Genome-wide identification of LcC2DPs gene family in Lotus corniculatus provides insights into regulatory network in response to abiotic stresses

Genome-wide identification of LcC2DPs gene family in Lotus corniculatus provides insights into regulatory network in response to abiotic stresses | 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 Article Genome-wide identification of LcC2DPs gene family in Lotus corniculatus provides insights into regulatory network in response to abiotic stresses Guangfen Yang, Yujie Liu, Zouxian Gong, Siya Chen, Li Song, Shihui Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5763738/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Low temperatures and drought reduce forage yield and quality, with protein kinases crucial for plant stress response. This study examines the LcC2DPs protein kinase family in Lotus corniculatus , identifying 90 members, with some tandemly distributed on chromosomes 2–6, and grouped into 5 subfamilies (I-V). 34 homologous gene pairs were found in Arabidopsis thaliana . LcC2DP genes promoters contain hormone and stress response elements. GO analysis highlights enrichment in hormone response and kinase activity. Transcriptomic analysis links 78 genes to environmental response and stress growth, with 10 validated by qRT-PCR after treatment with 100 µM ABA and IAA, 20% PEG6000, and 4°C. Protein interaction analysis identifies 5 core proteins (LcC2DP5, 11, 15, 38, and 58) activated by drought and cold stress. Gene analysis revealed that only LcC2DP5 and LcC2DP15 share co-expression transcription factors, with bZIP , bHLH , WRKY , NAC , MYB_related , MYB , C3H , and C2H2 being prominent. These proteins are expressed under drought and cold conditions, highlighting LcC2DP5 and LcC2DP15 activity. NAC and C2H2 are vital for drought response, while bZIP and MYB_related are important for cold response. This suggests that various LcC2DPs in Lotus corniculatus respond to hormones and stress via a TF regulatory network. Biological sciences/Computational biology and bioinformatics Biological sciences/Plant sciences calcium-dependent protein kinase C2 domain Lotus corniculatus Signal transduction Drought response Cold stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Global climate change is increasing the frequency and severity of extreme weather, like droughts and cold spells 1 – 2 , which threaten ecosystems, agriculture, and forage production 3 – 4 . These conditions disrupt water supply, hinder forage growth, thus reduce yield and quality, sometimes leading to plant death 5 . For instance, summer droughts with high temperatures can cut forage yield by about 30% 6 . In plant cells, genes and their products manage signal perception and transduction, activating downstream genes to protect against stress 7 . Calcium-dependent protein kinases (CDPKs) are vital in forage grasses’ response to abiotic stress 8 , acting as key signaling effectors. They detect environmental changes and trigger intracellular pathways 9 . Under drought and low temperature stress, CDPKs enhance forage grass adaptability by influencing stomatal movement, antioxidant enzymes, osmoprotectants synthesis, and gene expression 9 – 10 . Protein kinases, through phosphorylation and dephosphorylation, are crucial for plant response to abiotic stresses such as drought and low temperature 11 . Phosphorylation of protein kinases amplifies intracellular signal cascades by altering its charge, conformation, and function, while dephosphorylation removes foreign signals in plant pathways 12 . Most protein kinases are calcium-dependent, playing a role in calcium signal responses 13 . Calcium-dependent (CD) factors, such CDPK, are crucial in plant stress responses, growth, and development 14 . Studies indicate that cells detect signals via Receptor-like kinase (RLK) and membrane hardening, which then activate Ca 2+ channels to raise cytoplasm Ca 2+ levels 14 . Ca 2+ receptors like Ca 2+ -dependent protein kinase (CDPK), Calmodulin (CaM), Calmodulin-like proteins (CMLs), and Calcineurin B-like proteins (CBLs), facilitate signal transduction 13 . CDPK with a C2 domain, crucial for binding Ca 2+ , are extensively involved in regulating cell signaling, perception, conduction, gene expression, growth, and development 15 . Additionally, hormones, particularly Abscisic acid (ABA), are vital in plant responses to various stresses, including low temperature 16 . Research indicates that ABA boosts salt and drought resistance in Suaeda liaotungensis through working with jasmonic acid (JA) under cold stress 17 . MAPK protein kinase is key in the signal transduction of indole-3-acetic acid (IAA) 18 , where G protein activates intracellular signals after IAA is detected by cell membrane receptors 19 , can be to induce the stransduction of IAA. Transcription factors (TFs) play a pivotal role in helping plants respond to harsh environments by binding to specific promoter and adjusting gene expression 20 . TFs generally have two domains: the DNA-binding domain (DBD) and the transcriptional activation or inhibition domain 21 . Based on DBD structure, plant TFs are categorized into families like MYB , WRKY , GARP , TCP , bZIP , bHLH , and HSF , etc. 22 . Among them, bHLH 23 , NAC 24 , MYB_related 25 , MYB 26 , C3H , and C2H2 27 are crucial in regulating plant stress responses to drought, low temperature, and salt. C2 domain protein (C2DP), a class of calcium-dependent binding proteins in eukaryotes, is involved in stress response and growth regulation 28 – 33 . CDPK proteins with C2 domain play a crucial role in plant growth, development, and stress responses like drought 34 . While their functions have been studied in model plants like Arabidopsis ( Arabidopsis thaliana ) 28 , tobacco ( Nicotiana tabacum ) 29 , and rice ( Oryza sativa ) 31 , they are under-researched in forage crops Lotus corniculatus , also known as five-leaf grass or cowhorn, is a high-quality perennial legume with strong stress resistance, widely cultivated for forage and soil conservation 35 – 36 . This study identified and characterized the LcC2DPs protein kinase gene family, analyzing their expression in response to ABA and IAA hormone treatments and stress conditions like drought and low temperature. The findings offer insights and potential gene candidates for enhancing stress resistance in Lotus corniculatus breeding. Materials and methods Plant materials Lotus corniculatus was planted at a key laboratory's experimental base of Education Ministry at Guizhou University (Guiyang, Guizhou, 106° 675 'E; 26° 427 ' N). Family members identification The research utilized genome and protein sequences, and annotation data from a Lotus corniculatus database ( http://www.kazusa.or.jp/lotus/ ) The Hidden Markov Model (HMM) profile (version 3.3) (PF00168) was used to identify C2DPs proteins by searching the Pfam database 37 . An initial domain search with an E-value < 1×10 − 10 was conducted to create a species-specific model for the Lotus corniculatus genome protein sequence. C2 domain protein sequences of Lotus corniculatus were then submitted to the Conserved Domain Database (CDD) ( https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi ), and screened with SMART ( http://smart.embl-heidelberg.de ) and Pfam ( http://pfam.xfam.org/ ) to exclude sequences without C2 domain 38 . This process identified high-cofidence C2 domain