impact on global agricultural yields, as it hinders plant establishment, development, and growth, ultimately leading to reduced crop yields[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDrought stress is also one of the most severe abiotic stresses facing plants worldwide[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. With global warming and the impact of human activities, soil drought is becoming increasingly severe and has become a global issue that constrains ecological conservation, plant distribution, and productivity. Under natural conditions, water content in plant tissues accounts for 75%\u0026ndash;90% of fresh tissue weight and plays a crucial role in various physiological processes such as growth, development, and metabolism. However, drought restricts water absorption and utilization capacity, thereby limiting crop growth and yield[\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The effects of drought on crops are multifaceted, disrupting various cellular physiological processes, including signal perception, photosynthesis, and all molecular and biochemical functions of cells[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. It does so by inhibiting processes such as cell differentiation, division, and elongation[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Disruption of these physiological processes can lead to delayed growth and development or even plant death, severely impacting agricultural production and hindering local economic development[\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eMedicago sativa L.\u003c/em\u003e is a perennial leguminous forage crop with a well-developed root system, offering natural advantages in improving the ecological environment and conserving soil and water. Due to its strong drought tolerance, cold tolerance, salt and alkali tolerance, dense foliage, palatability, and high nutritional value, alfalfa is not only used for genetic improvement but also as a high-quality forage to provide nutrition for livestock[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. It is an excellent forage widely cultivated in arid and semi-arid regions with promising prospects for industrial development. As one of the most widely distributed and extensively cultivated forage species globally, it is hailed as the \u0026ldquo;King of Forages\u0026rdquo; due to its high nutritional value and yield potential, and is among the world\u0026rsquo;s most economically valuable forage crops[\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. 'Xinjiangdaye' (\u003cem\u003eMedicago sativa\u003c/em\u003e) is recognized as a high-yielding and high-quality alfalfa variety. This variety is characterized by a well-developed root system, an upright growth habit, hollow square-shaped stems, exceptionally large leaves, purple flowers, prolonged longevity, and resistance to senescence. Therefore, there is an urgent need to breed salt-tolerant and drought-resistant alfalfa varieties to ensure the sustainable production of alfalfa worldwide.\u003c/p\u003e \u003cp\u003e4-Coumarate: CoA ligase (4CL) is a key enzyme in the biosynthesis of secondary metabolites such as lignin and flavonoids, and is closely related to plant stress resistance. Flavonoids are very important polyphenolic secondary metabolites in plants, involved in regulating physiological activities such as plant growth, flower color formation, and responses to biotic or abiotic stresses[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], playing a key role in helping plants adapt to their ecological environment or resist external invasions during growth and development. At the same time, they also possess various pharmacological effects, such as antitumor, antioxidant, antihypertensive, and anti-inflammatory activities[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. 4-Coumarate: CoA ligase (4CL) is a key enzyme linking the phenylpropanoid metabolic pathway with the lignin synthesis pathway. Located at the terminal end of the phenylalanine metabolic pathway, it catalyzes the conversion of cinnamic acid and its hydroxy or methoxy derivatives into corresponding coenzyme A esters. Intermediate products derived from different 4CL reactions are subsequently distributed to metabolic branches for the synthesis of phenylpropanoid derivatives such as flavonoids or lignans. Furthermore, they play a crucial regulatory role in the metabolic pathways of phenylpropanoids, including flavonoids, lignans, and coumarins. Based on the functions of the proteins encoded by 4CL genes, 4CLs can be classified into three categories: Class I primarily regulates the biosynthesis of plant lignan compounds; Class II primarily regulates the formation of flavonoid compounds; and Class III consists of 4CL-like proteins, whose specific functions remain unclear[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Taking \u003cem\u003eArabidopsis thaliana\u003c/em\u003e as an example, \u003cem\u003estudies have shown that At4CL1\u003c/em\u003e, \u003cem\u003eAt4CL2\u003c/em\u003e, \u003cem\u003eand At4CL4\u003c/em\u003e are involved in lignin formation in Arabidopsis[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], while \u003cem\u003eAt4CL3\u003c/em\u003e is involved in the biosynthesis of flavonoids[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Since 4CL genes are widely regulated during plant stress responses, research into their regulatory mechanisms and expression levels has become increasingly extensive[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. 4CL genes have been extensively identified across plant species. In 1981, the first 4CL gene was cloned and identified in \u003cem\u003ePetroselinumsativum\u003c/em\u003e. Subsequently, 13 \u003cem\u003eAt4CL\u003c/em\u003e genes were identified in Arabidopsis[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], and 14 \u003cem\u003eOs4CL\u003c/em\u003e genes were identified in rice[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], 10 4CL genes were identified in the potato genome[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], and 50 4CL genes were documented in cassava[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], as well as in soybean[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and other plant species. Similar studies in Arabidopsis, rice, and other species have shown that 4CL genes are closely associated with lignin synthesis, flavonoid biosynthesis, and stress responses[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, 43 members of the 4CL gene family were identified in 'Xinjiangdaye' (\u003cem\u003eMedicago sativa\u003c/em\u003e). The characteristics of the alfalfa 4CL gene family were systematically analyzed, and comprehensive analyses were conducted on gene structure, physicochemical properties, phylogenetic relationships, and \u003cem\u003ecis\u003c/em\u003e-acting elements. To elucidate its functional role in stress responses, this study investigated the expression profiles of the 4CL gene under two major abiotic stress conditions (drought and salt stress). The transcriptomic results were validated through RT-qPCR experiments, provided theoretical support for future research into the functions of the 4CL gene family and its role in alfalfa\u0026rsquo;s abiotic stress response.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eIdentification of members of the alfalfa\u003c/b\u003e \u003cb\u003eMs4CL\u003c/b\u003e \u003cb\u003egene family\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe complete genomic resources for alfalfa (\u003cem\u003eMedicago sativa L.\u003c/em\u003e), including protein sequences, gene annotation files (GFF format), and genomic DNA sequences, were obtained from the publicly available whole-genome sequencing project (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://fgshare.com/projects/whole_genome_sequencing_and_assembly_of_Medicago_sativa/66380\u003c/span\u003e\u003cspan address=\"https://fgshare.com/projects/whole_genome_sequencing_and_assembly_of_Medicago_sativa/66380\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Protein sequences of members of the 4CL gene family from \u003cem\u003eArabidopsis thaliana L.\u003c/em\u003e were downloaded from TAIR as reference sequences, \u003cem\u003eincluding At4CL1\u003c/em\u003e(At1g51680), \u003cem\u003eAt4CL2\u003c/em\u003e(At3g21240), \u003cem\u003eAt4CL3\u003c/em\u003e(At1g65060), \u003cem\u003eAt4CL4(At3g21230), AtACS1(At1g20480), AtACS2(At1g20490), AtACS3(At1g20500), AtOPCL1(At1g20510), AtACS5(At1g62940), AtACS6(At4g05160), AtACS7(At4g19010), AtACS8(At5g38120), AtACS9\u003c/em\u003e(At5g63380) and 13 other \u003cem\u003eAt4CL\u003c/em\u003e proteins. Initially, BLAST homology searches were performed on protein sequences from 'Xinjiangdaye' (\u003cem\u003eMedicago sativa\u003c/em\u003e) using 13 protein sequences, including \u003cem\u003eAt4CL1\u003c/em\u003e and \u003cem\u003eAtACS9\u003c/em\u003e from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, as query sequences, with a strict E-value cutoff of 1E\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e. Concurrently, the hidden Markov model (HMM) of the 4CL family conserved domain (PF00501)[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] was retrieved from the Pfam database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org/\u003c/span\u003e\u003cspan address=\"http://pfam.xfam.