Characterization of the BPC Genes in Alfalfa and Functional Verification of MsBPC10 in Salt Tolerance

Characterization of the BPC Genes in Alfalfa and Functional Verification of MsBPC10 in Salt Tolerance | 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 Characterization of the BPC Genes in Alfalfa and Functional Verification of MsBPC10 in Salt Tolerance Li Zhao, Xianyang Li, Xinyue Ma, Yuqi Zhang, Hao Liu, fei He, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7468142/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study systematically analyzed the BPC family in alfalfa (Medicago sativa) for the first time, identifying 18 MsBPC genes randomly distributed on 14 chromosomes. Their encoded proteins are mostly alkaline and classified into three subgroups. Cis-acting element analysis of promoters showed that the promoter region of MsBPC contains various cis-acting elements related to hormone response, growth and development, as well as stress response. Expression pattern analysis in different tissues revealed that 7 were expressed in all 6 tissues, 4 were only expressed in a single tissue, and the remaining genes were expressed in 3-5 tissues, suggesting that MsBPC genes may be involved in the regulation of different growth and developmental stages of Medicago sativa. Transcriptome analysis under salt, drought, and cold stresses showed that 12, 11, and 12 genes responded, respectively. RT-qPCR detection confirmed that MsBPC genes responded to salt and drought treatments, further verifying their important roles in abiotic stress responses Subcellular localization analysis revealed that MsBPC5 and MsBPC10 are localized in the nucleus, which is consistent with the predicted results. heterologous expression in yeast was employed to characterize the function of MsBPC10, which was upregulated in response to salt stress. The study conducted a comprehensive identification and analysis of the BPC gene family in Medicago sativa. Through the analysis of RNA-seq data, candidate genes related to abiotic stress were screened out, providing candidate genes for Medicago sativa stress-resistant breeding. BPC genes Medicago sativa L. Functional Verification Salt Tolerance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Key Message The BPC family in alfalfa was first systematically analyzed, identifying 18 MsBPC genes. Their proteins, promoter elements, tissue expression and abiotic stress responses were studied, and stress-resistant candidate genes were screened to assist stress-resistant breeding. Introduction During their lifecycle, plants encounter diverse biotic and abiotic stresses (Chen et al. 2021 ). To survive, plants have evolved multiple regulatory mechanisms (Liu et al.2021; Fichman et al.2021;Gong et al.2020). Transcription factors binding to specific DNA sequences to regulate target gene expression are a key strategy among them (Foley et al.2002). BPC (Basic Penta Cysteine) transcription factors are plant-specific proteins defined by five conserved cysteine residues at their C-terminus. These residues form a highly conserved DNA-binding zinc finger domain, a signature of BPC transcription factors (Santi et al.2003; Berger et al.2012). Currently, the BPC gene family has been systematically identified in various plant species. For example, 6 BPC members have been identified in Camellia japonica (Hu et al.2022), 7 in Arabidopsis thaliana (Bai et al.2017), 4 BPC members in Oryza sativa (Cao et al.2018),4 BPC members in Cucumis sativus (Huang et al.2019)], and 12 and 6 BPC family members in Chinese cabbage and Cucurbita pepo , respectively (Feng et al.2023; Hu et al.2022). BPC transcription factors are key players in regulating plant growth and development. For instance, Arabidopsis mutants with multiple BPC allele mutations display a range of developmental defects, such as dwarfism, production of small and curled leaves, early flowering, abnormal ovules, failure in flower bud differentiation, and even severe sterility. (Wang et al.2003; Black et al.1996; Monfared et al.2011). In Arabidopsis thaliana , BPC transcription factors activate the function of INO (Inner No Outer), a protein related to ovule development, by binding to GA/TC-rich dinucleotide sequences in promoters. Except for AtBPC5 , all other BPC genes are expressed in both vegetative and reproductive organs. (Gregis et al.2012). In Cucumis sativus , CsBPC2 participates in seed germination by regulating ABSCISIC ACID INSENSITIVE 3 (ABI3) (Meister et al.2004). In Oryza sativa , BPC transcription factors regulate growth and development through different mechanisms: OsGBP1 negatively regulates grain length by affecting the expression of grain-related genes, while also negatively regulating seedling growth and development; OsGBP3 affects plant height and positively regulates grain length, promoting the expression of grain-related genes (Kater et al.2014). Additionally, OsGBP1 can directly bind to the GAGA repeat sequences on the promoter elements of OsLFL1 , Ghd8 , and Hd3a , thereby delaying flowering time (Brand et al.2015; Bai et al.2017). These studies confirm the important role of BPC transcription factors in plant growth and development. These studies confirmed that BPC transcription factors play an important role in plant growth and development. BPC transcription factors also play an important role in plant responses to biotic and abiotic stresses. In Arabidopsis thaliana , it was found that the BPC1BPC2 double mutant exhibited reduced tolerance to salt stress compared to the wild type, and an accumulation of β-1,4-galactoside was also observed. BPC1/BPC2 bind to the β-1,4-galactoside synthase 1 ( GALS1 ) gene, recognizing the region rich in GAGA repeat sequences in its promoter region, directly regulating the synthesis of β-1,4-galactosyltransferase. When plants are subjected to salt stress, the expression of BPC1/BPC2 decreases, leading to impaired binding with GALS1 , increased synthesis of β-1,4-galactose, and inhibited cellulose synthesis, thereby reducing the plant's salt tolerance (Cao et al.2018;Li et al.2007;Li et al.2008).In Brassica napus , BnBPC6 regulates the formation of the plant wax layer by controlling the elongation process of fatty acids, thereby resisting biotic and abiotic stresses (Liu et al.2021c; Liu et al.2021b). In Cucumis sativus studies, overexpression of CsBPC2 in tobacco inhibits seed germination under salt, polyethylene glycol, and abscisic acid stress, thereby identifying it as a negative regulator of seed germination under osmotic stress (Liu et al.2021a). These studies indicate that BPC transcription factors play important roles in plant responses to biotic and abiotic stresses through diverse mechanisms. Medicago sativa L. (alfalfa) is a perennial leguminous forage crop characterized by strong stress tolerance, high nutritional value, and significant economic importance, earning it the widespread title of “King of Forage Crops” (Huang et al.2019). As a globally cultivated important forage crop, Medicago sativa plays a crucial role in livestock production and soil improvement.( Acharya et al.2020; Pontes et al.2007). To date, the BPC gene family has been identified and functionally studied in various plant species. Such as Arabidopsis thaliana, cucumber, and rice, but its identification and functional analysis in alfalfa have not been reported. This study comprehensively utilized bioinformatics methods to identify and characterize the BPC gene family in tetraploid alfalfa, and employed real-time fluorescent quantitative PCR (quantitative real-time PCR, RT-qPCR) technology to analyze the expression patterns of this gene family under salt and drought stress. This provides a theoretical basis for further studying the biological functions of alfalfa BPC genes and aims to offer theoretical references for stress-tolerant breeding in alfalfa. Materials and methods MsBPC gene family identification and chromosome distribution Download the protein sequences of the Arabidopsis thaliana BPC genes from the Arabidopsis thaliana information resource (TAIR) ( https://www.arabidopsis.org/ ). The genomic data of alfalfa "Xinjiang Da Ye" were taken from the Alfalfa Genome Project ( https://fgshare.com/projects/whole_genome_sequencing_and_assembly_of_Medicago_sativa/66380 ). Medicago truncatula genome-wide data are derived from the online website (). The conserved domain (PF06217) file of the BPC protein is downloaded from the Pfam database ( https://pfam.xfam.org/).(Finn et al.2014) The BPC protein sequence of Arabidopsis thaliana was used as the query sequence, and the blastp function in TBtools was used to compare the alfalfa protein database to identify the MsBPC gene. At the same time, the e-value ≤ e − 10 was set by the hmmsearch command of HMMER to screen out the BPC gene containing the typical domain of the BPC family. The protein sequences of candidate genes obtained from BLAST were submitted to NCBI-CDD ( https://www.ncbi.nlm.nih.gov/cdd ) and SMART ( http://smart.embl-heidelberg.de/a ) for further identification and removal of redundant sequences (Bork et al.1998). Finally, candidate genes are determined. Analysis of Physicochemical Properties of gene Family members Analysis of Physicochemical Properties of MsBPC gene Family members The genomic sequences, chromosomal locations and protein sequence lengths of MsBPC genes were extracted from the ‘Xinjiang Daye’ reference genome GFF file using TBtools software. The MsBPC protein sequences were subsequently submitted to the online WoLF PSORT tool ( https://wolfpsort.hgc.jp/ ) for subcellular localization prediction and determination of theoretical isoelectric points (pI) and molecular weights. Chromosomal mapping of MsBPC genes was performed using TBtools, followed by systematic renaming based on their chromosomal location. Phylogenetic analysis and Motif Analysis of MsBPC Protein sequences of MsBPC were obtained from the UniProt database ( https://www.uniprot.org ) for constructing a phylogenetic tree. Using the MEGA11 software, multiple sequence alignments of the protein sequences were performed, and a phylogenetic tree was constructed using maximum likelihood estimation (NJ) with specific parameters set to the Poisson model, and a Bootstrap repetition of 1000 times (Tamura et al.2021). The MEME online program ( http://meme.nbcr.net/meme/intro.html ) was used to obtain conserved motifs of the MsBPC protein, with parameters set to default values, defining the number of conserved motifs as 10, and restricting the optimal motif width between 6 to 50 residues. Gene Duplication Events and collinearity analysis of gene family Gene Duplication Events and collinearity analysis of MsBPC gene family The MCScanX tool was used to analyze the collinearity information of BPC genes within and between species (Guo et al.2012) Finally, the results of the collinearity information were visualized using TBtools (Debarry et al.2012). Additionally, the Ka/Ks Calculator function in TBtools was employed to calculate the number of nonsynonymous substitutions per nonsynonymous site (Ka) and the number of synonymous substitutions per synonymous site (Ks), as well as their ratio, to infer the selection pressure during the gene evolutionary process Analysis of acting elements in the Promoter of Gene Analysis of Cis- acting elements in the Promoter of MsBPC Gene The TBtools tool was used to extract the 2000 bp sequence upstream of the start codon of the MsBPC gene as the promoter region. The online website PlantCARE ( https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) was employed to predict the types and numbers of cis- acting elements in the promoter region (Déhais et al.2002). Visualization of these cis- acting elements was conducted using the GSDS2.0 website ( http://gsds.gao-lab.org/ ). Transcript abundance analysis using RNA-seq Transcriptome Data Transcriptome data of six different tissues (roots, root nodules, elongating stems, short stems, flowers, and leaves) of Medicago sativa were downloaded from the NCBI public database (SRP055547). Additionally, transcriptomic data of Medicago sativa under salt, cold, and drought stress conditions (SRR7091780 ~ SRR7091794, SRR7160313 ~ SRR7160357) were utilized to analyze the tissue-specific expression patterns of MsBPC and to investigate the expression changes of MsBPC under stress conditions.