family proteins in Lotus corniculatus from all non-redundant genes. Physicochemical and molecular analysis Expasy ( https://web.expasy.org/protparam ) and WoLF PSORT ( https://wolfpsort.hgc.jp ) software were used to analyze theoretical molecular weight (MW) 39 , isoelectric point (pI), and subcellular localization. MEME Suite 5.5.0 ( https://meme-suite.org/meme/tools/meme ) identified conserved motifs in Lotus corniculatus C2DP genes, with a maximum of 10 motifs 39 . TBtools' Visualize Gene Structure examined intron and exon regions of LcC2DPs , TBtools' Visualize Gene Structure examined intron and exon regions, and Gene Location Visualize from GFF mapped chromosome localization. A phylogenetic tree was created using the Neighbor-Joining (NJ) algorithm in MEGA 7.0, and final plot was visualized with iTOL ( https://itol.embl.de/ ) 5 . Collinearity analysis was performed with MCScanX. TBtools aided in visualizing collinearity analysis. A 2000 bp upstream DNA sequence from LcC2DPs was used for promoter analysis, with cis -acting elements predicted via the PlantCARE database ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html ) 40 . Function prediction GO and KEGG analyses were conducted using the DAVID tool ( https://david.ncifcrf.gov/ ), and figures were created with Microscopic Letter ( https://bioinformatics.com.cn/ ). A Lotus corniculatus protein-protein interaction network was constructed by importing Lotus corniculatus C2DPs sequences into the STRING database ( https://string-db.org/ ), saved as a TSV file, and visualized the PPI network with Cytoscape software. Using the Japonicus plant database ( https://planttfdb.gao-lab.org/prediction.php ) 41 . A Python script analyzed gene transcriptome FPKM values, and a coexpression network was built with DEGs and TF data Cytoscape v3.7.10 software was used for visual analysis. Gene expression analysis To study LcC2DP gene expression, 12-day-old hydroponic seedlings with healthy rhizomes were transplanted into soil for 2 weeks culture. During this time, they underwent drought, low temperature, and hormone treatment. Drought stress was simulated by applying respectively carried out during this period, 500 ml 20% PEG solution daily for 15 days. The Lotus corniculatus seedlings were placed in a growth incubator (Ningbo Southeast Instruments Co., Ltd.) at 4°C for 72 hours. For 15 days, 100 umol IAA and ABA were applied to the leaves and soil twice daily. Except for the low temperature group, the culture conditions were maintained at 25 ℃ with a 16-hour light and 8-hour dark cycle, 60% humidity, and 20000 Lk light intensity. Total RNA was extracted from each sample (drought and hormone, 0, 3, 6, 9,12, and 15 days; low temperature, 0, 6, 12, 24, 48, and 72 hours) using the OMEGA Plant RNA Kit (OMEGA, Guangzhou, China), and approximately 2 µg of RNA was used for reverse transcription with a StarScript II First-strand cDNA Synthesis Mix (CenStar, Beijing, China). LcC2DPs and LcUBI detection primers were designed with Primer Premier 5.0 software. The cDNA served as templates for quantitative real-time PCR (qRT-PCR) using these primers (Table S2), with cycling conditions of 94 C for 3 min, then 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72 C for 30 s on a qTower3G System (Analytik Jena AG, Jena city, Germany). LcUBI gene was the endogenous control for data normalization 42 , and gene expression was quantified using the comparative CT method 43 . All experiments included at least three independent biological replicates. Statistical analysis Data processing was performed using Microsoft Excel 2013, statistical analysis was carried out using SPSS24.0, and figures were generated using GraphPad Prism 8.4.3. In this research, all the experiments were carried out in three independent biological replicates. In each graph, error bars indicate the mean values ± standard error (SE) of three replicates. To test the statistical significance between measurements of different treatments, a multiple test selection with analysis of variance (ANOVA) and Student’s t-test were used. The significance level between the various time points has been placed (*P < 0.05, **P < 0.01, and ***P < 0.001). Results LcC2DPs family members Using HMMER, InterPro (PF00168), and SMART software, 90 LcC2DPs genes were identified after searching for sequences with typical C2 structural domains in Lotus corniculatus genome. Protein sequences revealed significant variation in the lengths and molecular weights of LcC2DP members. Their amino acids ranged from 60 (LcC2DP24) to 1,056 (LcC2DP3, LcC2DP27), and molecular weights varied from 6,681.80 (LcC2DP24) to 78,9062.1 (LcC2DP9) kDa. The LcC2DPs family included 34 basic proteins and 56 acidic members with isoelectric points below 7. Fat coefficients of the family ranged from 60.13 (LcC2DP49) to113.15 (LcC2DP61), with 87 proteins being hydrophilic. The LcC2DPs family consists of 42 stable and 48 unstable proteins, with LcC2DP62 having the highest instability index of 60.07. These proteins were found in various cellular locations, including the nucleus, cytoplasm, cytoplasmic membrane, chloroplast, endoplasmic reticulum, cytoskeleton, peroxisome, vesicles, and mitochondria. Specifically, 28 proteins were in the nucleus, 23 in the cytoplasm, and 14 in the plasma membrane (Table S1 ). This indicates that LcC2DPs family members are widely involved in various life processes. Motif, intron and exon of LcC2DPs To explore the diversity of LcC2DPs family members in Lotus corniculatus , the conserved motifs of LcC2DP proteins were analyzed using online MEME software. The analysis identified five subfamilies within LcC2DPs family (Fig. 1 A). Group I had at least 6 conserved motifs (motif 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) in 13 proteins. Group II primarily featured motif1 (77.8%) or motif1 + motif6 (22.2%). Group III mainly included motif1 (50%) or motif1 + motif6 (50%). Group IV, with 8 members, consisted of motif1, 4, 6, 9 (67%) and motif 1, 6 (33%). The co-occurrence of motif 1 and motif6 is crucial for Group IV members. In group V, the combination included motif1 (7.1%), motif6 (7.1%), motif1 + motif6 (75%), and motif1 + motif6 + motif7 (10.71%) (Fig. 1 B). This suggests that motif1 and 6 are the most conserved in LcC2DPs family, indicating the diverse and special roles in plant cell. According to the method 44 , the LcC2DPs gene structure was mapped using genome annotation, showed 1–18 exons and 1–15 introns (Fig. 1 C). An exon appeared in 38 genes across Groups I-IV, while an intron was found in 7 genes from Groups I-III and V. Exons were more frequent than introns, and the size of coding sequences (CDS) after intron removal varied significantly, reflecting differences in protein molecular weight and amino acid composition among LcC2DPs family members. Chromosome localization TBtools analysis showed that LcC2DPs genes in Lotus corniculatus are distributed across chromosomes chr0 to chr6. Chr0 contains the most genes, with 34. Chr1, 2, and 4 each have 13 genes. Chr5 has the fewest, with just 1 gene, while chr6 has 9 genes. The tandem gene distribution is common, with genes LcC2DP 39, 40, 41, and 65 on chr1; LcC2DP44 and LcC2DP51 on chr2; LcC2DP82 and LcC2DP84 on chr3; LcC2DP71 , 72, 74 , 75 , 76 , 77 and 78 on chr4 (Fig. 2 A). Phyletic evolution A phylogenetic tree was created using amino acid sequence alignment of C2DPs