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Finally, by integrating the screening results from BLAST and HMMER, 43 members of the \u003cem\u003eMs4CL\u003c/em\u003e gene family were identified. Subsequently, the Protein Parameter Calc plugin in TBtools software was used to predict the physicochemical properties of the identified \u003cem\u003eMs4CL\u003c/em\u003e proteins, including the number of amino acids, molecular weight (MW), isoelectric point (pI), and instability index. Finally, subcellular localization predictions for \u003cem\u003eMs4CL\u003c/em\u003e family members were conducted using the WoLF PSORT online prediction system (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wolfpsort.hgc.jp/\u003c/span\u003e\u003cspan address=\"https://wolfpsort.hgc.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eChromosomal localization analysis of the\u003c/b\u003e \u003cb\u003eMs4CL\u003c/b\u003e \u003cb\u003egene in alfalfa\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBased on the genome annotation file (GFF format) of 'Xinjiangdaye' (\u003cem\u003eMedicago sativa\u003c/em\u003e), this study determined the physical locations of \u003cem\u003eMs4CL\u003c/em\u003e genes on chromosomes and analyzed the distribution of \u003cem\u003eMs4CL\u003c/em\u003e gene family members across these chromosomal locations using TBtools software.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic tree analysis of the\u003c/b\u003e \u003cb\u003eMs4CL\u003c/b\u003e \u003cb\u003egene in alfalfa\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMultiple sequence alignments of the 4CL protein sequences from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003e\u0026zwnj;Glycine max\u003c/em\u003e and \u003cem\u003eMedicago sativa\u003c/em\u003e were performed using the MEGA11.0[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] software. Based on the alignment results, a phylogenetic tree was constructed using the Maximum likelihood (ML) estimation, and branch support was evaluated through 1,000 bootstrap repetitions. The evolutionary tree was then visualized using the online platform Evolview (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://evolgenius.info//evolview-v2/\u003c/span\u003e\u003cspan address=\"https://evolgenius.info//evolview-v2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCollinearity analysis of the\u003c/b\u003e \u003cb\u003eMs4CL\u003c/b\u003e \u003cb\u003egene in alfalfa\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUsing MCScanX[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] (E-value threshold: 1E\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e), we analyzed the colinearity of the alfalfa 4CL gene family between Arabidopsis and soybean. The colinearity results were visualized using TBtools software.\u003c/p\u003e \u003cp\u003eThe gene structure of \u003cem\u003eMs4CL\u003c/em\u003e was analyzed based on the genome annotation file (GFF format), and colinearity analysis between alfalfa and alfalfa was performed using MCScanX[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] (E-value cutoff: 1E-10). The results were visualized using Advanced Circos.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic structure and conserved motif analysis of the 4CL Genes in\u003c/b\u003e \u003cb\u003eMedicago sativa\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe 4CL gene sequence of alfalfa was obtained from the genome annotation file, and the structure and conserved motifs of the alfalfa 4CL gene were analyzed using the TBtools software and the MEME website. To investigate protein characteristics, the MEME suite[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] (Multi-Functional Motif Extractor) was used with default parameters to identify conserved motifs in the \u003cem\u003eMs4CL\u003c/em\u003e protein, predicting a total of 10 conserved motifs. TBtools[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] was used to visualize the gene structure and motif distribution of the \u003cem\u003eMs4CL\u003c/em\u003e protein.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCis\u003c/b\u003e \u003cb\u003e-acting element analysis of the alfalfa\u003c/b\u003e \u003cb\u003eMs4CL\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate potential regulatory elements in the promoter region of the \u003cem\u003eMs4CL\u003c/em\u003e gene, the TBtools software was used to retrieve the 2000 bp upstream sequences of alfalfa 4CL gene family members from the alfalfa genome database, which were designated as the putative promoter region. Subsequently, these sequences were submitted to the PlantCARE database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for comprehensive cis-regulatory element prediction, followed by further screening. Finally, the cis-regulatory elements were visualized using TBtools software to illustrate the cis-regulatory elements in the promoter region.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression profiling of alfalfa\u003c/b\u003e \u003cb\u003eMs4CL\u003c/b\u003e \u003cb\u003egenes in different tissues\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo characterize the expression patterns of the \u003cem\u003eMs4CL\u003c/em\u003e gene across different tissues, this study retrieved RNA-seq datasets for six tissue types (elongated stems, flowers, leaves, nodules, pre-elongated stems, and roots) from the NCBI database. In addition, transcriptomic data from alfalfa subjected to drought stress and salt stress were analyzed, and the results were visualized using HeatMap.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRT-qPCR analysis of the\u003c/b\u003e \u003cb\u003eMs4CL\u003c/b\u003e \u003cb\u003eGene Under Drought and Salt Stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe experiment utilized Zhongmu No. 4 alfalfa cultivar obtained from the Institute of Animal Science, Chinese Academy of Agricultural Sciences. Seeds were treated at 4\u0026deg;C for 3 days, followed by cultivation in a greenhouse for 2 weeks (16/8-hour photoperiod, 70\u0026ndash;80% relative humidity, day/night temperatures of 24\u0026deg;C/20\u0026deg;C). Two-week-old seedlings were subjected to two types of abiotic stress: salt stress and drought stress. Salt stress using 250 mM NaCl solution with leaf samples collected at 0, 0.5, 1, 3, 6, 12, and 24 h post-treatment, with 0 h serving as the control. Drought stress using 400 mM mannitol with leaf samples collected at 0, 1, 3, 6, 12, and 24 h, with 0 h serving as the control. Each stress treatment was set up in triplicate, with each replicate consisting of five seedlings; the untreated control plants were grown under normal conditions. Total RNA was extracted from leaf tissue using the Eastep\u0026reg; Super Total RNA Extraction Kit according to the manufacturer\u0026rsquo;s instructions, and the corresponding cDNA was synthesized using the \u003cem\u003eTransScript\u003c/em\u003e\u0026reg; Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal). Gene-specific primers for members of the \u003cem\u003eMs4CL\u003c/em\u003e gene family were designed using Primer-BLAST on the NCBI website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). Real-time quantitative PCR (qPCR) assays were performed on a Bio-Rad CFX96 real-time quantitative PCR system. Each sample included three technical replicates. The alfalfa actin gene served as the internal control, and relative gene expression levels were calculated using 2^(\u0026minus;ΔΔCT) method[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGenome-Wide Identification and Physicochemical Properties of 4CL Family Members in the\u003c/b\u003e \u003cb\u003eMedicago sativa\u003c/b\u003e \u003cb\u003eGenome.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis study analyzed the basic physicochemical properties of 43 members of the \u003cem\u003eMs4CL\u003c/em\u003e gene family, the final prediction results are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The number of amino acids ranges from 433 to 1120 AA, and the molecular weight (MW) of the proteins ranges from 47.59 kDa to 122.12 kDa. The average isoelectric point (pI) is 6.92, ranging from 5.1 to 9.06. Among the 43 \u003cem\u003eMs4CL\u003c/em\u003e proteins, 25 are acidic, 17 are basic, and 1 is neutral, with a roughly equal distribution of charge types. Approximately 67% of the genes encoding these proteins exhibit an instability index below 40, indicating that most are relatively stable. Predictions of subcellular location indicate that 29 \u003cem\u003eMs4CL\u003c/em\u003e genes are localized to the plasma membrane. Others are localized to the endoplasmic reticulum, cytoplasm, peroxisomes, and chloroplasts.