(Bucciarelli et al.2015; Deng et al.2021) The transcriptome data were processed, and visualization was carried out using the Heatmap of TBtools. RT-qPCR analysis of the MsCPP gene in response to drought and salt stress Alfalfa (Zhongmu No. 1) seeds were provided from the Institute of Animal Science of the Chinese Academy of Agricultural Sciences. Whole alfalfa seeds of the same size were selected, sterilized with 5% sodium hypochlorite solution for 10 min, rinsed repeatedly with distilled water for 4 ~ 5 times, the seeds were spread flat in a Petri dish containing filter paper, incubated in a light incubator (16 h light/8 h dark) for 28 days, Salt stress was simulated using 250 mM NaCl, with 0 h as the control group, and root tip samples were collected at 0.5, 1, 3, 6, 12, and 24 h. 400 mM mannitol was used to simulate drought stress, and root tip samples were collected at 1, 3, 6, 12 and 24 hours. Each stress treatment was set up with 3 replicates, and each replicate contained 5 seedlings. Total RNA was extracted using the TRIzol reagent, and cDNA was synthesized using the EasyScript First-Strand cDNA Synthesis Kit. Primers were designed using Primer 5.0, with the sequences shown in Table 1 . The RT-qPCR experiments were conducted using SYBR Premix Ex Taq (Takara, Japan) and a 7500 real-time fluorescence quantitative PCR system (Applied Biosystems, USA). Each sample was set up with three replicates, with the Medicago sativa L. actin gene (Actin2) used as the internal control. Relative expression levels were calculated using the 2 −ΔΔCT method (Harshitha et al.2021) Graph Pad Prism 8 software was used for graphing. MsBPC5 and MsBPC10 Subcellular localization The ORF of MsBPC5 and MsBPC10 genes were cloned into the Super 1300-GFP vector to construct Super- MsBPC5 -GFP and Super - MsBPC10 - GFP . The vectors Super-MsBPC5-GFP , Super-MsBPC10-GFP , and the vector control ( Super 1300-GFP ) were introduced into Agrobacterium tumefaciens GV3101 were introduced into Agrobacterium tumefaciens GV3101 and then transiently transformed into N. benthamiana leaves. The fluorescent signals were detected using confocal microscopy (Leica Microsystems, Wetzlar, Germany). Validation of Heterologous Expression in Yeast The coding sequence of the MsBPC10 gene was initially amplified from the alfalfa cultivar Zhong mu 4 via polymerase chain reaction (PCR). Subsequently, the target MsBPC 10 gene was ligated into the pYES2-NTB expression vector to generate the recombinant plasmid pYES2 - MsBPC 10-NTB. Using the Yeast Colony Rapid Detection Kit (Nanjing Ruian), the correctly constructed pYES2 - MsBPC 10-NTB recombinant plasmid and the empty pYES2-NTB vector were individually introduced into the Saccharomyces cerevisiae strain INVSC 1 through yeast transformation. During the transformation procedure, the yeast cells were resuspended in 200 µL of sterile water and gently mixed before being plated onto the corresponding synthetic dropout (SD) selection media. The plates were then incubated at 30°C for 3–5 days to allow for colony formation. Following incubation, the correct positive transformants from the experimental group (pYES2- BPC10-NTB) and the negative control group (pYES2-NTB) were identified and selected. These positive transformants were subsequently resuspended in 2 mL of SG-U liquid medium, and the optical density at 600 nm (OD600) was adjusted to 0.6. The cell suspensions were then subjected to serial dilution (10 0 , 10⁻¹, 10⁻²). The diluted cell suspensions were spotted onto SG-U plates supplemented with varying concentrations of NaCl (0 M, 0.5 M, 1.0 M, 1.3 M, 1.5 M). The plates were incubated at 30°C for 7 days to assess the growth and colony formation under different osmotic stress conditions. At the end of the incubation period, photographs were taken to document and analyze the colony morphology and growth status. Results Identification of the BPC Gene Family Medicago sativa L A total of 18 MsBPC genes with complete conserved domains were identified in Medicago sativa , distributed across 14 chromosomes. (Figure.1). Based on the position of the genes on the chromosomes, the BPC genes were named MsBPC 1 to MsBPC 18. The important characteristics of gene and protein sequences are shown in Table 1 and Table S1. Further analysis indicates significant differences in the protein sequence lengths, molecular weights, and theoretical isoelectric points of the MsBPC genes. The protein sequence length ranges from 195 amino acids ( MsBPC10 ) to 339 amino acids ( MsBPC 1 to 4), with molecular weights ranging from 22.26 kDa ( MsBPC10 ) to 37.78 kDa ( MsBPC 4), and theoretical isoelectric points ranging from 6.71 ( MsBPC10 ) to 9.68 ( MsBPC 15 to 17). Subcellular localization results show that 7 genes are localized in the nucleus; 4 genes in mitochondria; 2 genes in chloroplasts; 2 genes in cell membranes; MsBPC 3 in the cytoplasm; MsBPC 11 in vacuole; and MsBPC 1 is located extracellular. Table 1 Basic information analysis of MsBPC gene family Gene ID Gene name Protein Length (aa) MW (kDa) pI Subcellular Location MS.gene27035 MsBPC1 339 37.77 9.26 Extracellular MS.gene24195 MsBPC2 339 37.78 9.26 Nucleus MS.gene64186 MsBPC3 339 37.77 9.26 Cytoplasm MS.gene63981 MsBPC4 339 37.78 9.26 Nucleus MS.gene028699 MsBPC5 295 32.65 9.6 Nucleus MS.gene31269 MsBPC6 280 30.87 9.62 Nucleus MS.gene79458 MsBPC7 312 35.36 9.36 Plasma membrane MS.gene08364 MsBPC8 280 30.86 9.66 Chloroplast MS.gene030481 MsBPC9 312 35.37 9.36 Nucleus MS.gene030483 MsBPC10 195 22.26 6.71 Nucleus MS.gene028203 MsBPC11 280 30.91 9.58 Vacuole MS.gene000525 MsBPC12 312 35.36 9.36 Mitochondrion MS.gene023254 MsBPC13 280 30.88 9.66 Nucleus MS.gene34978 MsBPC14 280 30.89 9.66 Plasma membrane MS.gene000243 MsBPC15 289 32.12 9.68 Mitochondrion MS.gene27413 MsBPC16 289 32.12 9.68 Chloroplast MS.gene041646 MsBPC17 289 32.14 9.68 Mitochondrion MS.gene000299 MsBPC18 288 31.99 9.63 Mitochondrion chr: chromosome; aa: amino acid; MW: molecular weight; pI: isoelectric point Phylogenetic analysis and classification of the MsBPC gene family To explore the evolutionary relationship between the BPC gene of Medicago sativa L. and the BPC gene of Arabidopsis , this study utilized MEGA 11 software to construct a phylogenetic tree for Arabidopsis thaliana and Medicago sativa , which included 18 MsBPC sequences from Medicago sativa and 7 At BPC sequences from Arabidopsis thaliana (Figure.2). Based on gene structure and conserved motif analyses, the 25 BPC proteins were divided into three groups: Group I contains 9 members (6 MsBPC and 3 AtBPC) , Group II contains 11 members (8 MsBPC and 3 AtBPC ), and Group III contains 5 members (4 MsBPC and 1 AtBPC) . Analysis of the Gene Structure and Conserved Motifs of MsBPC gene family To further understand the structure of the MsBPC gene, analyses were conducted using TBtools software and the MEME online platform. The gene structure analysis indicates that the MsBPC gene is relatively short, with most members containing two exons and one intron. The coding region features a GAGA repeat sequence specifically recognized by BPC transcription factors. Ten conserved motifs were predicted via the MEME website, ranging in length from 6 to 50 amino acids. The results were visualized using TBtools software (Figure. 3). Figure 4 presents detailed information about the ten conserved motifs. The findings show that the MsBPC family members in alfalfa encompass a varying number of conserved motifs, ranging from 5 to 9, with motif 4 present in all MsBPC members, while motif 2 was not identified in MsBPC10 , suggesting that these two motifs are highly conserved within MsBPC proteins. Annotation of motifs 2 and 4 using NCBI's CD search tool revealed that both cover the BPC gene-specific motif GAGA-bind. Variations in motifs among different subgroups highlight the diversity of functions in MsBPC proteins. Similarities in motif composition within the same subgroup indicate that these proteins have related functions. Gene duplication events and chromosomal collinearity analysis of MsBPC gene family To understand the expansion mechanism of the MsBPC family in alfalfa, the interspecies evolutionary relationship of alfalfa was analyzed by using MCScanX. The results showed that there were 22 segmental duplications events and 1 tandem repeat event between these members (Figure.5 and Table S2). For example, MsBPC7 and MsBPC12 are located on chr4.1 and chr4.3, respectively. Segmental duplications events occur extensively in MsBPC genes, These results suggest that segmental duplication has played a major role in the expansion of the MsBPC gene family. In addition, The Ka/Ks of MsBPC8 and MsBPC11 were greater than 0.5 in the collinearity gene pairs, indicating that they underwent positive selection during evolution, while the Ka/Ks of the other gene pairs were less than 0.5 and underwent purification selection during evolution (Table 2 ). To explore the evolutionary relationship of BPC gene family in different species, a collinearity plot of Medicago sativa with Arabidopsis thaliana , Medicago truncatula , and Glycine max were constructed (Fig. 6 ). The results showed that 4 MsBPC genes in alfalfa had a collinearity relationship with Arabidopsis thaliana , and a total of 8 homologous gene pairs were identified. 7 MsBPC genes were collinearity with Medicago truncatula , and 7 homologous gene pairs were identified. 15 MsBPC genes were collinearity with Glycine max , and 26 homologous gene pairs were identified. It is worth noting that the degree of collinearity between BPC genes between Glycine max , and Medicago sativa is significantly higher than that of Arabidopsis thaliana , which may be due to the fact that soybean and alfalfa belong to the same leguminous plant, are more closely related, and the evolutionary pattern of gene families is more similar. Table 2 Evolutionary pressures between different gene pairs Gene 1 Gene 2 Ka Ks Ka/Ks MsBPC2 MsBPC3 0.00126609 0.017871285 0.070845 MsBPC14 MsBPC17 0.320670253 2.136703417 0.150077 MsBPC14 MsBPC12 0.652030359 2.644770393 0.246536 MsBPC14 MsBPC13 0.001546791 0.005203837 0.29724 MsBPC17 MsBPC12 0.681922855 3.096127244 0.22025 MsBPC17 MsBPC13 0.323082154 2.136703417 0.151206 MsBPC12 MsBPC13 0.652030359 2.644770393 0.246536 MsBPC11 MsBPC8 0.006988533 0.013060844 0.535075 MsBPC11 MsBPC9 0.668794505 2.911712094 0.229691 MsBPC2 MsBPC4 0.002534054 0.022414817 0.113053 MsBPC6 MsBPC5 0.003098378 0.010425885 0.297181 Analysis of cis- acting elements in the promoter regions of MsBPC genes family To explore the potential function of BPC gene in alfalfa, the cis- acting element of MsBPC gene was predicted using the PlantCARE database (Fig. 7 and Table S3). The results showed that a total of 13 cis- acting elements related to hormone response, stress response, and growth and development were screened. These elements include ABRE elements related to abscisic acid response, P-box, GARE-motif and TATC-box elements related to gibberellin response, TCA-element element related to salicylic acid response, TGACG-motif and CGTCA-motif elements related to methyl jasmonate response, LTR element related to low temperature response, and MBSI element related to the regulation of flavonoid biosynthesis. The cis- acting elements of each gene are unevenly distributed, which may be the reason why each gene has a different function. Analysis of the expression in different tissues of the MsBPC in Medicago sativa L. The expression level data of six tissues, including roots, leaves, flowers, elongated stems, Pre-elongated stems, and nodules of Medicago sativa , were obtained from public databases. The analysis results showed that 7 genes were expressed in all 6 tissues, 4 genes were only expressed in a single tissue, and the remaining genes were expressed in 3–5 tissues, suggesting that MsBPC genes may be involved in the regulation of different growth and developmental stages of Medicago sativa . (Fig. 8 and Table S4) Expression of MsBPC genes under abiotic stress response To explore the potential regulatory mechanisms of the MsBPC genes under different stresses, the RNA-seq data of alfalfa plants under the abiotic stresses (salt, drought, cold) were analyzed The results showed that 12 MsBPC s could respond to salt stress. 7 MsBPC s could respond to drought stress. 12 MsBPC genes could respond to cold stress (Fig. 9 and Table S5). These results suggest that BPC transcription factors play an important role in alfalfa response to abiotic stresses. To clarify validate the RNA-seq data, 2 genes were selected for RT-qPCR analysis. As shown in Fig. 10 , the expression of MsBPC10 decreased first, then increased, and finally decreased under drought stress. The expression of MsBPC5 decreased first and then increased, but was always lower than that of the control group. Under salt stress, the expression of MsBPC5 decreased first and then increased. The expression of MsBPC10 first increased, then decreased, and then increased, and was always higher than that of the control group. RT-qPCR results were consistent with RNA-seq data. MsBPC5 and MsBPC10 Subcellular localization The GV3101 strains harboring Super-MsBPC5-GFP and Super-MsBPC10-GFP were transiently transformed into Nicotiana benthamiana cells. the results show that both MsBPC5 and MsBPC10 are localized in the nucleus. (Fig. 11 ). the results are consistent with previous predictions. MsBPC10 improve Salt Tolerance in Yeast Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis conducted after treatment with 200 mM NaCl demonstrated that the transcriptional level of MsBPC10 initially increased, subsequently decreased, and then increased again relative to the control group (CK). These results suggest that the protein encoded by MsBPC10 may play a pivotal role in the salt stress response of Medicago sativa . To elucidate the effects of MsBPC10 on yeast growth and salt tolerance, the pYES2-BPC10-NTB construct was introduced into Saccharomyces cerevisiae cells. Under standard growth conditions, no significant differences in growth were observed between the transformed yeast strains and the corresponding empty vector control strains. However, when subjected to 1.5 M NaCl stress, the transformed yeast cells exhibited significant tolerance to high salinity. Notably, this enhanced salt tolerance was particularly evident at the 10⁻ 2 dilution level (Figure.12). Discussion BPC transcription factors are an important gene family in plants. They're known to regulate plant growth, development, and abiotic stress responses, as shown in many studies. This family has been identified in several plants, like Arabidopsis thaliana (Bai et al.2017), Oryza sativa (Cao et al.2018), Cucumis sativus (Huang et al.2019), and Chinese cabbage (Feng et al.2023), with 7, 4, 4, and 12 members found respectively. But in tetraploid Medicago sativa , this family hasn't been reported yet. In this study, focusing on tetraploid Medicago sativa , we found 18 MsBPC genes. This number is much higher than the BPC members reported in Arabidopsis thaliana , Oryza sativa , Cucumis sativus , and Chinese cabbage . This might be because Medicago sativa is a tetraploid with many homologous genes in its genome. We carried out a systematic analysis of the amplification and evolution of the MsBPC gene family in Medicago sativa . Compared with other species, the MsBPC family of alfalfa underwent significant gene family expansion, and fragment replication and tandem replication