proteins from Lotus corniculatus and Arabidopsis (Fig. 2 B), dividing them into five groups (I-V). Both species showed similarities: Group I includes 26 LcC2DPs and 11 AtC2DPs, some with only the C2 domain and others with an additional phospholipase D domain, PLDc, which produces phosphatidic acid from phosphatidylcholine, may play a role in forming transport vesicles or act as a component in signaling pathway trafficking. Group II comprises 20 LcC2DPs and 21 AtC2DPs members, some with a C2 domain and others with a C2_ArfGAP domain related to glycosylation. Group III includes 8 LcC2DPs and 16 AtC2DPs members, featuring either a C2 domain or a C2B_MCTP_PRT domain. The PRT-c domain, often found with a calcium-dependent C2 domain, is located at the C-terminus of phosphoribosyl transferases and similar proteins, and typically includes a potential transmembrane region. Group IV consists of 8 LcC2DPs and 5 AtC2DPs, with some members having only a C2 domain and others having an additional VWA domain. Group V includes 27 LcC2DPs and 22 AtC2DPs, with some members having only a C2 domain, while others have an additional GRAM domain. The GRAM domain is mainly found in membrane-associated proteins and may play a role in protein and lipid binding. Collinearity genes Gene duplication and divergence contribute to gene family expansion and the evolution of new functions 45 – 46 . A collinear gene analysis was conducted within Lotus corniculatus to explore the origin and evolutionary history of the LcC2DPs family. The LcC2DPs family contains 10 pairs of homologous genes on chromosomes 1 to 6 (Fig. 2 C). Collinearity analysis between Lotus corniculatus and Arabidopsis revealed 34 homologous relationships among 24 Lotus corniculatus and 25 genes Arabidopsis genes (Fig. 2 D). Gene pairings vary, with some Arabidopsis genes, like AtC2DP 22, corresponding to multiple Lotus corniculatus genes ( LcC2DP57 , 79 , 87) . Cis-acting elements of promoter Cis-acting elements in gene promoter affect the expression patterns. To understand the function of LcC2DPs gene family promoters, 2000 bp upstream sequences were analyzed by bioinformatics. These promoters are mostly enriched with elements responsive to light (GT1-motif), auxin (AuxRR-core), and abscisic acid (ABRE), related to the growth and development. They also contain elements for stress regulation, including responses to cold (MYB), drought (MYC or MBS), hypoxia (ARE), and defense (TC-rich repeats) (Fig. 3 ). GO and KEGG enrichment pathways GO analysis revealed that 90 LcC2DP genes were categorized into 30 groups, spanning cellular components, molecular functions, and biological processes. The cellular components primarily involved endosome, cytoplasm, endoplasmic reticulum, membrane, lysosomal membrane, plasma desmosis, vesicles, and membranes. Molecular functions of the endoplasmic reticulum mainly included calcium ion binding, protein binding, calmodulin binding, enzyme regulator activity, GTPase activation, kinase activity, protein serine/threonine kinase activity, catalytic activity, organic acid and carboxylic acid binding. Biological processes covered abscisic acid response, hormone signaling pathways, cell signal transduction, growth and developmental regulation, abiotic stimulus response, oxidative stress and defense response regulation. GO analysis revealed that the pathways of hormone response, protein binding activation, and kinase activity are crucial for the biologically active of C2DPs family members, including responses to environmental stress, growth regulation and other functions (Fig. 4 A). KEGG enrichment analysis identified 90 LcC2DPs genes across 13 pathways, primarily involving material metabolism: substance metabolism and transportation, ubiquitin system, coenzyme and protein metabolism, genetic information processing, pantothenic acid biosynthesis, gene transcription. This indicates that the metabolism, ubiquitin system, and gene transcription mechanism are key pathways enriched for LcC2DPs genes (Fig. 4 B). Gene clustering group Under abiotic stress, 90 LcC2DP genes were grouped into different clusters 47 . In stem node tissue, gene expression showed three patterns: 53 genes were up-regulated (red), 25 were down-regulated (blue), and 12 showed no change (yellow), representing 58.89%, 27.8%, and 13.33% of the total, respectively. Over 0, 3, 6, 9, and 12 days, up-regulated genes numbered 36, 28, 43, 39, and 19, while down-regulated genes were 42, 49, 39, 35, and 39. Twelve genes showed no significant expression change over 12 days (Fig. 4 C). The LcC2DPs family, expressed in various plant tissues, plays diverse roles in cellular stress response, mainly by regulating pathways. Gene expression pattern qRT-PCR analysis of 10 LcC2DPs genes screened in the root, stem, and leaf of Lotus corniculatus confirmed their expression in all these organs (Fig. 5 A). The expression levels of certain genes, like LcC2DP1 , 2 , 24 , 43 , 60 , 61 , and 62 (p < 0.05, p < 0.01 or p < 0.001), were notably higher in stems and leaves compared to roots. LcC2DP8 (p < 0.001) and LcC2DP 31, 36 (p < 0.001) were primarily expressed in stems and leaves, respectively. This suggests that LcC2DPs genes play a crucial roles across root, stem, and leaf. Analysis of ABA-responsive (ABRE) and IAA-responsive (AuxRR-core) elements in Lotus corniculatus C2DPs family showed that treating plants with 100 µM ABA and IAA improved the growth over 15 days (Fig. 5 B). Samples were collected at various growth stages for qRT-PCR analysis (Fig. 5 C). Results showed that LcC2DPs family members respond to ABA and IAA hormones, with expression patterns varying over 0 to 15 days, forming four groups. The first group ( LcC2DP 1, 24, and 31) had higher early expression with ABA (0–9 or 0–6 days) than IAA. The second group ( LcC2DP 2, 8, 36, 43, and 61) showed opposite, higher expression trends for IAA and ABA throughout. The third group, LcC2DP 60, exhibited notable transcription fluctuations in response to both hormones across all stages (0–6, 6–12, and 12–15 days). The fourth group, LcC2DP 62, showed higher gene expression under ABA treatment than IAA throughout the 0–15 period. Cis-acting elements in gene promoters can help analyze gene expression patterns and functions. To investigate the LcC2DPs gene family's functions, Lotus corniculatus was exposed to PEG6000 for drought simulation (0, 3, 6, 9, 12, and 15 days) and 4°C for cold stress (0, 6, 12, 24, 48, and 72 hours). Under drought, plant showed leaf curling, poor growth, and slow height increase (Fig. 6 A-a). In cold conditions, wilting began after 12 hours, worsening in the upper leaves, and by 72 hours, the Lotus corniculatus drooped (Fig. 6 A-b). qRT-PCR analysis of LcC2DP genes in young leaves under drought and low-temperature conditions revealed differences in the expression of 10 genes (Fig. 6 B). Compared to the initial expression, the LcC2DP1 gene showed fluctuating expression, being up-regulated in the early stages (3–6 days, p < 0.001) and down-regulated later (12–15 days, p < 0.001 or p < 0.01). LcC2DP8 was up-regulated during the middle to later phase (6–15 days, p < 0.01 or p < 0.001), while LcC2DP24, 31, 43 , and 60 consistently showed increased expression throughout (p < 0.05, p < 0.01 or p < 0.001). LcC2DP60 and LcC2DP61 were up-regulated in the early to middle stages (3–12 days, p < 0.05, p < 0.01 or p < 0.001). Under the 4 ℃ environment, the expression of LcC2DPs family genes in Lotus corniculatus varied (Fig. 6 C). LcC2DP1 was significantly up-regulated between 12 and 48 hours (p < 0.05), while LcC2DP24 showed up-regulation only at 72 hours (p < 0.01). LcC2DP8 and LcC2DP36 exhibited an ‘increase-decrease-rise’ pattern. LcC2DP43 and LcC2DP60 were continuously down-regulated from 6 to 72 and 12 to 72 hours, respectively. Notably, LcC2DP61 showed no significant expression changes, indicating that LcC2DP61 is not induced by low temperature for transcription. LcC2DP62 in plant cell was up-regulated at 6–72 hours. Results showed that in addition to LcC2DP61 , 9 other genes are involved in cold response in Lotus corniculatus . Regulatory pathways The C2DPs protein kinase family creates complex networks for cellular adaptation to stress 48 . To explore the cooperative or division of labor among C2DPs family members, they were analyzed using STRING (STRING://cn.string-db.org) for PCC statistics. Results showed that five core proteins, LcC2DP5, 11, 15, 38 and 58, primarily control the regulatory pathways among 90 family members, with 27 members interacting to varying extents with these core proteins (Fig. 7 A). Results indicates that LcC2DP1, 20, and 67 proteins interact with XLG2 protein from the G-alpha superfamily, which acts as an energy-releasing engine in signal transduction 49 . The activation and conformational changes in LcC2DPs depend on XLG2 activation. To confirm the response of five core genes to drought and low-temperature stress, qRT-PCR was used to measure the expression of LcC2DP5 , 11 , 15 , 38 , and 58 . The study found that all five core genes responded to drought stress, initially increasing and then decreasing. Gene expression for LcC2DP5 and 15 increased continuously from 0 to 9 days, LcC2DP11 and 38 from 0 to 6 days, and LcC2DP58 from 0 to 3 days. LcC2DP11 showed a significant decrease at 12 days compared to day 0 (p < 0.05). LcC2DP11 , 38 , and 58 had notable expression changes from 3 to 6 days (P < 0.01, or P < 0.05). LcC2DP5 showed the most significant expression from 9 to 15 days (p < 0.001), while LcC2DP15 was significantly expressed from 6–12 days (P < 0.001) (Fig. 7 B). Five core genes also responded to low-temperature. LcC2DP5 , 11 , and 38 continuously increased from 0 to 72 hours at 4 ℃, while LcC2DP15 and LcC2DP58 peaked at 72 and 24 hours, respectively. LcC2DP5 , 11 , and 38 showed differential expression from 6 to 48 hours (P < 0.001, P < 0.01 or P < 0.05) and decreased after 72 hours. LcC2DP11 and LcC2DP15 were significantly expressed from 48 to 72 hours (P < 0.001). LcC2DP58 was significantly expressed only at 24 hours (P < 0.001) (Fig. 7 C). Co-expression transcription factor Cell regulates its physiological process by TFs acting as mediators 5 . To explore the abiotic stress response mechanism of LcC2DPs family members, a Python script was used to identify co-expressed TFs for five core proteins. Only LcC2DP5 and LcC2DP15 provided co-expressed data, suggesting they are key in regulating abiotic stress responses within the LcC2DPs family (Fig. 7 D). This analysis was based on FPKM data from our existing transcriptome using a Python script 47 , we conducted co-expression analysis of transcription factors (r > 0.9). The study identified 49 types TFs, totaling 134, co-expressed with LcC2DP5 gene. The top eight TF types by member count include bHLH (8), WRKY (7), C3H (7), bZIP (7), MYB (5), MYB_related (5), C2H2 (5), and NAC (5). Analysis showed the LcC2DP15 gene is co-expressed with 38 types, totaling 125, including bHLH (15), WRKY (14), MYB_related (10), NAC (9), bZIP (9), C2H2 (7), and C3H (7) (Table S3). These TF families are known to play roles in abiotic stress responses 23 , 50 – 56 , suggesting that bHLH , WRKY , C3H , bZIP , MYB , NAC , C2H2 , and MYB_related are key pathways for LcC2DPs family in mediating abiotic stress regulation in plants. Expression of transcription factors Differential expression analysis of key abiotic stress-related TFs ( bZIP , bHLH , WRKY , NAC , MYB_related , MYB , C3H , and C2H2 ) in Lotus corniculatus under PEG-induced drought showed that all eight TFs were up-regulated (Fig. 8 A). The bHLH was significantly expressed only at the initial stage (3 days) (P < 0.001) before decreasing. MYB_related was significantly expressed during both the initial and middle stages (3–9 days) (P < 0.001 or P < 0.01), with reduced expression after 9 days (P < 0.01). In contrast, other TFs like bZIP , WRKY , NAC , MYB , C3H , and C2H2 were up-regulated throughout the stress period (3–15 days) (P < 0.001, P < 0.01, or P < 0.05), with bZIP , NAC , and C2H2 TFs showing the most significant differential expression in response to drought (P < 0.001). In Lotus corniculatus exposed to low temperature (4°C), all eight TFs ( bZIP , bHLH , WRKY , NAC , MYB_related , MYB , C3H , and C2H2 ) co-expressed with LcC2DPs were induced by cold stress (Fig. 8 B). MYB_related showed differential expression in early and middle stages (6–24 hours) (P < 0.001), and MYB in initial stage (6–12 hours), both peaking at 6 hours (P < 0.001). NAC and C3H were active during early and middle phases (6–48 hours) (P < 0.01 or P < 0.05). Analysis of core TFs linked to LcC2DPs under drought and low temperature stress showed that bZIP was consistently up-regulated throughout both conditions (3–12 days for drought and 6–48 hours for low temperature) (P < 0.001). bHLH was up-regulated early in drought (3 days) (P < 0.001) and entire low temperature exposure (6–72 hours) but down-regulated later in both stresses (15 days for drought and 72 hours for low temperature) (P < 0.001 or P < 0.05). WRKY was up-regulated during 3–6 and 12–15 days of drought, and at 6 and 24–72 hours of low temperature treatment (P < 0.001, P < 0.01 or P < 0.05). NAC was consistently up-regulated during drought period (3–15 days) (P < 0.001) but down-regulated in later stage (72 hours) under low temperature, showing different regulatory patterns for each stress. MYB_related had varied expression early on, then gradually decreased. The transcription of MYB differed between stresses, increasing under drought and decreasing under low temperature (P < 0.001, P < 0.01, or P < 0.05). C3H and C2H2 showed differential expression patterns under drought and low temperature stress. C3H was up-regulated early and mid-stress (P < 0.001 or P < 0.01), while C2H2 was consistently up-regulated (P < 0.001 or P C2H2 > WRKY > bZIP > C3H > MYB > MYB_related > bHLH. In low temperatures, it was: bZIP > MYB_related > C2H2 > C3H > NAC > bHLH > MYB > WRKY . NAC and C2H2 were key under drought, while bZIP and MYB_related were crucial under low temperature. Discussion The continuing warming of global climate has severely affected grass growth and development. Research into genes involved in stress regulation will be essential for grass molecular breeding. This paper identifies and analyzes 90 C2DP genes in Lotus corniculatus genome, which are linked to stress responses in rice ( Oryza sativa L) 57 , cotton ( Gossypium hirsutum L) 58 and Arabidopsis ( Arabidopsis thaliana ) 59 were found in of Lotus corniculatus (Table S1 ). The LcC2DP gene family is larger than in rice, cotton and Arabidopsis , which have 82, 31, and 16 C2DP genes, respectively. Rich functionality in a gene family is contingent upon its member numbers 60 . The complexity and diversity of C2DPs