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical properties and predicted subcellular location of the \u003cem\u003eMs4CL\u003c/em\u003e gene family\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSequence ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber of Amino Acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMw (kDa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTheoretical pI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInstability Index\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eChr Location\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSubcellular Location\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene98031.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e511\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e38.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e109656917-11054\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene27109.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e553\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e39.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.1135253-138331\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene051439.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e513\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.147712126-47715162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene051440.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e521\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e56.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.147741135-47745007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene004713.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e534\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e57.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.155144807-55150997\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003echloroplast\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene032245.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e570\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e61.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.166251979-66254285\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene032243.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e570\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e61.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.166266001-66268307\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene029771.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e540\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e58.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e39.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.248165949-48173152\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene029775.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e545\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e59.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.248218079-48222552\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene029773.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.248260290-48264767\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eEndoplasmic Reticulum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene029776.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e556\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.248290193-48294705\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eEndoplasmic Reticulum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene32098.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e547\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e59.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.255821500-55826837\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene24088.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e553\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e39.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.260938-64016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene68419.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e571\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e61.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.268019361-68021669\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene035028.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e122.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e41.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.345587979-45602628\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene95463.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e547\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e59.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.352629427-52635128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene88935.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.354865-57948\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene68421.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e570\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e61.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.363534484-63536790\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene005769.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e570\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e62.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e41.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.363574277-63576583\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene88987.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e553\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e39.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.4462664-465742\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene40027.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e528\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e57.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e36.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.459775198-59782133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eperoxisome\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene21415.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e570\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e62.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr1.471967508-71969814\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene028660.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e541\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e59.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e29.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr2.17999944-8004267\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene43857.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e539\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e59.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e30.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr2.26260300-6264969\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene43858.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e35.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr2.26275274-6280133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene31229.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e539\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e59.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e30.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr2.47118015-7122682\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e(continued)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSequence ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber of Amino Acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMw (kDa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTheoretical pI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInstability Index\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eChr Location\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSubcellular Location\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene31230.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e507\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e33.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr2.47133513-7137610\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene98283.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e61.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e39.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr3.161512018-61517425\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eperoxisome\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene022444.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e38.