were one of the driving forces of gene family expansion (Danilevskaya et al.2016;Baumgarten et al.2004). In this study, We detected 22 segmental and 1 tandem duplication events in the MsBPC gene family suggesting a segmental duplication dominated evolutionary pattern. Tissue-specific expression analysis is an important method for predicting the biological function of genes in plant growth and development. Based on RNA-seq data, we systematically analyzed the expression patterns of 18 MsBPC genes in 6 different tissues. The results showed that 7 genes were expressed in all 6 tissues,4 in just one tissue, and the remaining 7 in 3–5 tissues. This indicates MsBPC genes likely regulate different growth and developmental stages of Medicago sativa. Cis - acting elements in promoter regions determine the spatiotemporal specificity of gene expression. They interact with corresponding transcription factors in different tissues and cells, enabling precise gene expression in specific tissues, developmental stages, and environmental conditions. (Dai et al.2014;Conery et al.2000). Cis- element analysis showed that MsBPC genes have diverse cis- elements, indicating complex regulation and possibly explaining the different expression patterns of these genes across tissues and under various abiotic stresses. in this study, MsBPC gene promoters were found to contain cis- elements related to hormone responses (gibberellin, salicylic acid, abscisic acid, and auxin) and abiotic stress responses (defense/stress, drought induction, and low-temperature responses). These results suggest that MsBPC may participate in plant responses to environmental stresses through multiple pathways, consistent with findings in cucumber, apple, and Chinese cabbage. (Huang et al.2019; Chen et al.2020; Huang et al.2024). Studies have shown that BPC genes play an important role in regulating plant growth, development, and responses to abiotic stress. Their functions are both conserved and diverse across different species. In Oryza sativa , OsGBP1 negatively regulates grain length and seedling development by regulating the expression of grain shape - related genes. Overexpressing OsGBP1 inhibits seedling growth, causes leaf yellowing, reduces biomass, and delays flowering, while gene silencing or mutation promotes plant growth (Cao et al.2018).In Malus domestica , overexpression of MdBPC2 decreases auxin content and inhibits plant growth and root development, while exogenous application of auxin can restore normal growth(Chen et al.2020).Overexpression of CsBPC2 in tobacco significantly inhibited seed germination under salt, polyethylene glycol, and abscisic acid stress (Huang et al.2019). In Chinese cabbage, BcBPC9 enhances antioxidant enzyme gene expression, increasing cadmium stress tolerance in yeast and tobacco (Huang et al.2024). In addition, BPC transcription factors regulate Arabidopsis ovule development by controlling the STK gene. Mutations cause dwarfism, leaf curling, ovule development abnormalities, and reduced lateral roots (Airoldi et al.2005). These studies show that while BPC genes have different specific functions in different species, they mainly focus on regulating growth, development, and responses to abiotic stress. Conclusions This study systematically identified a total of 18 MsBPC genes, which are distributed across 14 chromosomes. Phylogenetic analysis divided them into three evolutionary groups. Each group has similar gene structures and motif compositions. Segmental duplication was found to be the main driver of MsBPC gene family expansion. Expression profiling revealed that MsBPC genes exhibited different expression patterns across tissues: 7 were expressed in all 6 tissues, 4 were only expressed in a single tissue, and the remaining genes were expressed in 3–5 tissues, suggesting that MsBPC genes may be involved in the regulation of different growth and developmental stages of alfalfa. Stress response analysis revealed that 12, 11, and 12 MsBPC genes were significantly responsive to salt, drought, and cold stress, respectively. Promoter cis - element analysis revealed the potential regulatory mechanisms of MsBPC genes in hormone and stress responses. Collinearity analysis showed a higher collinearity between Medicago sativa and Glycine max BPC genes than Arabidopsis thaliana , BPC family evolution is relatively conserved in leguminous plants. indicating a more similar evolutionary pattern of the BPC gene family in leguminous plants. Subcellular localization assays showed MsBPC5 and MsBPC10 to be nuclear-localized, consistent with predictions. Yeast heterologous expression verified the salt tolerance function of MsBPC10 .The findings of this study provide theoretical support for elucidating MsBPC gene functions and offer important candidate genes for stress-resistant breeding in Medicago sativa. Abbreviations BPC /BASIC PENTACYSTEINE MW molecular weight HMM hidden Markov modeling pI isoelectric points aa amino acid NJ Neighbor-joining ABRE abscisic acid responsiveness MBS drought-responsive element LTR low-temperature-responsive element TC-rich repeats stress-defense-responsive regulatory element Declarations Author Contributions Li zhao, Xianyang Li, Hao Liu and Yuqi Zhang conceived and designed the research framework. Xinyue Ma, and Fei He were responsible for manuscript composition and preparation. Mingna Li and Xue Wang executed the experimental procedures. Ruicai Long, Junmei Kang, and Qingchuan Yang performed data analysis and interpretation. Lin Chen and Changhong Guo supervised the entire research project and provided scientific direction. All authors thoroughly reviewed the final manuscript and provided formal approval for its 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. Availability of data and materials Data is provided within the manuscript or supplementary information files. Conflicts of Interest The authors declare no conflicts of interest. References Acharya JP, Lopez Y, Gouveia BT et al (2020) Breeding Alfalfa ( Medicago sativa L.) Adapted to Subtropical Agroecosystems. Agronomy 10:742 Airoldi CA, Kooiker M, Losa A et al (2005) BASIC PENTACYSTEINE1, a GA binding protein that induces conformational changes in the regulatory region of the homeotic Arabidopsis gene SEEDSTICK. Plant Cell 17(3):722–729 Bai L, Liu Y, Mu Y et al (2017) Cucumber CsBPCs Regulate the Expression of CsABI3 during Seed Germination. Front Plant Sci 8:459 Baumgarten A, Cannon SB, Mitra A et al (2004) The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol 4:10 Berger N, Dubreucq B (2012) Evolution goes GAGA: GAGA binding proteins across kingdoms. Biochim Biophys Acta 1819(8):863–868 Black R, Ecker JR, Lehman A (1996) HOOKLESS1, an ethylene response gene, is required for differential cell elongation in the Arabidopsis hypocotyl. Cell 85(2):183–194 Bork P, Milpetz F, Schultz J et al (1998) SMART, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci USA 95(11):5857–5864 Brand LH, Hecker A, Peter et al (2015) The Arabidopsis GAGA-Binding Factor BASIC PENTACYSTEINE6 Recruits the POLYCOMB-REPRESSIVE COMPLEX1 Component LIKE HETEROCHROMATIN PROTEIN1 to GAGA DNA Motifs. Plant Physiol 168(3):1013–1024 Bucciarelli B, Fu F, O'Rourke JA et al (2015) The Medicago sativa gene index 1.2: a web-accessible gene expression atlas for investigating expression differences between Medicago sativa subspecies. BMC Genomics 16(1):502 Cao H, Gong R, Zhang J et al (2018) Divergent functions of the GAGA-binding transcription factor family in rice. Plant journal: cell Mol biology 94(1):32–47 Chen H, Yang Y, Zeng Y et al (2020) Allele-aware chromosome-level genome assembly and efficient transgene-free genome editing for the autotetraploid cultivated alfalfa. Nat Commun 11(1):2494 Chen X, Ding Y, Yang Y et al (2021) Protein kinases in plant responses to drought, salt, and cold stress. J Integr Plant Biol 63(1):53–78 Conery JS, Lynch M (2000) The evolutionary fate and consequences of duplicate genes. Sci (New York N Y) 290(5494):1151–1155 Dai W, Liu JH, Peng T (2014) Critical cis -Acting Elements and Interacting Transcription Factors: Key Players Associated with Abiotic Stress Responses in Plants. Plant Mol Biol Rep 32:303–317 Danilevskaya ON, Meng X, Thatcher SR et al (2016) Genome-Wide Analysis of Alternative Splicing during Development and Drought Stress in Maize. Plant Physiol 170(1):586–599 Debarry JD, Tang H, Wang Y et al (2012) MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res, 40(7), e49 Déhais P, Lescot M, Thijs G et al (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30(1):325–327 Deng H, Dong X, Ma W et al (2021) Genome-wide identification of the MADS-box transcription factor family in autotetraploid cultivated alfalfa ( Medicago sativa L.) and expression analysis under abiotic stress. BMC Genomics 22(1):603 Feng Y, Wang J, Zhang S et al (2023) Genome-wide identification of flowering Chinese cabbage BPC family genes and BcBPC9 functional analysis in Cd stress tolerance[J]. Plant Stress 10:100–120 Fichman., Mittler R, Zandalinas SI et al (2022) Reactive oxygen species signalling in plant stress responses. Nat Rev Mol Cell Biol 23(10):663–679 Finn RD, Bateman A, Clements J et al (2014) Pfam: the protein families database. Nucleic Acids Res 42:D223–D230 Foley RC, Singh K et al (2002) Transcription factors in plant defense and stress responses. Curr Opin Plant Biol 5(5):430–436 Gong Z, Shi H, Xiong L et al (2020) Plant abiotic stress response and nutrient use efficiency. Sci China Life Sci 63(5):635–674 Gregis V, Simonini R-V, S., I., et al (2012) Basic pentacysteine proteins mediate MADS domain complex binding to the DNA for tissue-specific expression of target genes in Arabidopsis. Plant Cell 24(10):4163–4172 Harshitha R, Arunraj DR (2021) Real-time quantitative PCR: A tool for absolute and relative quantification. Biochem Mol Biol 49:800–812 Hu Z, Ma X, Yu Y et al (2022) Characterizations of a Class-I BASIC PENTACYSTEINE Gene Reveal Conserved Roles in the Transcriptional Repression of Genes Involved in Seed Development. Curr Issues Mol Biol 44(9):4059–4069 Huang B, Li S, Miao L et al (2019) Genome-Wide Identification and Characterization of Cucumber BPC Transcription Factors and Their Responses to Abiotic Stresses and Exogenous Phytohormones. Int J Mol Sci 20(20):5048 Huang Y, Wan S, Zhao H et al (2024) The transcription factor MdBPC2 alters apple growth and promotes dwarfing by regulating auxin biosynthesis. Plant Cell 36(3):585–604 Huang B, Li S, Miao L et al (2019) Genome-Wide Identification and Characterization of Cucumber BPC Transcription Factors and Their Responses to Abiotic Stresses and Exogenous Phytohormones. Int J Mol Sci 20(20):5048 Kater MM, Simonini S (2014) Class I BASIC PENTACYSTEINE factors regulate HOMEOBOX genes involved in meristem size maintenance. J Exp Bot 65(6):1455–1465 Kumar S, Stecher G, Tamura K (2021) MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol 38(7):3022–3027 Li L, Shen GZ, Zhang JL et al (2007) Ectopic expression of OsLFL1 in rice represses Ehd1 by binding on its promoter. Biochem Biophys Res Commun 360(1):251–256 Li L, Peng LT, Shi ZY et al (2008) Overexpression of transcription factor OsLFL1 delays flowering time in Oryza sativa. J Plant Physiol 165(8):876–885 Liu J, Long S, Zhang W et al (2021c) Maintenance of Cell Wall Integrity under High Salinity. Int J Mol Sci 22(6):3260 Liu M, Zhang Q, Zhao S et al (2021b) Regulation of Plant Responses to Salt Stress. Int J Mol Sci 22(9):4609 Liu Y, Yan J, Yang L et al (2021a) Cell wall β-1,4-galactan regulated by the BPC1/BPC2-GALS1 module aggravates salt sensitivity in Arabidopsis thaliana. Mol Plant 14(3):411–425 Meister RJ, Monfared MM, Williams LA et al (2004) Definition and interactions of a positive regulatory element of the Arabidopsis INNER NO OUTER promoter. Plant journal: cell Mol biology 37(3):426–438 Meister RJ, Monfared MM, Simon MK et al (2011) Overlapping and antagonistic activities of BASIC PENTACYSTEINE genes affect a range of developmental processes in Arabidopsis. Plant journal: cell Mol biology 66(6):1020–1031 Monfared MM, Simon MK et al (2011) Overlapping and antagonistic activities of BASIC PENTACYSTEINE genes affect a range of developmental processes in Arabidopsis. Plant journal: cell Mol biology 66(6):1020–1031 Pontes LDS, Soussana JF, Louault F et al (2007) Leaf traits affect the above ground productivity and Blackwell Publishing Ltd quality of pasture grasses. Funct Ecol 21:844–853 Santi L, Stile MR, Wang Y et al (2003) The GA octodinucleotide repeat binding factor BBR participates in the transcriptional regulation of the homeobox gene Bkn3. Plant journal: cell Mol biology 34(6):813–826 Cite Share Download PDF Status: Posted Version 1 posted 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-7468142","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512757552,"identity":"27715458-e082-4baf-bb1c-702e5eaff2cf","order_by":0,"name":"Li Zhao","email":"","orcid":"","institution":"Harbin Normal University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Zhao","suffix":""},{"id":512757553,"identity":"b859a328-1a82-4d35-8388-ba7c2b81db14","order_by":1,"name":"Xianyang Li","email":"","orcid":"","institution":"Harbin Normal 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Institute of Animal Science","correspondingAuthor":true,"prefix":"","firstName":"Lin","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-08-27 06:01:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7468142/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7468142/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91609643,"identity":"45bde78f-db76-443d-aac2-e61a65df57c1","added_by":"auto","created_at":"2025-09-18 09:42:54","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1402833,"visible":true,"origin":"","legend":"\u003cp\u003eChromosome distribution of the \u003cem\u003eMsBPC\u003c/em\u003egenes.