gene family suggest that variations among plant species may result from gene loss and duplication, key factors in gene family evolution. These processes can lead to the emergence of new genes with unique functions 61 . After identifying LcC2DPs family, their physicochemical properties were examined (Table S1 ). Subcellular localization showed that LcC2DPs proteins in the cell nucleus and cytoplasm, similar to CDPK in other plant 62 . This suggests that LcC2DP proteins activate transcription factors and regulate gene expression in response to signals, like other protein kinases. The division of labor family members is linked to their nuclear and cytoplasmic localization, affecting their role in signal transduction. LcC2DP genes primarily cluster on chromosomes 1, 2, 3, and 4 (Fig. 2 A), indicating possible tandem duplications in these regions of the Lotus corniculatus genome 63 . Such duplications are key in stress resistance, membrane function in rice and Arabidopsis , and signal transduction in legumes 64 – 66 . This implies that many LcC2DP genes play roles in stress responses and cellular signal transduction, potentially explaining the large number of family members in plants like rice and sweet potato, and suggesting interactions between chromosomes. Phylogenetic analysis based on gene structure classified 90 family members into five groups (Fig. 2 B). The C2DP genes of Arabidopsis were similarly divided into five groups 59 , confirming our analysis. C2DPs genes in Lotus corniculatus outnumber those in cotton and rice 57 – 58 . C2DPs were grouped into 5 in Arabidopsis and 7 in rice, with rice having fewer members than Lotus corniculatus . Gene functional redundancy has emerged during evolution. Gene duplication is a key driver of new gene functions but can cause redundancy. This redundancy helps maintain metabolic balance across environmental changes and development 67 . The study indicates that under purifying selection, duplicated genes varied expression or specialization supports plant adaptation to diverse environments 68 . Genome analysis shows that tandem duplication plays a crucial role in gene family expansion by generating new genes and clusters 69 , In this study, tandem duplication analysis identified 10 pairs of collinear homologous genes within Lotus corniculatus and 34 pairs between Lotus japonicas and Arabidopsis (Fig. 2 D). This suggests that tandem duplication has contributed to gene expansion in the Lotus corniculatus genome, potentially aiding its adaptation to complex environmental conditions and physiological functions compared to plant like potato 48 . Collinearity analysis revealed only 20 homologous gene pairs between Cucumis melo , Vitis vinifera , and Arabidopsis thaliana 41 . Our findings indicate a strong evolutionary relationship between the LcC2DPs gene family and Arabidopsis, suggesting similar functions. Overall, Lotus corniculatus shares a similar evolutionary process and genetic trait variation with Arabidopsis and other organisms. Cis-acting elements in promoters can elucidate gene expression regulation, cellular signaling network, and gene-environment interactions 70 . The prediction of cis-acting elements in LcC2DP promoter regions identified multiple hormone (AuxRR-core and ABRE) and stress response elements (MYC, MBS, ARE, TC-rich, and MYB) were identified (Fig. 3 ). Evidence suggests that AtCDPKs is involved in external stimuli responses with ABA 71– 72 . In Arabidopsis , CDPKs have been linked to abiotic stresses like drought, cold, heat, and salinity 10 , 72 . This indicates LcC2DP family genes may play a complex role in stress responses related to plant hormone regulation. Changes in LcC2DPs gene expression under hormone and abiotic stress were analyzed using qRT-PCR. This study confirms that LcC2DPs is involved in plant hormone and abiotic stress responses, including ABA, IAA, drought, and low temperature. CDPK regulate ABA-mediated signaling and influence phytohormones responses, such as IAA and cytokinins (CTK) 73 – 74 . qRT-PCR results showed LcC2DP 's sensitivity to 100 µM ABA and IAA in Lotus corniculatus (Fig. 5 C), leading to its classification into four groups based on the hormones response differences over time. Specifically, LcC2DP1 , 24 , 31 , LcC2DP2 , 8 , 36 , 43 , 61 , LcC2DP60 , and LcC2DP62 belong to groups 1, 2, 3, 4, respectively, according to RNA-seq data 47 . CDPK proteins are crucial for signal transduction, gene expression, growth, and development by binding to Ca 2+ , and they enhance plant resilience to various stresses 75 – 76 . Our study highlights the diverse biological roles of protein kinases. In grape and rice 73 , 30 , VpCDPK16 and OsCPK21 respond to ABA signaling, while IAA affect CDPK expression and activity, with NtCDPK1 transcripts increasing upon IAA treatment 77 . The expression level of different LcC2DP genes in Lotus corniculatus , treated with ABA and IAA, suggest a potential role in hormone signaling regulation. The cis-acting elements analysis and qRT-PCR assays on tender leaves exposed to PEG6000 and low temperature (4 ℃) revealed that 10 gene showed expression differences under drought and cold conditions. These genes exhibited varying expression patterns during drought treatment, categorized into four models: LcC2DP1 , 24, 31 ; LcC2DP2, 8, 36, 43 , and 61 ; LcC2DP60 ; LcC2DP62 groups (Fig. 6 B). CDPK are crucial for Arabidopsis under drought and other stresses 71 . For example, GhCDPK60 enhances drought resistance 78 , while GhCDPK4 boosts tobacco's drought tolerance 79 . In conclusion, LcC2DPs family is closely linked to drought response and exhibits complex regulation in Lotus corniculatus. CDPK proteins have diverse roles in responding to abiotic stresses like cold, salinity, and heat in plants 34 , 71 . LcC2DP1, 2, 24 , 8 , 31, 36 , 60 , and 62 genes show varied expression at different stages under low-temperatures (Fig. 6 C). LcC2DP61 showed no significant changes throughout the process and was unaffected by low temperature. LcC2DP61 's expression did not align with its promoter's ‘defense and stress responsiveness’ elements, possibly due to other regulatory like trans-acting factor 80 . Besides, LcC2DP43 expression significantly decreased at each stage in 4 ℃ environment, indicating a unique transcription model pattern. ShCDPK12 and ShCDPK30 function differently in response to cold stress in Solanum habrochaites and Arabidopsis 34 . CDPKs respond to abiotic stresses like drought, heat, salinity, and cold 71 . This study reveals that LcC2DPs reacts differently to low-temperature, possibly due to their abundance of stress-related cis-elements and family members. Protein-protein interactions are crucial in biological pathways 81 . Using the online tool STRING (available at //cn.string-db.org) with Cytoscape for predicting and visualizing the interactions among LcC2DPs family members. Analysis revealed that this family has five core proteins ( LcC2DP5 , 11 , 15 , 38 , and 58 ). LcC2DP1 , LcC2DP20 , and LcC2DP67 interact with XLG2, a G-alpha superfamily member (Fig. 7 A). XLG2 and G-alpha protein positively influence hormone signal transduction like IAA, ABA, and gibberellin (GA), affecting cell division, developmental, and stress response in plants 82 – 83 . Thus, LcC2DP family genes are crucial in plant hormone signaling and stress response pathways. The TFs bind to cis-regulatory elements in target