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr3.267600791-67607624\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eperoxisome\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene29781.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e39.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr3.364732306-64741053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eperoxisome\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene35477.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e433\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e47.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e36.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr3.473563592-73574480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eperoxisome\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene35476.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e38.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr3.473577439-73583135\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eperoxisome\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene09098.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e479\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e52.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e34.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr4.140440362-40452390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene09104.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e58.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr4.140575186-40578911\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene09107.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e581\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e63.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e38.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr4.140668323-40673618\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene049799.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e451\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e49.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e35.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr4.245978451-45982896\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene049798.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e59.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e38.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr4.246006941-46011662\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eEndoplasmic Reticulum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene09153.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e59.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr4.343371506-43374762\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eEndoplasmic Reticulum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene007715.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e59.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr4.445661522-45664778\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eEndoplasmic Reticulum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene016573.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e564\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e61.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e35.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr8.164342047-64346426\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene61077.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e564\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e61.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e35.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr8.260542431-60546713\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene88460.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e551\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e36.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr8.357778400-57782757\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS.gene61755.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMs4CL43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e563\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e61.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e36.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003echr8.458607134-58611518\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eplasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e\u003cstrong\u003eChromosomal localization Analysis of\u003c/strong\u003e\u003cstrong\u003eMs4CLs\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eIn this study, we performed chromosomal localization analysis on members of the \u003cem\u003eMs4CL\u003c/em\u003e gene family. Based on their sequence order and chromosomal positions, they were named \u003cem\u003eMs4CL1 to Ms4CL4\u003c/em\u003e3. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Of the 43 \u003cem\u003eMs4CL\u003c/em\u003e genes, 21 are located on chromosome 1, accounting for 48.84% of the total \u003cem\u003eMs4CL\u003c/em\u003e genes. Among these, the highest number of genes is found on chr1.2, with 7 \u003cem\u003eMs4CL\u003c/em\u003e genes located there. Only 4 \u003cem\u003eMs4CL\u003c/em\u003e genes are located on chromosome 8, representing the smallest proportion. Additionally, the \u003cem\u003eMs4CL1\u003c/em\u003e gene has not been anchored.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic and collinearity analyses of\u003c/b\u003e \u003cb\u003eMs4CLs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the phylogenetic relationships of 4CL proteins in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003e\u0026zwnj;Glycine max\u003c/em\u003e and \u003cem\u003eMedicago sativa\u003c/em\u003e, a phylogenetic tree was constructed in this study. The results showed that among the 60 4CL proteins analyzed, 13 were from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, 4 were from \u003cem\u003e\u0026zwnj;Glycine max\u003c/em\u003e, and 43 were from \u003cem\u003eMedicago sativa\u003c/em\u003e. The phylogenetic analysis classified the 60 4CL protein sequences from these three species into four distinct subfamilies (I, II, III, and IV). Specifically, Group I contains six \u003cem\u003eMs4CL\u003c/em\u003e members, Group II contains twenty \u003cem\u003eMs4CL\u003c/em\u003e members, Group III contains nine \u003cem\u003eMs4CL\u003c/em\u003e members, Group IV contains eight \u003cem\u003eMs4CL\u003c/em\u003e members. The shortest branch of the phylogenetic tree consists of ten 4CL proteins, containing only one \u003cem\u003eAtACS9\u003c/em\u003e gene, with the remaining members being \u003cem\u003eMs4CL\u003c/em\u003e proteins. Additionally, all four \u003cem\u003eGm4CL\u003c/em\u003e proteins are classified in Group I (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Phylogenetic analysis indicates that \u003cem\u003eMedicago sativa\u003c/em\u003e 4CL genes are more closely related to \u003cem\u003e\u0026zwnj;Glycine max\u003c/em\u003e genes than to \u003cem\u003eArabidopsis thaliana\u003c/em\u003e genes. To further validate this finding, we analyzed the colinearity relationships among \u003cem\u003eMedicago sativa\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, and \u003cem\u003e\u0026zwnj;Glycine max\u003c/em\u003e. The \u003cem\u003eMedicago sativa Ms4CL\u003c/em\u003e gene exhibits homological synteny with both \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003e\u0026zwnj;Glycine max\u003c/em\u003e. There are 4 syntenic pairs between \u003cem\u003eMs4CL\u003c/em\u003e and the \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (\u003cem\u003eAt4CL\u003c/em\u003e) gene, whereas there are 32 syntenic pairs between \u003cem\u003eMs4CL\u003c/em\u003e and the \u003cem\u003e\u0026zwnj;Glycine max\u003c/em\u003e (\u003cem\u003eGm4CL\u003c/em\u003e) gene, indicating a closer evolutionary relationship between the \u003cem\u003eMedicago sativa\u003c/em\u003e 4CL gene and \u003cem\u003e\u0026zwnj;Glycine max\u003c/em\u003e, a plant of the same family (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGene structure and conserved motifs of\u003c/b\u003e \u003cb\u003eMs4CL\u003c/b\u003e \u003cb\u003egene family\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the structure-function relationship of \u003cem\u003eMs4CL\u003c/em\u003e proteins, this study systematically analyzed their gene architectures and conserved motifs. Using the MEME suite analysis, ten distinct motifs were identified among 43 \u003cem\u003eMs4CL\u003c/em\u003e proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Among these, Motifs 1, 2, 3, 5, 6, and 8 were present in all 43 \u003cem\u003eMs4CL\u003c/em\u003e genes, indicating that these motifs exhibit a group-specific pattern of conservation. Phylogenetic tree-based clustering classified these motifs into four major groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), with motifs within each group exhibiting