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7468142/v1/5d43baf773ce11be83104c4b.jpg"},{"id":91611503,"identity":"013de199-57c9-4e6c-a990-35d32f13bb09","added_by":"auto","created_at":"2025-09-18 09:58:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1368879,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of \u003cem\u003eBPC\u003c/em\u003e genes in \u003cem\u003eMedicago sativa\u003c/em\u003e and\u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7468142/v1/e322857c90ebd2600ffba232.jpg"},{"id":91609641,"identity":"a082c092-66d0-4f8b-a683-311ccb3d6276","added_by":"auto","created_at":"2025-09-18 09:42:54","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1012874,"visible":true,"origin":"","legend":"\u003cp\u003eBasic structures and motifs of the \u003cem\u003eMsBPC\u003c/em\u003egenes. (A) Phylogenetic tree of the \u003cem\u003eMsBPC\u003c/em\u003egenes. (B) Basic structures of the \u003cem\u003eMsBPC\u003c/em\u003egenes. (C) The structure of the \u003cem\u003eMsBPC\u003c/em\u003eprotein motif.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7468142/v1/67d4cbb2c99263ba00f84888.jpg"},{"id":91610239,"identity":"aa4250ab-290b-46aa-ad23-547ae802ad74","added_by":"auto","created_at":"2025-09-18 09:50:54","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":13167273,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of conserved motifs in the \u003cem\u003eMsBPC\u003c/em\u003e gene family of \u003cem\u003eMedicago sativa\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7468142/v1/d85a34023030519f194a2741.jpg"},{"id":91609640,"identity":"1f4dde33-a428-4023-9d9d-2799ac6d1c73","added_by":"auto","created_at":"2025-09-18 09:42:54","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3011273,"visible":true,"origin":"","legend":"\u003cp\u003eCollinearity analysis of \u003cem\u003eMsBPC \u003c/em\u003egenes\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7468142/v1/1e3035aa1b659cfa25c57d0c.jpg"},{"id":91611502,"identity":"92b473db-3219-4c99-8442-5a6bd74c0d85","added_by":"auto","created_at":"2025-09-18 09:58:54","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3847039,"visible":true,"origin":"","legend":"\u003cp\u003eCollinearity analysis of the \u003cem\u003eMsBPC\u003c/em\u003e genes with those of\u003cem\u003eArabidopsis thalian,\u003c/em\u003e \u003cem\u003eMedicago truncatula\u003c/em\u003e and \u003cem\u003eGlycine max.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7468142/v1/f24503bfc30922f5893a341f.jpg"},{"id":91610234,"identity":"ed688973-7f52-451d-aa59-8d30a70df567","added_by":"auto","created_at":"2025-09-18 09:50:54","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1299817,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of \u003cem\u003ecis-\u003c/em\u003eacting elements related to hormone response in alfalfa \u003cem\u003eMsBPC\u003c/em\u003e gene promoter region\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7468142/v1/aaad0b33e50f8e5adc6688e9.jpg"},{"id":91612033,"identity":"a913779f-c654-41dd-b70c-0b881b9289be","added_by":"auto","created_at":"2025-09-18 10:06:54","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":395962,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of \u003cem\u003eMsBPC\u003c/em\u003e gene expression in different tissues of alfalfa\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7468142/v1/8098610ad509c7885972d1fb.jpg"},{"id":91609648,"identity":"ad034144-6784-48e1-9817-de437dddbc73","added_by":"auto","created_at":"2025-09-18 09:42:54","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":965041,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of \u003cem\u003eBPC\u003c/em\u003e gene in alfalfa under salt, drought, and cold stress\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7468142/v1/5a2c767d17d3c81818bce89b.jpg"},{"id":91609646,"identity":"90ecb285-a6c6-40ed-8f52-edaae904c073","added_by":"auto","created_at":"2025-09-18 09:42:54","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":651692,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMsBPC\u003c/em\u003e expression under drought and salt stress conditions according to RT–qPCR. CK, control treatment; M, drought stress; S, salt stress; M1-5: 0.5h, 1h, 3h, 6h, 12h, 24h; S1-6: 0.5h, 1h, 3h, 6h, 12h, 24h.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7468142/v1/13e75461370673996ec5a2aa.jpg"},{"id":91609652,"identity":"c5210595-b63f-43dd-880d-59b455eb1e61","added_by":"auto","created_at":"2025-09-18 09:42:54","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":12626225,"visible":true,"origin":"","legend":"\u003cp\u003eSubcellular localization of the \u003cem\u003eSuper-MsBPC5-GFP\u003c/em\u003eand\u003cem\u003e Super-MsBPC10-GFP\u003c/em\u003e fusion protein in \u003cem\u003eNicotiana benthamiana\u003c/em\u003eleaf epidermal cells. Leaf epidermal cells transformed with \u003cem\u003eSuper-1300-GFP\u003c/em\u003ewere used as a control. Scale bars: 20μm\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7468142/v1/a27206d46a168191884b8173.jpg"},{"id":91609656,"identity":"d4389c75-e5a2-4839-a32e-a2268944a81c","added_by":"auto","created_at":"2025-09-18 09:42:54","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":259327,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of NaCl treatment of \u003cem\u003eMsBPC10 \u003c/em\u003egenes in yeast heterologous expression compared to the blank pYES2 line. After 36h of resuspension under control and different concentrations of NaCl, consecutive dilutions (10\u003csup\u003e0\u003c/sup\u003e, 10\u003csup\u003e−1\u003c/sup\u003e,10\u003csup\u003e-2\u003c/sup\u003e) of yeast were spotted onto SG−Ura solid medium. Sterile water was used as the control.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7468142/v1/d5912d2f107686cd0d0f574d.jpg"},{"id":92192343,"identity":"d63a2cb4-031b-4b42-8cd3-959789df24fd","added_by":"auto","created_at":"2025-09-25 15:22:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":41358221,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7468142/v1/9b7d8b26-5307-4cab-a477-7b4cb54ea651.pdf"}],"financialInterests":"","formattedTitle":"Characterization of the BPC Genes in Alfalfa and Functional Verification of MsBPC10 in Salt Tolerance","fulltext":[{"header":"Key Message","content":"\u003cp\u003eThe BPC family in alfalfa was first systematically analyzed, identifying 18 MsBPC genes. Their proteins, promoter elements, tissue expression and abiotic stress responses were studied, and stress-resistant candidate genes were screened to assist stress-resistant breeding.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eDuring their lifecycle, plants encounter diverse biotic and abiotic stresses (Chen et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To survive, plants have evolved multiple regulatory mechanisms (Liu et al.2021; Fichman et al.2021;Gong et al.2020). Transcription factors binding to specific DNA sequences to regulate target gene expression are a key strategy among them (Foley et al.2002). \u003cem\u003eBPC\u003c/em\u003e (Basic Penta Cysteine) transcription factors are plant-specific proteins defined by five conserved cysteine residues at their C-terminus. These residues form a highly conserved DNA-binding zinc finger domain, a signature of \u003cem\u003eBPC\u003c/em\u003e transcription factors (Santi et al.2003; Berger et al.2012). Currently, the \u003cem\u003eBPC\u003c/em\u003e gene family has been systematically identified in various plant species. For example, 6 \u003cem\u003eBPC\u003c/em\u003e members have been identified in \u003cem\u003eCamellia japonica\u003c/em\u003e (Hu et al.2022), 7 in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Bai et al.2017), 4 \u003cem\u003eBPC\u003c/em\u003e members in \u003cem\u003eOryza sativa\u003c/em\u003e (Cao et al.2018),4 \u003cem\u003eBPC\u003c/em\u003e members in \u003cem\u003eCucumis sativus\u003c/em\u003e (Huang et al.2019)], and 12 and 6 \u003cem\u003eBPC\u003c/em\u003e family members in \u003cem\u003eChinese cabbage\u003c/em\u003e and \u003cem\u003eCucurbita pepo\u003c/em\u003e, respectively (Feng et al.2023; Hu et al.2022).\u003c/p\u003e\u003cp\u003e\u003cem\u003eBPC\u003c/em\u003e transcription factors are key players in regulating plant growth and development. For instance, Arabidopsis mutants with multiple \u003cem\u003eBPC\u003c/em\u003e allele mutations display a range of developmental defects, such as dwarfism, production of small and curled leaves, early flowering, abnormal ovules, failure in flower bud differentiation, and even severe sterility. (Wang et al.2003; Black et al.1996; Monfared et al.2011). In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eBPC\u003c/em\u003e transcription factors activate the function of INO (Inner No Outer), a protein related to ovule development, by binding to GA/TC-rich dinucleotide sequences in promoters. Except for \u003cem\u003eAtBPC5\u003c/em\u003e, all other BPC genes are expressed in both vegetative and reproductive organs. (Gregis et al.2012). In \u003cem\u003eCucumis sativus\u003c/em\u003e, \u003cem\u003eCsBPC2\u003c/em\u003e participates in seed germination by regulating ABSCISIC ACID INSENSITIVE 3 (ABI3) (Meister et al.2004). In \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eBPC\u003c/em\u003e transcription factors regulate growth and development through different mechanisms: \u003cem\u003eOsGBP1\u003c/em\u003e negatively regulates grain length by affecting the expression of grain-related genes, while also negatively regulating seedling growth and development; \u003cem\u003eOsGBP3\u003c/em\u003e affects plant height and positively regulates grain length, promoting the expression of grain-related genes (Kater et al.2014). Additionally, \u003cem\u003eOsGBP1\u003c/em\u003e can directly bind to the GAGA repeat sequences on the promoter elements of \u003cem\u003eOsLFL1\u003c/em\u003e, \u003cem\u003eGhd8\u003c/em\u003e, and \u003cem\u003eHd3a\u003c/em\u003e, thereby delaying flowering time (Brand et al.2015; Bai et al.2017). These studies confirm the important role of \u003cem\u003eBPC\u003c/em\u003e transcription factors in plant growth and development. These studies confirmed that \u003cem\u003eBPC\u003c/em\u003e transcription factors play an important role in plant growth and development.\u003c/p\u003e\u003cp\u003e\u003cem\u003eBPC\u003c/em\u003e transcription factors also play an important role in plant responses to biotic and abiotic stresses. In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, it was found that the \u003cem\u003eBPC1BPC2\u003c/em\u003e double mutant exhibited reduced tolerance to salt stress compared to the wild type, and an accumulation of β-1,4-galactoside was also observed. \u003cem\u003eBPC1/BPC2\u003c/em\u003e bind to the β-1,4-galactoside synthase 1 (\u003cem\u003eGALS1\u003c/em\u003e) gene, recognizing the region rich in GAGA repeat sequences in its promoter region, directly regulating the synthesis of β-1,4-galactosyltransferase. When plants are subjected to salt stress, the expression of \u003cem\u003eBPC1/BPC2\u003c/em\u003e decreases, leading to impaired binding with \u003cem\u003eGALS1\u003c/em\u003e, increased synthesis of β-1,4-galactose, and inhibited cellulose synthesis, thereby reducing the plant's salt tolerance (Cao et al.2018;Li et al.2007;Li et al.2008).In \u003cem\u003eBrassica napus\u003c/em\u003e, \u003cem\u003eBnBPC6\u003c/em\u003e regulates the formation of the plant wax layer by controlling the elongation process of fatty acids, thereby resisting biotic and abiotic stresses (Liu et al.2021c; Liu et al.2021b). In \u003cem\u003eCucumis sativus\u003c/em\u003e studies, overexpression of \u003cem\u003eCsBPC2\u003c/em\u003e in tobacco inhibits seed germination under salt, polyethylene glycol, and abscisic acid stress, thereby identifying it as a negative regulator of seed germination under osmotic stress (Liu et al.2021a). These studies indicate that \u003cem\u003eBPC\u003c/em\u003e transcription factors play important roles in plant responses to biotic and abiotic stresses through diverse mechanisms.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cem\u003eMedicago sativa\u003c/em\u003e L. (alfalfa) is a perennial leguminous forage crop characterized by strong stress tolerance, high nutritional value, and significant economic importance, earning it the widespread title of \u0026ldquo;King of Forage Crops\u0026rdquo; (Huang et al.2019). As a globally cultivated important forage crop, \u003cem\u003eMedicago sativa\u003c/em\u003e plays a crucial role in livestock production and soil improvement.( Acharya et al.2020; Pontes et al.2007). To date, the \u003cem\u003eBPC\u003c/em\u003e gene family has been identified and functionally studied in various plant species. Such as Arabidopsis thaliana, cucumber, and rice, but its identification and functional analysis in alfalfa have not been reported. This study comprehensively utilized bioinformatics methods to identify and characterize the \u003cem\u003eBPC\u003c/em\u003e gene family in tetraploid alfalfa, and employed real-time fluorescent quantitative PCR (quantitative real-time PCR, RT-qPCR) technology to analyze the expression patterns of this gene family under salt and drought stress. This provides a theoretical basis for further studying the biological functions of alfalfa \u003cem\u003eBPC\u003c/em\u003e genes and aims to offer theoretical references for stress-tolerant breeding in alfalfa.