gene regions to regulate plant growth, development, and stress responses 84 . A Python script identified co-expressed TFs for five core proteins, revealing that only LcC2DP5 and LcC2DP15 provided insights with co-expressed TFs. This suggests they are the central genes in the LcC2DPs family involved in plant abiotic stress (Fig. 7 D). Co-expression analysis identified eight TFs ( bZIP , bHLH , WRKY , NAC , MYB_related , MYB , C3H , and C2H2 ) in the regulation network of LcC2DP5 and LcC2DP15 for drought and 4 ℃ responses. Among them, bHLH and MYB regulate drought, NAC and MYB_related regulate low temperature, while bZIP , C2H2 , WRKY , and C3H form a common regulatory network under these stresses. The qRT-PCR analysis confirmed these findings after PEG6000 and 4 ℃ treatments. NAC (NAM, ATAF1/2, and CUC2) and MYB proteins are important factors in the stress response 11 , 85 . Our study showed that NAC and MYB expression increased after 20% PEG treatment for 15 days in Lotus corniculatus (Fig. 8 A), highlighting their role as key stress response regulators. As a crucial regulator in heat and drought stress responses, SNAC3 , in particular, enhances plant tolerance to heat and drought 85 . Overexpressing TaMYB33 and GbMYB5 genes enhances the drought tolerance in Arabidopsis and tobacco 86 , 26 , indicating that NAC and MYB transcription factors are vital for drought resistance in Lotus corniculatus . Low temperature pose significant environmental challenges, impacting plant physiology and yield 87 . This study shows that bHLH and MYB-related are highly expressed at 4°C in Lotus corniculatus (Fig. 8 B), with bHLH family members playing a key role in responding to cold stress by regulating cold -responsive genes like COR (cold-responsive) and CBF (cold-regulated binding factor) 88 – 90 . Simultaneously, the MYB_related family is crucial in responding to low temperature stress. Research shows that AhMYB30 from peanut MYB_related family enhances the freezing tolerance in transgenic Arabidopsis via DREB/CBF and ABA pathways 50 . Our findings indicate that bHLH and MYB_related transcription factors are key in regulating Lotus 's response to low temperature. Notably, high expression of bZIP , WRKY, C3H , and C2H2 was detected in both drought and cold conditions, suggesting their role in relating these stresses in Lotus corniculatus (Fig. 8 A, B). Various TF families, such as bZIP 91 , WRKY 56 C3H 92 and C2H2 27 , are crucial in regulating plant responses to stress, particularly cold and drought, by enhancing tolerance. Our findings support these roles. bZIP , WRKY, C3H , and C2H2 genes are activated by cold and drought stress. While bHLH 93 , bZIP 94 , NAC 54 , MYB_related 22 , MYB 5 , and C2H2 95 genes are found in both the nucleus and cytoplasm, WRKY 96 and C3H 92 genes are localized in the nucleus. This study implicates that NAC , C2H2 , bHLH , MYB , bZIP , and MYB_related TFs move to the nucleus after cytoplasmic activation, whereas WRKY and C3H are activated in the nucleus to regulate gene expression. C2DP , a calcium-dependent protein kinase, is activated by Ca 2+ during stress signal transduction, leading to phosphorylation and related TFs expression for stress response 97 . ZmNAC84 phosphorylation activates downstream genes by CDPK to participate in ABA-induced antioxidant defense 98 . Additionally, MYB is involved in the ABA signaling pathway for drought tolerance, regulated by PYR/PYL/RCARs, protein phosphate 2C (PP2Cs), and SNF1-related protein kinase 2s (nRK2s) in Arabidopsis 60 , 99 . bHLH plays a role in plant's response to low temperatures through CDPK phosphorylation pathways 100 . MAPK (mitogen-activated protein kinase) activation enhances the cold resistance via MYB-related under low temperature stress 101 . It is suggested that LcC2PDs phosphorylate activates NAC , MYB , and bHLH , MYB-related to improve drought and cold stress tolerance in Lotus plants. Additionally, bZIP , WRKY , C3H , and C2H2 are involved in both drought and cold stress responses (Fig. 9 ). Understanding these TFs can provide insights into plant abiotic regulation, aiding in the functional identification and analysis of stress regulation genes in forage crops. Conclusion This study used bioinformatics and experiments to identify and analyze the LcC2DP protein kinase family in Lotus corniculatus , finding 90 LcC2DP genes divided into five subgroups. Homologous genes pairs were present in both in Lotus corniculatus and Arabidopsis chromosomes. The LcC2DPs promoters contained hormone response elements (AuxRR-core, ABRE) and stress response elements (MYB, MYC, MBS) for cold and drought. GO enrichment revealed hormone response and kinase activity functions, and 13 KEGG pathways were identified. RNA-seq analysis showed 78 LcC2DPs related to stress response, with 10 genes linked to ABA, IAA, drought, and low temperature through qRT-PCR. Protein-protein interaction analysis identified five core proteins, with LcCDP5 and LcC2DP15 co-expressed with various TFs, including bZIP , bHLH , WRKY , NAC , MYB_related , MYB , C3H , and C2H2 , under stress conditions. NAC and C2H2 primarily respond to drought stress, while bZIP and MYB_related respond to low temperature. These findings form a basis for exploring LcC2DP family gene mechanisms in drought and cold stress responses. Declarations Declaration of Competing Interest The authors declare no competing interests. Funding This research was funded by the National Natural Science Foundation of China (No.32260338) and (No.31660685), and the Guizhou Province Science and Technology Project (2023ZK119). Author Contribution Author contribution LS conceived and designed the experiments. GY performed the experiments. GY carried out the bioinformatics analysis. GY wrote the manuscript. YL and SC gave insightful suggestions. LS improved the manuscript errors and English language. All authors read and approved the manuscript. Acknowledgement The authors thank master student Zouxian Gong (Clinical Medical College of Guizhou Medical University, Guiyang 550004, Guizhou Province, China.) for participating in the English revision. Data Availability The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, Beijing Institute of Genomics (China National Center for Bioinformation), Chinese Academy of Sciences, under accession number CRA002426 that are publicly accessible at https://bigd.big.ac.cn/gsa. References Pizzorni, M., Innocent, A & Tollin, N. Droughts and floods in a changing climate and implications for multi-hazard urban planning: A review. City Environ Interac 24 .https://doi.org/10.1016/j.cacint.2024.100169 (2024) Kim, J.S., Kidokoro, S., Yamaguchi-Shinozaki, K. & Shinozaki, K. Regulatory networks in plant responses to drought and cold stress . 