significant sequence homology. Variations in gene structure may influence functional differences. Therefore, this study characterized the gene structures of all identified \u003cem\u003eMs4CLs\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The results of the gene structure analysis indicate that \u003cem\u003eMs4CLs\u003c/em\u003e contain between four and thirteen exons. Specifically, there are six \u003cem\u003eMs4CL\u003c/em\u003e genes containing four exons, ten \u003cem\u003eMs4CL\u003c/em\u003e genes containing five exons, fourteen \u003cem\u003eMs4CL\u003c/em\u003e genes containing six exons, eleven \u003cem\u003eMs4CL\u003c/em\u003e genes containing seven exons, one \u003cem\u003eMs4CL\u003c/em\u003e gene containing eight exons, and one \u003cem\u003eMs4CL\u003c/em\u003e gene containing thirteen exons. \u003cem\u003eMs4CL15\u003c/em\u003e has the highest number of exons, with thirteen. It is worth noting that some 4CL genes contain longer introns, particularly \u003cem\u003eMs4CL15\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGene duplication events and collinearity analysis of\u003c/b\u003e \u003cb\u003eMs4CL\u003c/b\u003e \u003cb\u003egenes in\u003c/b\u003e \u003cb\u003eMedicago sativa\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the role of gene duplication events in the evolution of the \u003cem\u003eMs4CL\u003c/em\u003e gene family, this study identified tandem and segmental duplication events across chromosomes. A total of five tandem duplication events were identified (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), involving the gene pairs \u003cem\u003eMs4CL3\u003c/em\u003e/\u003cem\u003eMs4CL4\u003c/em\u003e, \u003cem\u003eMs4CL24\u003c/em\u003e/\u003cem\u003eMs4CL25\u003c/em\u003e, \u003cem\u003eMs4CL26\u003c/em\u003e/\u003cem\u003eMs4CL27\u003c/em\u003e, \u003cem\u003eMs4CL31\u003c/em\u003e/\u003cem\u003eMs4CL32\u003c/em\u003e, \u003cem\u003eMs4CL36\u003c/em\u003e/\u003cem\u003eMs4CL37\u003c/em\u003e. These tandem duplication events are primarily located on five chromosomes: chr1.1, chr2.2, chr2.4, chr3.4, and chr4.2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the \u003cem\u003eMs4CL\u003c/em\u003e gene family, we identified 32 segmental duplication events (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) unevenly distributed across chromosomes. Data analysis revealed that the chr1 region contains the largest number of genes and exhibits the highest density, the \u003cem\u003eMs4CL\u003c/em\u003e gene clustered on chr1 shows stronger homology and colinearity with itself and other chromosomes. No repetitive events were detected on chr2.3, chr5.1-chr5.4, chr6.1-chr6.4, chr7.1-chr7.4, indicating an uneven distribution of genes and selective collinearity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePrediction of cis-elements in the promoter sequences of\u003c/b\u003e \u003cb\u003eMs4CL\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the regulatory potential of the \u003cem\u003eMs4CL\u003c/em\u003e gene, this study analyzed \u003cem\u003ecis\u003c/em\u003e-acting elements within the 2000 bp promoter region of alfalfa (\u003cem\u003eMedicago sativa) Ms4CL\u003c/em\u003e gene family members (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). A total of eight elements were identified, including plant growth regulators, MeJA responsive, light responsive, salicylic acid responsive, anaerobic induction, low temperature responsive, drought responsive, involvement in defense and stress response. Furthermore, members of the alfalfa 4CL gene family are likely to respond to these stimuli. Jasmonic acid plays a crucial role by regulating numerous key processes in plant growth and development. As a signaling molecule, jasmonic acid responds to abiotic stresses (such as salt stress) to regulate the expression of a large number of genes and promote the initiation of specific defense mechanisms[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Among the 43 genes, 35 contain MeJA responsive elements, indicating that the \u003cem\u003eMs4CL\u003c/em\u003e gene family may participate in the salt stress response mediated by jasmonic acid. Additionally, 21 genes contain drought responsive, suggesting that the \u003cem\u003eMs4CL\u003c/em\u003e gene family may respond to drought stress. Furthermore, all 43 members of the \u003cem\u003eMs4CL\u003c/em\u003e gene family possess light responsive elements and plant growth regulators elements (including auxin, GA, and ABA). The \u003cem\u003eMs4CL\u003c/em\u003e gene family occupies a central hub position in the plant hormone regulatory network and plays a key regulatory role in balancing plant growth and development with stress responses. Among them, \u003cem\u003eMs4CL23\u003c/em\u003e contains the fewest response elements (only 13), while \u003cem\u003eMs4CL35\u003c/em\u003e contains the most (up to 33) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Response elements associated with low temperature, drought, defense, and salicylic acid are widely distributed across the \u003cem\u003eMs4CL\u003c/em\u003e gene family, indicating its significant role in stress response regulatory networks. The types and numbers of stress-related elements vary among different \u003cem\u003eMs4CL\u003c/em\u003e gene members, suggesting functional differentiation among family members in response to different stresses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTissue-Specific Gene Expression of\u003c/b\u003e \u003cb\u003eMs4CLs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the biological functions of \u003cem\u003eMs4CL\u003c/em\u003e genes in alfalfa (\u003cem\u003eMedicago sativa\u003c/em\u003e), this study examined their in elongated stems, flowers, leaves, nodules, pre-elongated stems, and roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e) across six different tissues. Among the 43 \u003cem\u003eMs4CL\u003c/em\u003e genes, only 30 showed detectable expression levels. Among these, 13 genes showed no detectable expression in any of the analyzed tissues. Specifically, \u003cem\u003eMs4CL8 and Ms4CL42\u003c/em\u003e were expressed only in roots, \u003cem\u003eMs4CL22\u003c/em\u003e were expressed only in flowers, and \u003cem\u003eMs4CL37\u003c/em\u003e was elongated stems-specific. These four genes exhibit tissue-specific expression patterns, while the remaining 26 \u003cem\u003eMs4CL\u003c/em\u003e genes show a multi-tissue expression profile. Among the six different tissues, the highest number of highly expressed genes was found in leaves tissue. In elongated stems, flowers, and pre-elongated stems, the expression levels of most \u003cem\u003eMs4CL\u003c/em\u003e genes were relatively low, with localized high expression observed only at a few gene loci.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression analysis of 4CL genes\u003c/b\u003e \u003cb\u003eMedicaogo sativa\u003c/b\u003e \u003cb\u003eleaves under drought and salt stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the functional divergence of \u003cem\u003eMs4CL\u003c/em\u003e genes under abiotic stress (Tables S5-S6), this study analyzed the expression profiles of alfalfa (\u003cem\u003eMedicago sativa\u003c/em\u003e) \u003cem\u003eMs4CL\u003c/em\u003e genes under drought and salt stress using RNA-seq data. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, the \u003cem\u003eMs4CL\u003c/em\u003e genes exhibited differential responses to both drought stress and salt stress. Among these, the \u003cem\u003eMs4CL34\u003c/em\u003e gene was statistically significant (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) upregulation only under salt stress. The eleven genes \u003cem\u003eMs4CL1\u003c/em\u003e, \u003cem\u003eMs4CL3-5\u003c/em\u003e, \u003cem\u003eMs4CL12-13\u003c/em\u003e, \u003cem\u003eMs4CL15-17\u003c/em\u003e, and \u003cem\u003eMs4CL20-21\u003c/em\u003e all exhibited statistically significant (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) differences under both drought stress and salt stress. Notably, \u003cem\u003eMs4CL13\u003c/em\u003e was specifically activated only under salt and drought stress, its expression was undetected in the non-stressed control group but was significantly induced under both salt and drought conditions (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The remaining \u003cem\u003eMs4CL\u003c/em\u003e genes did not exhibit statistically significant changes in expression under the tested conditions (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression of the\u003c/b\u003e \u003cb\u003eMs4CL\u003c/b\u003e \u003cb\u003egene in alfalfa under abiotic stress based on RT-qPCR\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBased on differential gene expression analysis, three representative genes (\u003cem\u003eMs4CL3\u003c/em\u003e, \u003cem\u003eMs4CL5\u003c/em\u003e, and \u003cem\u003eMs4CL13\u003c/em\u003e) that responded to both drought and salt stress were selected for real-time quantitative PCR (qPCR) analysis to ensure the reliability and biological representativeness of the results. qPCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e) revealed distinct stress response patterns, the expression levels of the \u003cem\u003eMs4CL5\u003c/em\u003e and \u003cem\u003eMs4CL13\u003c/em\u003e genes fluctuated (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), while the expression level of the \u003cem\u003eMs4CL3\u003c/em\u003e gene remained consistently lower than that of the control group. Under the drought stress, the gene expression levels of \u003cem\u003eMs4CL3\u003c/em\u003e, \u003cem\u003eMs4CL5\u003c/em\u003e, and \u003cem\u003eMs4CL13\u003c/em\u003e all peaked during the M2 and M3 periods, after which they began to decline, under salt stress, gene expression levels during the S3 and S4 periods were also significantly higher than during other periods (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). The peak expression levels of \u003cem\u003eMs4CL13\u003c/em\u003e under salt and drought stress occurred relatively later compared to those of \u003cem\u003eMs4CL5\u003c/em\u003e. For \u003cem\u003eMs4CL5\u003c/em\u003e, gene expression reached a peak at 3 hours under both drought and salt stress, with a 15.19-fold (drought) and 21.33-fold (salt) increase compared to the control group. For \u003cem\u003eMs4CL13\u003c/em\u003e, gene expression reached a peak at 6 hours under both drought and salt stress, with a 4.07-fold (drought) and 10.44-fold (salt). This is consistent with expression at the transcriptome level, validating the accuracy of the transcriptome data.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFlavonoids are an important class of secondary metabolites produced in plants via the phenylpropanoid pathway. 4CL is one of the key enzyme genes in the phenylpropanoid metabolic pathway and represents the final step in phenylpropanoid biosynthesis[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Members of the 4CL gene family not only play a crucial role in the complex processes of plant growth and development, but their activity levels also significantly influence the accumulation of plant compounds such as flavonoids, lignans and lignin, playing an important role in plant growth and development as well as in responses to biotic and abiotic stresses[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, through genome-wide analysis, 43 4CL gene members were identified in the genome of 'Xinjiangdaye' (\u003cem\u003eMedicago sativa\u003c/em\u003e) and systematically named \u003cem\u003eMs4CL1\u0026ndash;Ms4CL43\u003c/em\u003e according to the \u003cem\u003eArabidopsis thaliana\u003c/em\u003e nomenclature standards. This study comprehensively identified 43 \u003cem\u003eMs4CL\u003c/em\u003e genes and systematically analyzed their basic characteristic. By examining the expression patterns of these genes in different tissues and under drought and salt stress, the aim was to screen for \u003cem\u003eMs4CL\u003c/em\u003e genes with stress resistance potential, thereby providing candidate targets for the breeding of salt stress and drought stress alfalfa varieties.\u003c/p\u003e \u003cp\u003eSubcellular localization predictions indicate that most \u003cem\u003eMs4CLs\u003c/em\u003e are localized to the plasma membrane (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e), while \u003cem\u003eJr4CLs\u003c/em\u003e and \u003cem\u003eJm4CLs\u003c/em\u003e are predominantly distributed between the plasma membrane and chloroplasts, \u003cem\u003eGh4CLs\u003c/em\u003e exhibit widespread distribution, whereas \u003cem\u003eCit4CLs\u003c/em\u003e are confined to the cytoplasm, which may be related to differences in the functional sites of 4CLs[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on phylogenetic relationships (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the identified 4CL genes can be classified into four distinct subfamilies (I, II, III, and IV). Group II contains the highest number of \u003cem\u003eMs4CL\u003c/em\u003e genes, indicating that the alfalfa 4CL gene family underwent extensive duplication during evolution, likely to adapt to its unique environmental stresses and the demands of lignin or flavonoid synthesis. The overlapping distribution of genes across three species in Group I indicates that the core function of 4CLs is highly conserved in legumes and crucifers, suggesting involvement in fundamental phenylpropanoid pathways (such as lignin synthesis). The species-specific branches in Groups II and IV suggest that these genes may have evolved new, species-specific functions, such as stress resistance and the synthesis of specialized secondary metabolites in alfalfa.\u003c/p\u003e \u003cp\u003eThe number of 4CL genes varies significantly among different species[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], which may be related to gene duplication events experienced by different species during evolution. Collinearity analysis revealed genomic clustering, with approximately 74.4% of \u003cem\u003eMs4CL\u003c/em\u003e genes localized to nine chromosomal regions (chr1.1\u0026ndash;chr1.4, chr2.2, chr2.4, chr3.4, chr4.1\u0026ndash;chr4.2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These regions show a significant enrichment of tandem and segmental duplication events. From an evolutionary perspective, segmental duplication events are the core drivers of the expansion and functional diversification of this gene family. Tandem duplication events (such as \u003cem\u003eMs4CL3\u003c/em\u003e and \u003cem\u003eMs4CL4\u003c/em\u003e) and numerous segmental duplication events (such as \u003cem\u003eMs4CL3\u003c/em\u003e and \u003cem\u003eMs4CL13\u003c/em\u003e) have collectively shaped the genomic distribution of family members. Gene copies generated by duplication may undergo subfunctionalization or functionalization during evolution, resulting in some copies (such as \u003cem\u003eMs4CL3\u003c/em\u003e, \u003cem\u003eMs4CL5\u003c/em\u003e, and \u003cem\u003eMs4CL13\u003c/em\u003e) retain and enhance stress response capabilities, while others have acquired tissue-specific expression patterns. Chromosomal mapping (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and colinearity analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) further corroborate this finding. The genes are clustered on chromosomes or distributed across chromosomes, and exhibit conserved colinearity with \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003e\u0026zwnj;Glycine max\u003c/em\u003e, indicating that this family possesses conserved domains during evolution. Comparative synteny analysis further indicates that the \u003cem\u003eMedicago sativa Ms4CL\u003c/em\u003e gene shares homologous synteny with both \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003e\u0026zwnj;Glycine max\u003c/em\u003e, but with more syntenic pairs in \u003cem\u003e\u0026zwnj;Glycine max\u003c/em\u003e, suggesting a closer evolutionary relationship between the \u003cem\u003eArabidopsis thaliana\u003c/em\u003e 4CL gene and \u003cem\u003e\u0026zwnj;Glycine max\u003c/em\u003e, a plant of the same family.\u003c/p\u003e \u003cp\u003eChanges in gene structure and conserved domains affect the function of the gene[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. The results show (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) that \u003cem\u003eMs4CLs\u003c/em\u003e have 4\u0026ndash;13 exons, a phenomenon also observed in most species of the 4CL gene family. For example, the number of conserved motifs in \u003cem\u003eMd4CL\u003c/em\u003e proteins ranges from 0 to 18[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], while the number of exons in \u003cem\u003eTa4CL\u003c/em\u003e genes ranges from 1 to 18[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. In plants, an increase in the number of introns within a gene may generally be advantageous. As non-coding regions, introns can protect genes from mutations, thereby better preserving gene function[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTranscription factors in the promoter region of genes are used to predict the biological processes in which they may be involved[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e], and they also participate in the regulation of gene expression. The various transcription factors in gene promoters may be associated with different gene functions[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e].In other species, 4CL promoters are similarly rich in various stress and hormone response elements. For example, the 4CL promoter in Juglans species contains multiple elements associated with plant hormones (such as MeJA) and abiotic stress[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In