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003eMsBPC\u003c/em\u003e gene family identification and chromosome distribution\u003c/h2\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003ch2\u003eDownload the protein sequences of the \u003cem\u003eArabidopsis thaliana BPC\u003c/em\u003e genes from the \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section4\"\u003e\u003ch2\u003e\u003cem\u003ethaliana\u003c/em\u003e information resource (TAIR) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The genomic data of alfalfa\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\"Xinjiang Da Ye\" were taken from the Alfalfa Genome 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). \u003cem\u003eMedicago truncatula\u003c/em\u003e genome-wide\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003edata are derived from the online website (). The conserved\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003edomain (PF06217) file of the \u003cem\u003eBPC\u003c/em\u003e protein is downloaded from the Pfam database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pfam.xfam.org/).(Finn\u003c/span\u003e\u003cspan address=\"https://pfam.xfam.org/).(Finn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e et al.2014) The \u003cem\u003eBPC\u003c/em\u003e protein sequence of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e was used as the query sequence, and the blastp function in TBtools was used to compare the alfalfa protein database to identify the \u003cem\u003eMsBPC\u003c/em\u003e gene. At the same time, the e-value\u0026thinsp;\u0026le;\u0026thinsp;e\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e was set by the hmmsearch command of HMMER to\u003c/p\u003e\u003cp\u003escreen out the \u003cem\u003eBPC\u003c/em\u003e gene containing the typical domain of the \u003cem\u003eBPC\u003c/em\u003e family. The protein sequences of candidate genes obtained from BLAST were submitted to NCBI-CDD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/cdd\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/cdd\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and SMART (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl-heidelberg.de/a\u003c/span\u003e\u003cspan address=\"http://smart.embl-heidelberg.de/a\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for further identification and removal of\u003c/p\u003e\u003cp\u003eredundant sequences (Bork et al.1998). Finally, candidate genes are determined.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eAnalysis of Physicochemical Properties of gene Family members\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eAnalysis of Physicochemical Properties of \u003cem\u003eMsBPC\u003c/em\u003e gene Family members\u003c/div\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe genomic sequences, chromosomal locations and protein sequence lengths of \u003cem\u003eMsBPC\u003c/em\u003e genes were extracted from the \u0026lsquo;Xinjiang Daye\u0026rsquo; reference genome GFF file using TBtools software. The \u003cem\u003eMsBPC\u003c/em\u003e protein sequences were subsequently submitted to the online WoLF PSORT tool (\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) for subcellular localization prediction and determination of theoretical isoelectric points (pI) and molecular weights. Chromosomal mapping of \u003cem\u003eMsBPC\u003c/em\u003e genes was performed using TBtools, followed by systematic renaming based on their chromosomal location.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic analysis and Motif Analysis of \u003cem\u003eMsBPC\u003c/em\u003e\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eProtein sequences of \u003cem\u003eMsBPC\u003c/em\u003e were obtained from the UniProt database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for constructing a phylogenetic tree. Using the MEGA11 software, multiple sequence alignments of the protein sequences were performed, and a phylogenetic tree was constructed using maximum likelihood estimation (NJ) with specific parameters set to the Poisson model, and a Bootstrap repetition of 1000 times (Tamura et al.2021). The MEME online program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme.nbcr.net/meme/intro.html\u003c/span\u003e\u003cspan address=\"http://meme.nbcr.net/meme/intro.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to obtain conserved motifs of the \u003cem\u003eMsBPC\u003c/em\u003e protein, with parameters set to default values, defining the number of conserved motifs as 10, and restricting the optimal motif width between 6 to 50 residues.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGene Duplication Events and collinearity analysis of gene family\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eGene Duplication Events and collinearity analysis of \u003cem\u003eMsBPC\u003c/em\u003e gene family\u003c/div\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe MCScanX tool was used to analyze the collinearity information of \u003cem\u003eBPC\u003c/em\u003e genes within and between species (Guo et al.2012) Finally, the results of the collinearity information were visualized using TBtools (Debarry et al.2012). Additionally, the Ka/Ks Calculator function in TBtools was employed to calculate the number of nonsynonymous substitutions per nonsynonymous site (Ka) and the number of synonymous substitutions per synonymous site (Ks), as well as their ratio, to infer the selection pressure during the gene evolutionary process\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eAnalysis of acting elements in the Promoter of Gene\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eAnalysis of \u003cem\u003eCis-\u003c/em\u003eacting elements in the Promoter of \u003cem\u003eMsBPC\u003c/em\u003e Gene\u003c/div\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe TBtools tool was used to extract the 2000 bp sequence upstream of the start codon of the \u003cem\u003eMsBPC\u003c/em\u003e gene as the promoter region. The online website PlantCARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"https://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed to predict the types and numbers of \u003cem\u003ecis-\u003c/em\u003eacting elements in the promoter region (D\u0026eacute;hais et al.2002). Visualization of these \u003cem\u003ecis-\u003c/em\u003eacting elements was conducted using the GSDS2.0 website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.gao-lab.org/\u003c/span\u003e\u003cspan address=\"http://gsds.gao-lab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eTranscript abundance analysis using RNA-seq\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTranscriptome Data Transcriptome data of six different tissues (roots, root nodules, elongating stems, short stems, flowers, and leaves) of \u003cem\u003eMedicago sativa\u003c/em\u003e were downloaded from the NCBI public database (SRP055547). Additionally, transcriptomic data of \u003cem\u003eMedicago sativa\u003c/em\u003e under salt, cold, and drought stress conditions (SRR7091780\u0026thinsp;~\u0026thinsp;SRR7091794, SRR7160313\u0026thinsp;~\u0026thinsp;SRR7160357) were utilized to analyze the tissue-specific expression patterns of \u003cem\u003eMsBPC\u003c/em\u003e and to investigate the expression changes of \u003cem\u003eMsBPC\u003c/em\u003e under stress conditions.(Bucciarelli et al.2015; Deng et al.2021) The transcriptome data were processed, and visualization was carried out using the Heatmap of TBtools.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eRT-qPCR analysis of the \u003cem\u003eMsCPP\u003c/em\u003e gene in response to drought and salt stress\u003c/h2\u003e\u003cp\u003eAlfalfa (Zhongmu No. 1) seeds were provided from the Institute of Animal Science of the Chinese Academy of Agricultural Sciences. Whole alfalfa seeds of the same size were selected, sterilized with 5% sodium hypochlorite solution for 10 min, rinsed repeatedly with distilled water for 4\u0026thinsp;~\u0026thinsp;5 times, the seeds were spread flat in a Petri dish containing filter paper, incubated in a light incubator (16 h light/8 h dark) for 28 days, Salt stress was simulated using 250 mM NaCl, with 0 h as the control group, and root tip samples were collected at 0.5, 1, 3, 6, 12, and 24 h. 400 mM mannitol was used to simulate drought stress, and root tip samples were collected at 1, 3, 6, 12 and 24 hours. Each stress treatment was set up with 3 replicates, and each replicate contained 5 seedlings.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTotal RNA was extracted using the TRIzol reagent, and cDNA was synthesized using the EasyScript First-Strand cDNA Synthesis Kit. Primers were designed using Primer 5.0, with the sequences shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The RT-qPCR experiments were conducted using SYBR Premix Ex Taq (Takara, Japan) and a 7500 real-time fluorescence quantitative PCR system (Applied Biosystems, USA). Each sample was set up with three replicates, with the \u003cem\u003eMedicago sativa L.\u003c/em\u003e actin gene (Actin2) used as the internal control. Relative expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method (Harshitha et al.2021) Graph Pad Prism 8 software was used for graphing.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003eMsBPC5\u003c/em\u003e and \u003cem\u003eMsBPC10\u003c/em\u003e Subcellular localization\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe ORF of \u003cem\u003eMsBPC5\u003c/em\u003e and \u003cem\u003eMsBPC10\u003c/em\u003e genes were cloned into the \u003cem\u003eSuper 1300-GFP\u003c/em\u003e vector to construct Super-\u003cem\u003eMsBPC5\u003c/em\u003e-GFP and \u003cem\u003eSuper\u003c/em\u003e-\u003cem\u003eMsBPC10\u003c/em\u003e-\u003cem\u003eGFP\u003c/em\u003e. The vectors \u003cem\u003eSuper-MsBPC5-GFP\u003c/em\u003e, \u003cem\u003eSuper-MsBPC10-GFP\u003c/em\u003e, and the vector control (\u003cem\u003eSuper 1300-GFP\u003c/em\u003e) were introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101 were introduced into Agrobacterium tumefaciens GV3101 and then transiently transformed into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. The fluorescent signals were detected using confocal microscopy (Leica Microsystems, Wetzlar, Germany).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eValidation of Heterologous Expression in Yeast\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe coding sequence of the \u003cem\u003eMsBPC10\u003c/em\u003e gene was initially amplified from the alfalfa cultivar Zhong mu 4 via polymerase chain reaction (PCR). Subsequently, the target \u003cem\u003eMsBPC\u003c/em\u003e10 gene was ligated into the pYES2-NTB expression vector to generate the recombinant plasmid pYES2 -\u003cem\u003eMsBPC\u003c/em\u003e10-NTB. Using the Yeast Colony Rapid Detection Kit (Nanjing Ruian), the correctly constructed pYES2 -\u003cem\u003eMsBPC\u003c/em\u003e10-NTB recombinant plasmid and the empty pYES2-NTB vector were individually introduced into the Saccharomyces cerevisiae strain INVSC 1 through yeast transformation. During the transformation procedure, the yeast cells were resuspended in 200 \u0026micro;L of sterile water and gently mixed before being plated onto the corresponding synthetic dropout (SD) selection media. The plates were then incubated at 30\u0026deg;C for 3\u0026ndash;5 days to allow for colony formation.\u003c/p\u003e\u003cp\u003eFollowing incubation, the correct positive transformants from the experimental group (pYES2- BPC10-NTB) and the negative control group (pYES2-NTB) were identified and selected. These positive transformants were subsequently resuspended in 2 mL of SG-U liquid medium, and the optical density at 600 nm (OD600) was adjusted to 0.6. The cell suspensions were then subjected to serial dilution (10\u003csup\u003e0\u003c/sup\u003e, 10⁻\u0026sup1;, 10⁻\u0026sup2;). The diluted cell suspensions were spotted onto SG-U plates supplemented with varying concentrations of NaCl (0 M, 0.5 M, 1.0 M, 1.3 M, 1.5 M). The plates were incubated at 30\u0026deg;C for 7 days to assess the growth and colony formation under different osmotic stress conditions. At the end of the incubation period, photographs were taken to document and analyze the colony morphology and growth status.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eIdentification of the\u003c/b\u003e \u003cb\u003eBPC\u003c/b\u003e \u003cb\u003eGene Family\u003c/b\u003e \u003cb\u003eMedicago sativa\u003c/b\u003e \u003cb\u003eL\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 18 \u003cem\u003eMsBPC\u003c/em\u003e genes with complete conserved domains were identified in \u003cem\u003eMedicago sativa\u003c/em\u003e, distributed across 14 chromosomes. (Figure.1). Based on the position of the genes on the chromosomes, the \u003cem\u003eBPC\u003c/em\u003e genes were named \u003cem\u003eMsBPC\u003c/em\u003e1 to \u003cem\u003eMsBPC\u003c/em\u003e18. The important characteristics of gene and protein sequences are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table S1. Further analysis indicates significant differences in the protein sequence lengths, molecular weights, and theoretical isoelectric points of the \u003cem\u003eMsBPC\u003c/em\u003e genes. The protein sequence length ranges from 195 amino acids (\u003cem\u003eMsBPC10\u003c/em\u003e) to 339 amino acids (\u003cem\u003eMsBPC\u003c/em\u003e1 to 4), with molecular weights ranging from 22.26 kDa (\u003cem\u003eMsBPC10\u003c/em\u003e) to 37.78 kDa (\u003cem\u003eMsBPC\u003c/em\u003e4), and theoretical isoelectric points ranging from 6.71 (\u003cem\u003eMsBPC10\u003c/em\u003e) to 9.68 (\u003cem\u003eMsBPC\u003c/em\u003e15 to 17). Subcellular localization results show that 7 genes are localized in the nucleus; 4 genes in mitochondria; 2 genes in chloroplasts; 2 genes in cell membranes; \u003cem\u003eMsBPC\u003c/em\u003e3 in the cytoplasm; \u003cem\u003eMsBPC\u003c/em\u003e11 in vacuole; and \u003cem\u003eMsBPC\u003c/em\u003e1 is located extracellular.\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\u003eBasic information analysis of \u003cem\u003eMsBPC\u003c/em\u003e gene family\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\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=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene 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\u003eProtein Length (aa)\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\u003epI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\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.gene27035\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e339\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e37.