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GhCDPK60 positively regulates drought stress tolerance in both transgenic Arabidopsis and cotton by regulating proline content and ROS level . Front Plant Sci 13, 1072584. https://10.3389/fpls.2022.1072584 (2022). Kong, H. et al. Calcium-dependent protein kinase GhCDPK4 plays a role in drought and abscisic acid stress responses . Plant Sci 332, 111704. https://10.1016/j.plantsci.2023.111704 (2023). Lynch, T., Erickson, B.J. & Finkelstein, R.R. Direct interactions of ABA-insensitive(ABI)-clade protein phosphatase(PP)2Cs with calcium-dependent protein kinases and ABA response element-binding bZIPs may contribute to turning off ABA response . Plant Mol Biol 80, 647-58. https://10.1007/s11103-012-9973-3 (2012). Zhu, P. et al. Computational identification of protein-protein interactions in rice based on the predicted rice interactome network . Genom Proteom Bionf 9, 128-37. https://10.1016/s1672-0229(11)60016-8 (2011). Ma, M. et al. A surface-receptor-coupled G protein regulates plant immunity through nuclear protein kinases . Cell Host Microbe 30, 1602-1614.e5. https://10.1016/j.chom.2022.09.012 (2022). Maruta, N. et al. GTP binding by Arabidopsis extra-large G protein 2 is not essential for its functions . Plant Physiol 186, 1240-1253. https://10.1093/plphys/kiab119 (2021). Hajheidari, M. & Huang, S.C. Elucidating the biology of transcription factor-DNA interaction for accurate identification of cis-regulatory elements . Curr Opin Plant Biol 68, 102232. https://10.1016/j.pbi.2022.102232 (2022). Fang, Y. et al. A stress-responsive NAC transcription factor SNAC3 confers heat and drought tolerance through modulation of reactive oxygen species in rice . J Exp Bot 66, 6803-17. https://10.1093/jxb/erv386 (2015). Qin, Y. et al. Over-expression of TaMYB33 encoding a novel wheat MYB transcription factor increases salt and drought tolerance in Arabidopsis. Mol Biol Rep 39, 7183-92. https://10.1007/s11033-012-1550-y (2012). Kidokoro, S., Shinozaki, K. & Yamaguchi-Shinozaki, K. Transcriptional regulatory network of plant cold-stress responses . Trends Plant Sci 27, 922-935. https://10.1016/j.tplants.2022.01.008 (2022). Li, G., Jin, L. & Sheng, S. Genome-Wide Identification of bHLH Transcription Factor in Medicago sativa in Response to Cold Stress . Genes (Basel) 13, 2371. https://10.3390/genes13122371 (2022). Liu, J.-H., Peng, T. & Dai, W. Critical cis-acting elements and interacting transcription factors: key players associated with abiotic stress responses in plants . Plant Mol. Biol. Rep 32, 303-317. https://https://10.1261/rna.895308 (2014). Ritonga, F.N. & Chen, S. Physiological and Molecular Mechanism Involved in Cold Stress Tolerance in Plants . Plants (Basel) 9, 0560. https://10.3390/plants9050560 (2020). Ma, M. et al. Genome-wide identification and expression analysis of the bZIP transcription factors, and functional analysis in response to drought and cold stresses in pear ( Pyrus breschneideri ) . BMC Plant Biol 21, 583. https://10.1186/s12870-021-03356-0 (2021). Cheng, X. et al. Identification of the wheat C3H gene family and expression analysis of candidates associated with seed dormancy and germination . Plant Physiol Biochem 156, 524-537. https://10.1016/j.plaphy.2020.09.032 (2020). Greb-Markiewicz, B., Kazana, W., Zarębski, M. & Ożyhar, A. The subcellular localization of bHLH transcription factor TCF4 is mediated by multiple nuclear localization and nuclear export signals . Sci Rep 9, 15629. https://10.1038/s41598-019-52239-w (2019). Wiese, A.J. et al. Arabidopsis bZIP18 and bZIP52 Accumulate in Nuclei Following Heat Stress where They Regulate the Expression of a Similar Set of Genes . Int J Mol Sci 22, 0530. https://10.3390/ijms22020530 (2021). Shao, L. et al. Identification of C2H2 zinc finger genes through genome-wide association study and functional analyses of LkZFPs in response to stresses in Larix kaempferi. BMC Plant Biol 23, 298. https://10.1186/s12870-023-04298-5 (2023). Long, Q. et al. Transcription factor WRKY22 regulates canker susceptibility in sweet orange ( Citrus sinensis Osbeck ) by enhancing cell enlargement and CsLOB1 expression . Hortic Res 8, 50. https://10.1038/s41438-021-00486-2 (2021). Liese, A. & Romeis, T. Biochemical regulation of in vivo function of plant calcium-dependent protein kinases (CDPK) . Biochim Biophys Acta 1833, 1582-9. https://10.1016/j.bbamcr.2012.10.024 (2013). Zhu, Y. et al. Phosphorylation of a NAC Transcription Factor by a Calcium/Calmodulin-Dependent Protein Kinase Regulates Abscisic Acid-Induced Antioxidant Defense in Maize . Plant Physiol 171, 1651-64. https://10.1104/pp.16.00168 (2016). Li, J., Han, G., Sun, C. & Sui, N. Research advances of MYB transcription factors in plant stress resistance and breeding . Plant Signal Behav 14, 1613131. https://10.1080/15592324.2019.1613131 (2019). Praat, M., De Smet, I. & van Zanten, M. Protein kinase and phosphatase control of plant temperature responses . J Exp Bot 72. https://10.1093/jxb/erab345 (2021). Abdullah, S.N.A., Azzeme, A.M. & Yousefi, K. Fine-Tuning Cold Stress Response Through Regulated Cellular Abundance and Mechanistic Actions of Transcription Factors . Front Plant Sci 13, 850216. https://10.3389/fpls.2022.850216 (2022). Additional Declarations No competing interests reported. Supplementary Files Supplementarydata.docx Cite Share Download PDF Status: Published Journal Publication published 18 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 07 Mar, 2025 Reviews received at journal 05 Mar, 2025 Reviewers agreed at journal 20 Feb, 2025 Reviewers agreed at journal 17 Feb, 2025 Reviews received at journal 17 Feb, 2025 Reviewers agreed at journal 16 Feb, 2025 Reviewers invited by journal 15 Feb, 2025 Editor assigned by journal 09 Jan, 2025 Editor invited by journal 09 Jan, 2025 Submission checks completed at journal 09 Jan, 2025 First submitted to journal 04 Jan, 2025 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. 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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-5763738","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":400254331,"identity":"d06d89e7-3a92-4721-a37e-5cf854dfda88","order_by":0,"name":"Guangfen Yang","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Guangfen","middleName":"","lastName":"Yang","suffix":""},{"id":400254332,"identity":"d4f35a98-8c0a-4b63-b62c-59e707aab3c7","order_by":1,"name":"Yujie Liu","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Yujie","middleName":"","lastName":"Liu","suffix":""},{"id":400254333,"identity":"9d65111f-06b9-4b93-a92f-39e621aaf148","order_by":2,"name":"Zouxian Gong","email":"","orcid":"","institution":"Clinical Medical College of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zouxian","middleName":"","lastName":"Gong","suffix":""},{"id":400254334,"identity":"fce132a9-c55e-488d-b4df-d743e7930207","order_by":3,"name":"Siya Chen","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Siya","middleName":"","lastName":"Chen","suffix":""},{"id":400254336,"identity":"4abd966f-b8d0-4f3c-a119-f879dff78a99","order_by":4,"name":"Li Song","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApElEQVRIiWNgGAWjYBACPjBZYMPDz95ApBY2MGmQJiPZc4A0LYdtDG44EKtFuv3xxx8G53kYbjAwfviYQ4wWmTNm0jwGt3kYZzcwS87cRowWiRw2ZgagFmaZA2zMvMRpSQc57BwPm0QC0VoSDCR4DA7w8BCvBeKXZB4JnoPNxPmFHxxiFXb29sebD374SIwWBgk4i7GBGPUoWkbBKBgFo2AU4AAAnsstW0i9stMAAAAASUVORK5CYII=","orcid":"","institution":"Guizhou University","correspondingAuthor":true,"prefix":"","firstName":"Li","middleName":"","lastName":"Song","suffix":""},{"id":400254337,"identity":"f6070112-da94-4690-8644-68ce572ec2e6","order_by":5,"name":"Shihui Liu","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Shihui","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-01-04 13:23:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5763738/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5763738/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-97896-2","type":"published","date":"2025-04-18T15:57:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73635406,"identity":"cbda4a92-71d6-448e-bcb1-a472e23b6e3b","added_by":"auto","created_at":"2025-01-13 07:12:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":471770,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationship, gene structure, and motif composition of \u003cem\u003eLcC2DPs\u003c/em\u003e. (A) Rootless phylogenetic tree from MEGA7.0. (B) Motifs 1-10 shown in different colors. (C) Exon-intron structure: green for UTR, yellow for CDS, black for intron.