maize, the 4CL promoter contains \u003cem\u003ecis\u003c/em\u003e-acting elements responsive to drought, low temperature, light, MeJA, auxin, gibberellin, and salicylic acid[\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. This study found that the \u003cem\u003eMs4CL\u003c/em\u003e promoter exhibits similar compositional characteristics. A total of eight response elements were identified in the \u003cem\u003ecis\u003c/em\u003e-acting element analysis, with the promoter regions of most genes containing \u003cem\u003ecis\u003c/em\u003e-acting elements responsive to hormones such as MeJA and ABA, as well as abiotic stresses induced by drought (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This structural feature suggests that the expression of \u003cem\u003eMs4CL\u003c/em\u003e genes may be co-regulated by hormonal signaling and environmental stress. The \u003cem\u003eMs4CL\u003c/em\u003e family not only participates in basic lignin synthesis but also responds extensively to various stimuli, including light, hormones, low temperature, drought, and defense, serving as a key node linking growth and development with environmental adaptation. Further analysis revealed that the \u003cem\u003eMs4CL3\u003c/em\u003e and \u003cem\u003eMs4CL13\u003c/em\u003e genes contain not only drought response elements but also MeJA response elements, whereas the \u003cem\u003eMs4CL5\u003c/em\u003e gene contains only drought response elements. This suggests that \u003cem\u003eMs4CL3\u003c/em\u003e and \u003cem\u003eMs4CL13\u003c/em\u003e may be involved in both drought stress responses and MeJA-mediated salt stress responses, implying that they have acquired a broader regulatory network during evolution. In contrast, \u003cem\u003eMs4CL5\u003c/em\u003e primarily participates in drought stress responses and may have undergone functional specialization, focusing specifically on drought stress responses and being less likely to be regulated by MeJA. Thus, within the alfalfa 4CL gene family, different members may exhibit functional differentiation in stress responses, with some possessing multiple regulatory pathways and others exhibiting more specialized functions.\u003c/p\u003e \u003cp\u003eStress response elements dominate the promoters of \u003cem\u003eMs4CL\u003c/em\u003e genes. Among them, 14 \u003cem\u003eMs4CL\u003c/em\u003e genes contain TC-rich repetitive sequences associated with stress resistance, while 35 \u003cem\u003eMs4CL\u003c/em\u003e genes contain MeJA-responsive \u003cem\u003ecis\u003c/em\u003e-acting elements (CGTCA-motif and TGACG-motif), indicating that this gene family may be involved in adaptation to abiotic stress. Notably, all \u003cem\u003eMs4CL\u003c/em\u003e promoters contain elements associated with major plant hormone pathways, including gibberellin response elements (GARE motifs, P-box), abscisic acid response elements (ABRE), and auxin (TGA) response elements; additionally, 9 genes contain salicylic acid-related elements (TCA elements). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the presence of a large number of growth regulator-related elements suggests that the 4CL gene family may indirectly enhance stress resistance by participating in growth regulation, rather than being limited to direct responses to stress. This indicates that the 4CL gene family plays an important role in alfalfa\u0026rsquo;s adaptation to abiotic stress and in basic growth processes.\u003c/p\u003e \u003cp\u003e4CL is a regulatory gene. Studies on its expression regulation indicate that 4CL gene expression is primarily regulated by the plant developmental stage[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], and expression levels vary across different developmental stages and tissues. Among \u003cem\u003ethem\u003c/em\u003e, \u003cem\u003eAt4CL1 and At4CL2\u003c/em\u003e are most strongly expressed in seedling roots, \u003cem\u003ewhile At4CL3\u003c/em\u003e is highly expressed in flowers[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In rice, the \u003cem\u003eOs4CL2\u003c/em\u003e gene exhibits tissue-specific expression, with the highest expression levels observed in anthers[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In soybean, the \u003cem\u003eGm4CL3\u003c/em\u003e and \u003cem\u003eGm4CL4\u003c/em\u003e genes, which are associated with flavonoid biosynthesis, are highly expressed in roots and the hypocotyl[\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. A similar phenomenon has also been observed in alfalfa, where these stress-responsive genes exhibit distinct tissue specificity: the \u003cem\u003eMs4CL3\u003c/em\u003e gene is highly expressed in roots, while the \u003cem\u003eMs4CL5\u003c/em\u003e gene is highly expressed in nodules, with relatively low expression in tissues such as stems and flowers (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This suggests that their functions are not limited to stress responses but also involve the regulation of tissue differentiation during normal plant growth and development.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that 4CL genes can respond to plant stress, but different members of the same 4CL gene family within a species may respond differently to stress[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In Eucommia ulmoides, all 35 \u003cem\u003eEuc4CLs\u003c/em\u003e responded to salt stress, and the expression levels of \u003cem\u003emost Euc4CLs\u003c/em\u003e significantly increased after salt treatment, \u003cem\u003ewith Euc4CL9\u003c/em\u003e, \u003cem\u003eEuc4CL17, and Euc4CL27\u003c/em\u003e[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] showing the highest expression levels. In mulberry, all \u003cem\u003e4 Ma4CLs\u003c/em\u003e responded to salt stress. Under salt stress, all \u003cem\u003eMa4CL1\u0026ndash;3\u003c/em\u003e showed overall upregulation, while \u003cem\u003eMa4CL4\u003c/em\u003e exhibited a trend of upregulation in the stem and downregulation in the roots following salt stress[\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. In the potato, \u003cem\u003eSt4CL6\u003c/em\u003e and \u003cem\u003eSt4CL7\u003c/em\u003e were upregulated under PEG stress following PEG-induced drought treatment simulation, whereas \u003cem\u003eSt4CL4\u003c/em\u003e and \u003cem\u003eSt4CL5\u003c/em\u003e expression was suppressed[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Numerous studies have shown that 4CL genes play a crucial role in responding to and regulating processes related to drought and salt stress[\u003cspan additionalcitationids=\"CR76 CR77\" citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. However, systematic studies on the function of 4CL in alfalfa\u0026rsquo;s response to abiotic stresses are currently lacking. To elucidate the functional relevance of \u003cem\u003eMs4CL\u003c/em\u003e genes under abiotic stress conditions, this study systematically characterized the expression patterns of \u003cem\u003eMs4CL\u003c/em\u003e genes under drought and salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). Three genes (\u003cem\u003eMs4CL3\u003c/em\u003e, \u003cem\u003eMs4CL5\u003c/em\u003e, and \u003cem\u003eMs4CL13\u003c/em\u003e) were selected and analyzed using real-time quantitative PCR (qPCR). \u003cem\u003eMs4CL5\u003c/em\u003e was upregulated under both drought and salt stress, indicating the importance of this gene in abiotic stress responses. The \u003cem\u003eMs4CL13\u003c/em\u003e gene was not upregulated in all treatment groups under drought and salt stress, but rather reached its peak only during specific time points (such as M3, S4). The three target genes exhibited distinct functional differentiation under drought and salt stress, \u003cem\u003eMs4CL5\u003c/em\u003e and \u003cem\u003eMs4CL13\u003c/em\u003e were stress-induced, while \u003cem\u003eMs4CL3\u003c/em\u003e was stress-repressed, possibly related to growth maintenance. Combined with real-time quantitative PCR (qPCR) results, it can be observed that the genes identified in this study exhibit largely consistent responses to abiotic stress. The qPCR results for \u003cem\u003eMs4CL3\u003c/em\u003e, \u003cem\u003eMs4CL5\u003c/em\u003e, and \u003cem\u003eMs4CL13\u003c/em\u003e generally align with the expression patterns observed under salt and drought stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e4-Coumarate:CoA ligase (4CL) catalyze the production of various phenolic secondary metabolites, particularly lignin and flavonoids, which play a key role in the regulation of plant stress resistance[\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Although this study has revealed the expression profiles of \u003cem\u003eMs4CLs\u003c/em\u003e, their specific functions in the metabolic pathways of lignin, flavonoids, and other metabolites require further