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eExtracellular\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene24195\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e339\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e37.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene64186\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e339\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e37.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene63981\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC4\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e339\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e37.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene028699\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC5\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e295\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e32.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene31269\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC6\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e280\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e30.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene79458\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC7\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e312\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e35.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePlasma membrane\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene08364\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC8\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e280\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e30.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChloroplast\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene030481\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC9\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e312\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e35.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene030483\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC10\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e195\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e22.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene028203\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC11\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e280\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e30.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eVacuole\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene000525\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC12\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e312\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e35.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMitochondrion\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene023254\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC13\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e280\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e30.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene34978\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC14\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e280\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e30.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePlasma membrane\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene000243\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC15\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e289\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e32.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMitochondrion\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene27413\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC16\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e289\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e32.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChloroplast\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene041646\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC17\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e289\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e32.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMitochondrion\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMS.gene000299\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC18\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e288\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e31.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMitochondrion\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003echr: chromosome; aa: amino acid; MW: molecular weight; pI: isoelectric point\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic analysis and classification of the \u003cem\u003eMsBPC\u003c/em\u003e gene family\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo explore the evolutionary relationship between the \u003cem\u003eBPC\u003c/em\u003e gene of \u003cem\u003eMedicago sativa\u003c/em\u003e L. and the \u003cem\u003eBPC\u003c/em\u003e gene of \u003cem\u003eArabidopsis\u003c/em\u003e, this study utilized MEGA 11 software to construct a phylogenetic tree for \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eMedicago sativa\u003c/em\u003e, which included 18 \u003cem\u003eMsBPC\u003c/em\u003e sequences from \u003cem\u003eMedicago sativa\u003c/em\u003e and 7 At\u003cem\u003eBPC\u003c/em\u003e sequences from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Figure.2). Based on gene structure and conserved motif analyses, the 25 \u003cem\u003eBPC\u003c/em\u003e proteins were divided into three groups: Group I contains 9 members (6 \u003cem\u003eMsBPC\u003c/em\u003e and 3 \u003cem\u003eAtBPC)\u003c/em\u003e, Group II contains 11 members (8 \u003cem\u003eMsBPC\u003c/em\u003e and 3 \u003cem\u003eAtBPC\u003c/em\u003e), and Group III contains 5 members (4 \u003cem\u003eMsBPC\u003c/em\u003e and 1 \u003cem\u003eAtBPC)\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of the Gene Structure and Conserved Motifs of\u003c/b\u003e \u003cb\u003eMsBPC\u003c/b\u003e \u003cb\u003egene family\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further understand the structure of the \u003cem\u003eMsBPC\u003c/em\u003e gene, analyses were conducted using TBtools software and the MEME online platform. The gene structure analysis indicates that the \u003cem\u003eMsBPC\u003c/em\u003e gene is relatively short, with most members containing two exons and one intron. The coding region features a GAGA repeat sequence specifically recognized by \u003cem\u003eBPC\u003c/em\u003e transcription factors. Ten conserved motifs were predicted via the MEME website, ranging in length from 6 to 50 amino acids. The results were visualized using TBtools software (Figure. 3). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e presents detailed information about the ten conserved motifs. The findings show that the \u003cem\u003eMsBPC\u003c/em\u003e family members in alfalfa encompass a varying number of conserved motifs, ranging from 5 to 9, with motif 4 present in all \u003cem\u003eMsBPC\u003c/em\u003e members, while motif 2 was not identified in \u003cem\u003eMsBPC10\u003c/em\u003e, suggesting that these two motifs are highly conserved within \u003cem\u003eMsBPC\u003c/em\u003e proteins. Annotation of motifs 2 and 4 using NCBI's CD search tool revealed that both cover the \u003cem\u003eBPC\u003c/em\u003e gene-specific motif GAGA-bind. Variations in motifs among different subgroups highlight the diversity of functions in \u003cem\u003eMsBPC\u003c/em\u003e proteins. Similarities in motif composition within the same subgroup indicate that these proteins have related functions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene duplication events and chromosomal collinearity analysis of\u003c/b\u003e \u003cb\u003eMsBPC\u003c/b\u003e \u003cb\u003egene family\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo understand the expansion mechanism of the \u003cem\u003eMsBPC\u003c/em\u003e family in alfalfa, the interspecies evolutionary relationship of alfalfa was analyzed by using MCScanX. The results showed that there were 22 segmental duplications events and 1 tandem repeat event between these members (Figure.5 and Table S2). For example, \u003cem\u003eMsBPC7\u003c/em\u003e and \u003cem\u003eMsBPC12\u003c/em\u003e are located on chr4.1 and chr4.3, respectively. Segmental duplications events occur extensively in \u003cem\u003eMsBPC\u003c/em\u003e genes, These results suggest that segmental duplication has played a major role in the expansion of the \u003cem\u003eMsBPC\u003c/em\u003e gene family. In addition, The Ka/Ks of \u003cem\u003eMsBPC8\u003c/em\u003e and \u003cem\u003eMsBPC11\u003c/em\u003e were greater than 0.5 in the collinearity gene pairs, indicating that they underwent positive selection during evolution, while the Ka/Ks of the other gene pairs were less than 0.5 and underwent purification selection during evolution (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo explore the evolutionary relationship of \u003cem\u003eBPC\u003c/em\u003e gene family in different species, a collinearity plot of \u003cem\u003eMedicago sativa\u003c/em\u003e with \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eMedicago truncatula\u003c/em\u003e, and \u003cem\u003eGlycine max\u003c/em\u003e were constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The results showed that 4 \u003cem\u003eMsBPC\u003c/em\u003e genes in alfalfa had a collinearity relationship with \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, and a total of 8 homologous gene pairs were identified. 7 \u003cem\u003eMsBPC\u003c/em\u003e genes were collinearity with \u003cem\u003eMedicago truncatula\u003c/em\u003e, and 7 homologous gene pairs were identified. 15 \u003cem\u003eMsBPC\u003c/em\u003e genes were collinearity with \u003cem\u003eGlycine max\u003c/em\u003e, and 26 homologous gene pairs were identified. It is worth noting that the degree of collinearity between \u003cem\u003eBPC\u003c/em\u003e genes between \u003cem\u003eGlycine max\u003c/em\u003e, and \u003cem\u003eMedicago sativa\u003c/em\u003e is significantly higher than that of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, which may be due to the fact that soybean and alfalfa belong to the same leguminous plant, are more closely related, and the evolutionary pattern of gene families is more similar.\u003c/p\u003e\u003cp\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 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEvolutionary pressures between different gene pairs\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGene 2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eKa\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eKs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eKa/Ks\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMsBPC2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00126609\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.017871285\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.070845\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMsBPC14\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC17\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.320670253\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.136703417\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.150077\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMsBPC14\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC12\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.652030359\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.644770393\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.246536\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMsBPC14\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC13\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.001546791\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.005203837\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.29724\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMsBPC17\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC12\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.681922855\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.096127244\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.22025\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMsBPC17\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC13\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.323082154\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.136703417\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.151206\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMsBPC12\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC13\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.652030359\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.644770393\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.246536\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMsBPC11\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC8\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.006988533\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.013060844\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.535075\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMsBPC11\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC9\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.668794505\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.911712094\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.229691\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMsBPC2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC4\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.002534054\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.022414817\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.113053\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMsBPC6\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMsBPC5\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.003098378\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.010425885\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.297181\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\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of\u003c/b\u003e \u003cb\u003ecis-\u003c/b\u003e\u003cb\u003eacting elements in the promoter regions of\u003c/b\u003e \u003cb\u003eMsBPC\u003c/b\u003e \u003cb\u003egenes family\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo explore the potential function of \u003cem\u003eBPC\u003c/em\u003e gene in alfalfa, the \u003cem\u003ecis-\u003c/em\u003eacting element of \u003cem\u003eMsBPC\u003c/em\u003e gene was predicted using the PlantCARE database (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and Table S3). The results showed that a total of 13 \u003cem\u003ecis-\u003c/em\u003eacting elements related to hormone response, stress response, and growth and development were screened. These elements include ABRE elements related to abscisic acid response, P-box, GARE-motif and TATC-box elements related to gibberellin response, TCA-element element related to salicylic acid response, TGACG-motif and CGTCA-motif elements related to methyl jasmonate response, LTR element related to low temperature response, and MBSI element related to the regulation of flavonoid biosynthesis. The \u003cem\u003ecis-\u003c/em\u003eacting elements of each gene are unevenly distributed, which may be the reason why each gene has a different function.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of the expression in different tissues of the\u003c/b\u003e \u003cb\u003eMsBPC\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eMedicago sativa\u003c/b\u003e \u003cb\u003eL.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe expression level data of six tissues, including roots, leaves, flowers, elongated stems, Pre-elongated stems, and nodules of \u003cem\u003eMedicago sativa\u003c/em\u003e, were obtained from public databases. The analysis results showed that 7 genes were expressed in all 6 tissues, 4 genes were only expressed in a single tissue, and the remaining genes were expressed in 3\u0026ndash;5 tissues, suggesting that \u003cem\u003eMsBPC\u003c/em\u003e genes may be involved in the regulation of different growth and developmental stages of \u003cem\u003eMedicago sativa\u003c/em\u003e. (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and Table S4)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression of\u003c/b\u003e \u003cb\u003eMsBPC\u003c/b\u003e \u003cb\u003egenes under abiotic stress response\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo explore the potential regulatory mechanisms of the \u003cem\u003eMsBPC\u003c/em\u003e genes under different stresses, the RNA-seq data of alfalfa plants under the abiotic stresses (salt, drought, cold) were analyzed The results showed that 12 \u003cem\u003eMsBPC\u003c/em\u003es could respond to salt stress. 7 \u003cem\u003eMsBPC\u003c/em\u003es could respond to drought stress. 12 \u003cem\u003eMsBPC\u003c/em\u003e genes could respond to cold stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e and Table S5). These results suggest that \u003cem\u003eBPC\u003c/em\u003e transcription factors play an important role in alfalfa response to abiotic stresses.\u003c/p\u003e\u003cp\u003eTo clarify validate the RNA-seq data, 2 genes were selected for RT-qPCR analysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, the expression of \u003cem\u003eMsBPC10\u003c/em\u003e decreased first, then increased, and finally decreased under drought stress. The expression of \u003cem\u003eMsBPC5\u003c/em\u003e decreased first and then increased, but was always lower than that of the control group. Under salt stress, the expression of \u003cem\u003eMsBPC5\u003c/em\u003e decreased first and then increased. The expression of \u003cem\u003eMsBPC10\u003c/em\u003e first increased, then decreased, and then increased, and was always higher than that of the control group. RT-qPCR results were consistent with RNA-seq data.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003eMsBPC5\u003c/em\u003e and \u003cem\u003eMsBPC10\u003c/em\u003e Subcellular localization\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe GV3101 strains harboring \u003cem\u003eSuper-MsBPC5-GFP\u003c/em\u003e and \u003cem\u003eSuper-MsBPC10-GFP\u003c/em\u003e were transiently transformed into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e cells. the results show that both \u003cem\u003eMsBPC5\u003c/em\u003e and \u003cem\u003eMsBPC10\u003c/em\u003e are localized in the nucleus. (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). the results are consistent with previous predictions.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMsBPC10\u003c/b\u003e \u003cb\u003eimprove Salt Tolerance in Yeast\u003c/b\u003e\u003c/p\u003e\u003cp\u003eQuantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis conducted after treatment with 200 mM NaCl demonstrated that the transcriptional level of \u003cem\u003eMsBPC10\u003c/em\u003e initially increased, subsequently decreased, and then increased again relative to the control group (CK). These results suggest that the protein encoded by \u003cem\u003eMsBPC10\u003c/em\u003e may play a pivotal role in the salt stress response of \u003cem\u003eMedicago sativa\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eTo elucidate the effects of \u003cem\u003eMsBPC10\u003c/em\u003e on yeast growth and salt tolerance, the pYES2-BPC10-NTB construct was introduced into Saccharomyces cerevisiae cells. Under standard growth conditions, no significant differences in growth were observed between the transformed yeast strains and the corresponding empty vector control strains. However, when subjected to 1.5 M NaCl stress, the transformed yeast cells exhibited significant tolerance to high salinity. Notably, this enhanced salt tolerance was particularly evident at the 10⁻\u003csup\u003e2\u003c/sup\u003e dilution level (Figure.12).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eBPC\u003c/em\u003e transcription factors are an important gene family in plants. They're known to regulate plant growth, development, and abiotic stress responses, as shown in many studies. This family has been identified in several plants, like \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Bai et al.2017), \u003cem\u003eOryza sativa\u003c/em\u003e (Cao et al.2018), \u003cem\u003eCucumis sativus\u003c/em\u003e (Huang et al.2019), and \u003cem\u003eChinese cabbage\u003c/em\u003e (Feng et al.2023), with 7, 4, 4, and 12 members found respectively. But in tetraploid \u003cem\u003eMedicago sativa\u003c/em\u003e, this family hasn't been reported yet. In this study, focusing on tetraploid \u003cem\u003eMedicago sativa\u003c/em\u003e, we found 18 \u003cem\u003eMsBPC\u003c/em\u003e genes. This number is much higher than the \u003cem\u003eBPC\u003c/em\u003e members reported in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eCucumis sativus\u003c/em\u003e, and \u003cem\u003eChinese cabbage\u003c/em\u003e. This might be because \u003cem\u003eMedicago sativa\u003c/em\u003e is a tetraploid with many homologous genes in its genome.\u003c/p\u003e\u003cp\u003eWe carried out a systematic analysis of the amplification and evolution of the \u003cem\u003eMsBPC\u003c/em\u003e gene family in \u003cem\u003eMedicago sativa\u003c/em\u003e. Compared with other species, the \u003cem\u003eMsBPC\u003c/em\u003e family of alfalfa underwent significant gene family expansion, and fragment replication and tandem replication were one of the driving forces of gene family expansion (Danilevskaya et al.2016;Baumgarten et al.2004). In this study, We detected 22 segmental and 1 tandem duplication events in the \u003cem\u003eMsBPC\u003c/em\u003e gene family suggesting a segmental duplication dominated evolutionary pattern. Tissue-specific expression analysis is an important method for predicting the biological function of genes in plant growth and development. Based on RNA-seq data, we systematically analyzed the expression patterns of 18 \u003cem\u003eMsBPC\u003c/em\u003e genes in 6 different tissues. The results showed that 7 genes were expressed in all 6 tissues,4 in just one tissue, and the remaining 7 in 3\u0026ndash;5 tissues. This indicates \u003cem\u003eMsBPC\u003c/em\u003e genes likely regulate different growth and developmental stages of \u003cem\u003eMedicago sativa.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eCis\u003c/em\u003e - acting elements in promoter regions determine the spatiotemporal specificity of gene expression. They interact with corresponding transcription factors in different tissues and cells, enabling precise gene expression in specific tissues, developmental stages, and environmental conditions. (Dai et al.2014;Conery et al.2000). \u003cem\u003eCis-\u003c/em\u003eelement analysis showed that \u003cem\u003eMsBPC\u003c/em\u003e genes have diverse \u003cem\u003ecis-\u003c/em\u003eelements, indicating complex regulation and possibly explaining the different expression patterns of these genes across tissues and under various abiotic stresses. in this study, \u003cem\u003eMsBPC\u003c/em\u003e gene promoters were found to contain \u003cem\u003ecis-\u003c/em\u003eelements related to hormone responses (gibberellin, salicylic acid, abscisic acid, and auxin) and abiotic stress responses (defense/stress, drought induction, and low-temperature responses). These results suggest that \u003cem\u003eMsBPC\u003c/em\u003e may participate in plant responses to environmental stresses through multiple pathways, consistent with findings in cucumber, apple, and Chinese cabbage. (Huang et al.2019; Chen et al.2020; Huang et al.2024).\u003c/p\u003e\u003cp\u003eStudies have shown that \u003cem\u003eBPC\u003c/em\u003e genes play an important role in regulating plant growth, development, and responses to abiotic stress. Their functions are both conserved and diverse across different species. In \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eOsGBP1\u003c/em\u003e negatively regulates grain length and seedling development by regulating the expression of grain shape - related genes. Overexpressing \u003cem\u003eOsGBP1\u003c/em\u003e inhibits seedling growth, causes leaf yellowing, reduces biomass, and delays flowering, while gene silencing or mutation promotes plant growth (Cao et al.2018).In \u003cem\u003eMalus domestica\u003c/em\u003e, overexpression of \u003cem\u003eMdBPC2\u003c/em\u003e decreases auxin content and inhibits plant growth and root development, while exogenous application of auxin can restore normal growth(Chen et al.2020).Overexpression of \u003cem\u003eCsBPC2\u003c/em\u003e in tobacco significantly inhibited seed germination under salt, polyethylene glycol, and abscisic acid stress (Huang et al.2019). In Chinese cabbage, \u003cem\u003eBcBPC9\u003c/em\u003e enhances antioxidant enzyme gene expression, increasing cadmium stress tolerance in yeast and tobacco (Huang et al.2024). In addition, \u003cem\u003eBPC\u003c/em\u003e transcription factors regulate Arabidopsis ovule development by controlling the \u003cem\u003eSTK\u003c/em\u003e gene. Mutations cause dwarfism, leaf curling, ovule development abnormalities, and reduced lateral roots (Airoldi et al.2005). These studies show that while \u003cem\u003eBPC\u003c/em\u003e genes have different specific functions in different species, they mainly focus on regulating growth, development, and responses to abiotic stress.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis study systematically identified a total of 18 \u003cem\u003eMsBPC\u003c/em\u003e genes, which are distributed across 14 chromosomes. Phylogenetic analysis divided them into three evolutionary groups. Each group has similar gene structures and motif compositions. Segmental duplication was found to be the main driver of \u003cem\u003eMsBPC\u003c/em\u003e gene family expansion. Expression profiling revealed that \u003cem\u003eMsBPC\u003c/em\u003e genes exhibited different expression patterns across tissues: 7 were expressed in all 6 tissues, 4 were only expressed in a single tissue, and the remaining genes were expressed in 3\u0026ndash;5 tissues, suggesting that \u003cem\u003eMsBPC\u003c/em\u003e genes may be involved in the regulation of different growth and developmental stages of alfalfa. Stress response analysis revealed that 12, 11, and 12 \u003cem\u003eMsBPC\u003c/em\u003e genes were significantly responsive to salt, drought, and cold stress, respectively. Promoter \u003cem\u003ecis\u003c/em\u003e - element analysis revealed the potential regulatory mechanisms of \u003cem\u003eMsBPC\u003c/em\u003e genes in hormone and stress responses. Collinearity analysis showed a higher collinearity between \u003cem\u003eMedicago sativa\u003c/em\u003e and \u003cem\u003eGlycine max BPC\u003c/em\u003e genes than \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, BPC family evolution is relatively conserved in leguminous plants. indicating a more similar evolutionary pattern of the \u003cem\u003eBPC\u003c/em\u003e gene family in leguminous plants. Subcellular localization assays showed \u003cem\u003eMsBPC5\u003c/em\u003e and \u003cem\u003eMsBPC10\u003c/em\u003e to be nuclear-localized, consistent with predictions. Yeast heterologous expression verified the salt tolerance function of \u003cem\u003eMsBPC10\u003c/em\u003e.The findings of this study provide theoretical support for elucidating \u003cem\u003eMsBPC\u003c/em\u003e gene functions and offer important candidate genes for stress-resistant breeding in \u003cem\u003eMedicago sativa.