validation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study identified a total of 43 \u003cem\u003eMs4CL\u003c/em\u003e genes in the 'Xinjiangdaye' (\u003cem\u003eMedicago sativa\u003c/em\u003e) genome, classified into four subfamilies, with Group II having the largest number of members, indicating that this family has undergone extensive duplication and diversification during evolution. Genomic structure analysis revealed that \u003cem\u003eMs4CLs\u003c/em\u003e contain 4 to 13 exons. \u003cem\u003eCis\u003c/em\u003e-acting element analysis indicated that the promoters of most \u003cem\u003eMs4CL\u003c/em\u003e genes contain elements responsive to MeJA, ABA, and drought stress. Notably, \u003cem\u003eMs4CL3 and Ms4CL13\u003c/em\u003e contain both drought and MeJA response elements, whereas \u003cem\u003eMs4CL\u003c/em\u003e5 contains only drought response elements, suggesting functional differentiation among family members. Tissue-specific expression analysis revealed that \u003cem\u003eMs4CL3\u003c/em\u003e is highly expressed in roots, while \u003cem\u003eMs4CL5\u003c/em\u003e is highly expressed in root nodules. qPCR results indicated that \u003cem\u003eMs4CL5\u003c/em\u003e was significantly upregulated under both drought and salt stress, \u003cem\u003eMs4CL13\u003c/em\u003e reached its expression peak during a specific time window, \u003cem\u003eand Ms4CL3\u003c/em\u003e exhibited a repressed expression pattern, demonstrating distinct functional differentiation among the three genes. This study preliminarily reveals the functional characteristics of alfalfa 4CL genes, laying the foundation for subsequent functional studies of the 4CL gene family. Future experiments will investigate the 4CL genes and elucidate their mechanisms of action in plant abiotic stress resistance, further validating the specific functions of \u003cem\u003eMs4CLs\u003c/em\u003e in the synthesis of metabolites such as lignin and flavonoids, as well as in stress resistance regulation, thereby providing a theoretical basis and candidate targets for the molecular breeding of salt- and drought-tolerant alfalfa varieties.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMW: Molecular weight\u003c/p\u003e\n\u003cp\u003epI: isoelectric points\u003c/p\u003e\n\u003cp\u003eAA: amino acid\u003c/p\u003e\n\u003cp\u003eML: Maximum likelihood estimation\u003c/p\u003e\n\u003cp\u003eHMM: The hidden Markov model\u003c/p\u003e\n\u003cp\u003eMEME: Multiple em for motif elicitation\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaiyue Lei and Shuyan Liu jointly conceived and designed the study and drafted the original manuscript. Yuqi Zhang, Xinyue Ma, and Li Zhao were responsible for the visualization and production of the figures and tables. Fei He and Ruicai Long handled data organization and manuscript revision. Tiejun Zhang and Qingchuan Yang conducted data analysis and verified data accuracy. Min Lu and Lin Chen supervised the entire research project and provided scientific guidance. All authors reviewed and approved the final manuscript prior to submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32371757,32441018), the major demonstration project \u0026ldquo;The Open Competition\u0026rdquo; for Seed Industry Science and Technology Innovation in Inner Mongolia (No. 2022JBGS0016).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eField and laboratory studies were conducted by local legislation. This article does not contain any studies with human participants or animals and does not involve any endangered or protected species. The plant materials sampled and experiments performed in this research complied with institutional, national, and international guidelines and legislation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32371757,32441018), the major demonstration project \u0026ldquo;The Open Competition\u0026rdquo; for Seed Industry Science and Technology Innovation in Inner Mongolia (No. 2022JBGS0016).\u003c/p\u003e"},{"header":"References ","content":"\u003col\u003e\n\u003cli\u003eZhu JK. 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BMC Plant Biol 2020, 20(1):125.\u003c/li\u003e\n\u003cli\u003eZhang CH, Ma T, Luo WC, et al. Identification of 4CL Genes in Desert Poplars and Their Changes in Expression in Response to Salt Stress. Genes (Basel) 2015, 6(3):901-917.\u003c/li\u003e\n\u003cli\u003eLiu Q, Le Luo, Zheng L. Lignins: Biosynthesis and Biological Functions in Plants. International Journal of Molecular Sciences 2018, 19(2):335.\u003c/li\u003e\n\u003cli\u003eAida S, Susmita D, Namira A, et al. Diverse Physiological Roles of Flavonoids in Plant Environmental Stress Responses and Tolerance. Plants (Basel) 2022, 11(22):3158.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Medicago sativa L., Ms4CL, abiotic stress, expression analysis","lastPublishedDoi":"10.21203/rs.3.rs-9384279/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9384279/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e4-Coumarate: CoA ligase (4CL) is a pivotal enzyme in the phenylpropanoid metabolic pathway and plays a crucial role in plant growth, development, and stress responses. In this study, a genome-wide analysis was conducted to identify a total of 43 \u003cem\u003eMs4CL\u003c/em\u003e genes (\u003cem\u003eMs4CLs\u003c/em\u003e) in 'Xinjiangdaye' (\u003cem\u003eMedicago sativa\u003c/em\u003e). The fundamental structure, phylogenetic relationships, \u003cem\u003ecis\u003c/em\u003e-acting element analysis, and expression patterns of these genes were systematically analysed. The results indicate that \u003cem\u003eMs4CL\u003c/em\u003e genes can be classified into four subfamilies, and subcellular localization predictions suggest that most genes are localized to the plasma membrane. The number of exons ranges from four to thirteen, consistent with the characteristics of most 4CL gene families. The promoter regions are rich in stress response elements associated with MeJA, ABA, and drought, implying that the expression of these genes may be regulated by stress-induced hormonal signals. Collinearity analyses revealed that segmental duplications are the primary mechanisms driving the expansion and functional diversification of this family. Analysis of tissue-specific expression revealed that the four genes \u003cem\u003eMs4CL8\u003c/em\u003e, \u003cem\u003eMs4CL22\u003c/em\u003e, \u003cem\u003eMs4CL37\u003c/em\u003e, and \u003cem\u003eMs4CL42\u003c/em\u003e exhibit significant tissue-specific expression patterns. An analysis of responses to abiotic stresses indicates that \u003cem\u003eMs4CL5\u003c/em\u003e and \u003cem\u003eMs4CL13\u003c/em\u003e are stress-induced genes in response to salt and drought stresses, whereas \u003cem\u003eMs4CL3\u003c/em\u003e is a stress-repressed gene in response to salt and drought stresses. These three genes exhibit distinct functional differentiation in stress responses. \u003cem\u003eCis\u003c/em\u003e-acting element analysis further revealed promoter enrichment in defense/stress-responsive and phytohormone-related elements, consistent with their roles in environmental adaptation. This study provides a theoretical foundation for elucidating the mechanisms of salt and drought tolerance in alfalfa and supports its genetic improvement.\u003c/p\u003e","manuscriptTitle":"Genome-wide Identification and Expression Pattern Analysis of the Medicago sativa 4-Coumarate: CoA ligase (4CL) Gene Family","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-29 18:04:58","doi":"10.21203/rs.3.rs-9384279/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-19T02:27:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"121228339532012821060628162058327753149","date":"2026-05-14T04:28:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-28T08:02:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"337306812634668971446323998428339148555","date":"2026-04-23T15:46:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-21T00:24:24+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-16T09:35:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-15T11:28:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-15T11:27:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-04-11T03:49:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5fc8006f-fa05-4be5-adab-06fb6b2ad4cd","owner":[],"postedDate":"April 29th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-19T02:27:20+00:00","index":121,"fulltext":""},{"type":"reviewerAgreed","content":"121228339532012821060628162058327753149","date":"2026-05-14T04:28:07+00:00","index":116,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-29T18:04:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-29 18:04:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9384279","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9384279","identity":"rs-9384279","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}