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBPC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e/BASIC PENTACYSTEINE\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMW\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emolecular weight\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHMM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehidden Markov modeling\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003epI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eisoelectric points\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eaa\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eamino acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNJ\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNeighbor-joining\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eABRE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eabscisic acid responsiveness\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003edrought-responsive element\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLTR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003elow-temperature-responsive element\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTC-rich repeats\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003estress-defense-responsive regulatory element\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLi zhao, Xianyang Li, Hao Liu and Yuqi Zhang conceived and designed the research framework. Xinyue Ma, and Fei He were responsible for manuscript composition and preparation. Mingna Li and Xue Wang executed the experimental procedures. Ruicai Long, Junmei Kang, and Qingchuan Yang performed data analysis and interpretation. Lin Chen and Changhong Guo supervised the entire research project and provided scientific direction. All authors thoroughly reviewed the final manuscript and provided formal approval for its 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 “The Open Competition” 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"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAcharya JP, Lopez Y, Gouveia BT et al (2020) Breeding Alfalfa (\u003cem\u003eMedicago sativa\u003c/em\u003e L.) Adapted to Subtropical Agroecosystems. Agronomy 10:742\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAiroldi CA, Kooiker M, Losa A et al (2005) BASIC PENTACYSTEINE1, a GA binding protein that induces conformational changes in the regulatory region of the homeotic Arabidopsis gene SEEDSTICK. Plant Cell 17(3):722\u0026ndash;729\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBai L, Liu Y, Mu Y et al (2017) Cucumber \u003cem\u003eCsBPCs\u003c/em\u003e Regulate the Expression of \u003cem\u003eCsABI3\u003c/em\u003e during Seed Germination. Front Plant Sci 8:459\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaumgarten A, Cannon SB, Mitra A et al (2004) The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol 4:10\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBerger N, Dubreucq B (2012) Evolution goes GAGA: GAGA binding proteins across kingdoms. Biochim Biophys Acta 1819(8):863\u0026ndash;868\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBlack R, Ecker JR, Lehman A (1996) HOOKLESS1, an ethylene response gene, is required for differential cell elongation in the Arabidopsis hypocotyl. Cell 85(2):183\u0026ndash;194\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBork P, Milpetz F, Schultz J et al (1998) SMART, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci USA 95(11):5857\u0026ndash;5864\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrand LH, Hecker A, Peter et al (2015) The Arabidopsis GAGA-Binding Factor BASIC PENTACYSTEINE6 Recruits the POLYCOMB-REPRESSIVE COMPLEX1 Component LIKE HETEROCHROMATIN PROTEIN1 to GAGA DNA Motifs. Plant Physiol 168(3):1013\u0026ndash;1024\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBucciarelli B, Fu F, O'Rourke JA et al (2015) The Medicago sativa gene index 1.2: a web-accessible gene expression atlas for investigating expression differences between Medicago sativa subspecies. BMC Genomics 16(1):502\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCao H, Gong R, Zhang J et al (2018) Divergent functions of the GAGA-binding transcription factor family in rice. Plant journal: cell Mol biology 94(1):32\u0026ndash;47\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen H, Yang Y, Zeng Y et al (2020) Allele-aware chromosome-level genome assembly and efficient transgene-free genome editing for the autotetraploid cultivated alfalfa. Nat Commun 11(1):2494\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen X, Ding Y, Yang Y et al (2021) Protein kinases in plant responses to drought, salt, and cold stress. J Integr Plant Biol 63(1):53\u0026ndash;78\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eConery JS, Lynch M (2000) The evolutionary fate and consequences of duplicate genes. Sci (New York N Y) 290(5494):1151\u0026ndash;1155\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDai W, Liu JH, Peng T (2014) Critical \u003cem\u003ecis\u003c/em\u003e-Acting Elements and Interacting Transcription Factors: Key Players Associated with Abiotic Stress Responses in Plants. Plant Mol Biol Rep 32:303\u0026ndash;317\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDanilevskaya ON, Meng X, Thatcher SR et al (2016) Genome-Wide Analysis of Alternative Splicing during Development and Drought Stress in Maize. Plant Physiol 170(1):586\u0026ndash;599\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDebarry JD, Tang H, Wang Y et al (2012) MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res, 40(7), e49\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eD\u0026eacute;hais P, Lescot M, Thijs G et al (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30(1):325\u0026ndash;327\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeng H, Dong X, Ma W et al (2021) Genome-wide identification of the MADS-box transcription factor family in autotetraploid cultivated alfalfa (\u003cem\u003eMedicago sativa\u003c/em\u003e L.) and expression analysis under abiotic stress. BMC Genomics 22(1):603\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFeng Y, Wang J, Zhang S et al (2023) Genome-wide identification of flowering Chinese cabbage BPC family genes and BcBPC9 functional analysis in Cd stress tolerance[J]. Plant Stress 10:100\u0026ndash;120\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFichman., Mittler R, Zandalinas SI et al (2022) Reactive oxygen species signalling in plant stress responses. Nat Rev Mol Cell Biol 23(10):663\u0026ndash;679\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFinn RD, Bateman A, Clements J et al (2014) Pfam: the protein families database. Nucleic Acids Res 42:D223\u0026ndash;D230\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFoley RC, Singh K et al (2002) Transcription factors in plant defense and stress responses. Curr Opin Plant Biol 5(5):430\u0026ndash;436\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGong Z, Shi H, Xiong L et al (2020) Plant abiotic stress response and nutrient use efficiency. Sci China Life Sci 63(5):635\u0026ndash;674\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGregis V, Simonini R-V, S., I., et al (2012) Basic pentacysteine proteins mediate MADS domain complex binding to the DNA for tissue-specific expression of target genes in Arabidopsis. Plant Cell 24(10):4163\u0026ndash;4172\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHarshitha R, Arunraj DR (2021) Real-time quantitative PCR: A tool for absolute and relative quantification. Biochem Mol Biol 49:800\u0026ndash;812\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHu Z, Ma X, Yu Y et al (2022) Characterizations of a Class-I BASIC PENTACYSTEINE Gene Reveal Conserved Roles in the Transcriptional Repression of Genes Involved in Seed Development. Curr Issues Mol Biol 44(9):4059\u0026ndash;4069\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang B, Li S, Miao L et al (2019) Genome-Wide Identification and Characterization of Cucumber BPC Transcription Factors and Their Responses to Abiotic Stresses and Exogenous Phytohormones. Int J Mol Sci 20(20):5048\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang Y, Wan S, Zhao H et al (2024) The transcription factor \u003cem\u003eMdBPC2\u003c/em\u003e alters apple growth and promotes dwarfing by regulating auxin biosynthesis. Plant Cell 36(3):585\u0026ndash;604\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang B, Li S, Miao L et al (2019) Genome-Wide Identification and Characterization of Cucumber BPC Transcription Factors and Their Responses to Abiotic Stresses and Exogenous Phytohormones. Int J Mol Sci 20(20):5048\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKater MM, Simonini S (2014) Class I BASIC PENTACYSTEINE factors regulate HOMEOBOX genes involved in meristem size maintenance. J Exp Bot 65(6):1455\u0026ndash;1465\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKumar S, Stecher G, Tamura K (2021) MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol 38(7):3022\u0026ndash;3027\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi L, Shen GZ, Zhang JL et al (2007) Ectopic expression of OsLFL1 in rice represses Ehd1 by binding on its promoter. Biochem Biophys Res Commun 360(1):251\u0026ndash;256\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi L, Peng LT, Shi ZY et al (2008) Overexpression of transcription factor OsLFL1 delays flowering time in Oryza sativa. J Plant Physiol 165(8):876\u0026ndash;885\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu J, Long S, Zhang W et al (2021c) Maintenance of Cell Wall Integrity under High Salinity. Int J Mol Sci 22(6):3260\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu M, Zhang Q, Zhao S et al (2021b) Regulation of Plant Responses to Salt Stress. Int J Mol Sci 22(9):4609\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu Y, Yan J, Yang L et al (2021a) Cell wall β-1,4-galactan regulated by the BPC1/BPC2-GALS1 module aggravates salt sensitivity in Arabidopsis thaliana. Mol Plant 14(3):411\u0026ndash;425\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeister RJ, Monfared MM, Williams LA et al (2004) Definition and interactions of a positive regulatory element of the Arabidopsis INNER NO OUTER promoter. Plant journal: cell Mol biology 37(3):426\u0026ndash;438\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeister RJ, Monfared MM, Simon MK et al (2011) Overlapping and antagonistic activities of BASIC PENTACYSTEINE genes affect a range of developmental processes in Arabidopsis. Plant journal: cell Mol biology 66(6):1020\u0026ndash;1031\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMonfared MM, Simon MK et al (2011) Overlapping and antagonistic activities of BASIC PENTACYSTEINE genes affect a range of developmental processes in Arabidopsis. Plant journal: cell Mol biology 66(6):1020\u0026ndash;1031\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePontes LDS, Soussana JF, Louault F et al (2007) Leaf traits affect the above ground productivity and Blackwell Publishing Ltd quality of pasture grasses. Funct Ecol 21:844\u0026ndash;853\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSanti L, Stile MR, Wang Y et al (2003) The GA octodinucleotide repeat binding factor BBR participates in the transcriptional regulation of the homeobox gene Bkn3. Plant journal: cell Mol biology 34(6):813\u0026ndash;826\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"BPC genes, Medicago sativa L., Functional Verification, Salt Tolerance","lastPublishedDoi":"10.21203/rs.3.rs-7468142/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7468142/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"This study systematically analyzed the BPC family in alfalfa (Medicago sativa) for the first time, identifying 18 MsBPC genes randomly distributed on 14 chromosomes. Their encoded proteins are mostly alkaline and classified into three subgroups. Cis-acting element analysis of promoters showed that the promoter region of MsBPC contains various cis-acting elements related to hormone response, growth and development, as well as stress response. Expression pattern analysis in different tissues revealed that 7 were expressed in all 6 tissues, 4 were only expressed in a single tissue, and the remaining genes were expressed in 3-5 tissues, suggesting that MsBPC genes may be involved in the regulation of different growth and developmental stages of Medicago sativa. Transcriptome analysis under salt, drought, and cold stresses showed that 12, 11, and 12 genes responded, respectively. RT-qPCR detection confirmed that MsBPC genes responded to salt and drought treatments, further verifying their important roles in abiotic stress responses Subcellular localization analysis revealed that MsBPC5 and MsBPC10 are localized in the nucleus, which is consistent with the predicted results. heterologous expression in yeast was employed to characterize the function of MsBPC10, which was upregulated in response to salt stress. The study conducted a comprehensive identification and analysis of the BPC gene family in Medicago sativa. Through the analysis of RNA-seq data, candidate genes related to abiotic stress were screened out, providing candidate genes for Medicago sativa stress-resistant breeding.","manuscriptTitle":"Characterization of the BPC Genes in Alfalfa and Functional Verification of MsBPC10 in Salt Tolerance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-18 09:42:49","doi":"10.21203/rs.3.rs-7468142/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2ff2a52f-db22-480a-a432-f90b15774c2e","owner":[],"postedDate":"September 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-06T11:38:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-18 09:42:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7468142","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7468142","identity":"rs-7468142","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}