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5763738/v1/da055b41a8037d49d441502b.png"},{"id":73635404,"identity":"721c86d4-c37e-4e2c-99f5-bbb346ce631d","added_by":"auto","created_at":"2025-01-13 07:12:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":556221,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal localization, phylogenetic, and collinearity analysis of \u003cem\u003eLcC2DPs\u003c/em\u003efamily. (A) Chromosomal positions with length scale. (B) Phylogenetic tree with colored groups using Neighbor-joining (NJ) method and 1000 Bootstrap replications. (C) Intra-specific collinearity with red lines for duplicated genes; colored rectangles for chromosomes; blue circle for gene density. (D) Inter-specific collinearity: Chr1-5 for \u003cem\u003eArabidopsis\u003c/em\u003e, chr1-6 for \u003cem\u003eLotus\u003c/em\u003e, red lines for duplicated \u003cem\u003eLcC2DPs\u003c/em\u003e and \u003cem\u003eAtC2DPs\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5763738/v1/93aff5f543434dca00c62bb4.png"},{"id":73635783,"identity":"02a9d9e7-48a0-413d-9f14-2d0c948ebec3","added_by":"auto","created_at":"2025-01-13 07:20:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":662469,"visible":true,"origin":"","legend":"\u003cp\u003eCis-regulatory element analysis of \u003cem\u003eLcC2DPs\u003c/em\u003e promoter regions.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5763738/v1/a6ace00787b2e88e37b1e9b4.png"},{"id":73635416,"identity":"1af3871f-44e9-4a89-8370-89cc14e67774","added_by":"auto","created_at":"2025-01-13 07:12:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":402914,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of \u003cem\u003eLcC2DPs\u003c/em\u003egene family with GO (A) and KEGG (B) enrichment, where dots size and color indicategene numbers and q-value. (C) Heatmap of \u003cem\u003eLcC2DPs\u003c/em\u003eexpression under submergence stress over time.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5763738/v1/a9952c2874797aa45d65d911.png"},{"id":73635785,"identity":"404c5ef0-aec6-47de-a2b1-f985680f3c88","added_by":"auto","created_at":"2025-01-13 07:20:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":356359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eLcC2DPs\u003c/em\u003e gene expression profiles. (A) Expression in roots, stems, and leaves. (B) \u003cem\u003eLotus \u003c/em\u003ephenotype after 100 mM ABA and IAA treatment. (C) qRT-PCR expression levels post hormone treatment. Data are mean ± SD (n=3). Significance assessed by t-test. *, p\u0026lt;0.05; **, p\u0026lt;0.01; ***, p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5763738/v1/b6964798431638f96674a43e.png"},{"id":73635412,"identity":"e38b3b0b-cc3d-45f4-bf52-432dcd59da70","added_by":"auto","created_at":"2025-01-13 07:12:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":384995,"visible":true,"origin":"","legend":"\u003cp\u003ePlant phenotypes and \u003cem\u003eLcC2DPs \u003c/em\u003eexpression with 20% PEG and 4 °C treatments. (A) Phenotype comparison. (B) Expression under drought. (C) Expression in low temperatures. Data are mean ± SD (n=3). Significance assessed byt-test: *, p\u0026lt;0.05; **, p\u0026lt;0.01; ***, p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5763738/v1/7cc0d17e98e086ffc2fd2fc6.png"},{"id":73635417,"identity":"436b2f5b-b443-4415-abd6-3a030a8275b4","added_by":"auto","created_at":"2025-01-13 07:12:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":351778,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of protein-protein interactions, core gene expression, and co-expression. (A) Protein interactions. Expression of \u003cem\u003eLcC2DP5\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e, \u003cem\u003e38\u003c/em\u003e, and\u003cem\u003e 58\u003c/em\u003e under drought (B) and low temperature (C). (D) Co-expression of \u003cem\u003eLcC2DP5\u003c/em\u003e and \u003cem\u003eLcC2DP15\u003c/em\u003e, with solid lines for positive and dashed for negative correlations.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5763738/v1/691c5cd99ae2371d57da8ba5.png"},{"id":73635789,"identity":"e17cf2dd-ae87-40dd-92f7-1404b7d91cc4","added_by":"auto","created_at":"2025-01-13 07:20:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":271612,"visible":true,"origin":"","legend":"\u003cp\u003eTF expression via qRT-PCR under drought (A) and low temperature (B), with significance assessed by T- test. Data are mean ± SD (n=3). Significance: *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5763738/v1/aee8a94a1c01f69fb4191f3a.png"},{"id":73635425,"identity":"59f58b4c-25d9-4ad9-ad39-2a5c69e11888","added_by":"auto","created_at":"2025-01-13 07:12:53","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":238486,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptional regulatory network of \u003cem\u003eLcC2DP5\u003c/em\u003e and \u003cem\u003eLcC2DP15\u003c/em\u003e under drought and low temperature.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e When drought and low-temperature signals are detected by membrane receptors, calcium ions are released into the cytoplasm, activating protein kinases \u003cem\u003eLcC2DP5\u003c/em\u003e and \u003cem\u003eLcC2DP15\u003c/em\u003e. This triggers phosphorylation cascades that activate transcription factors like bZIP, bHLH, WRKY, NAC, MYB-related, MYB, C3H, and C2H2, leading to the transcription of stress-related genes and enhancing plant resilience.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e \u003cem\u003eNAC\u003c/em\u003e, \u003cem\u003eC2H2\u003c/em\u003e, \u003cem\u003ebHLH\u003c/em\u003e, \u003cem\u003eMYB\u003c/em\u003e, \u003cem\u003ebZIP\u003c/em\u003e, and \u003cem\u003eMYB_related\u003c/em\u003e TFs move to the nucleus after activation, while \u003cem\u003eWRKY \u003c/em\u003eand \u003cem\u003eC3H\u003c/em\u003e are activated in the nucleus.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003e \u003cem\u003eNAC\u003c/em\u003e and \u003cem\u003eC2H2\u003c/em\u003e are key in \u003cem\u003eLcC2DP5\u003c/em\u003e and \u003cem\u003eLcC2DP15\u003c/em\u003e pathways during drought, whereas \u003cem\u003ebHLH\u003c/em\u003e and \u003cem\u003eMYB_related\u003c/em\u003e are crucial in low temperature.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5763738/v1/f07208893d246cd0b9f10fc4.png"},{"id":81050816,"identity":"9f05074d-2d7d-46c3-8b23-5e288f23523b","added_by":"auto","created_at":"2025-04-21 16:05:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4869557,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5763738/v1/0d251bc5-97de-4d40-823c-8e7472d1c590.pdf"},{"id":73635410,"identity":"ac4afa6f-d1b0-4a52-8f2d-24da20b3db87","added_by":"auto","created_at":"2025-01-13 07:12:52","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":66518,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-5763738/v1/e6398d60e16816788f50b06c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide identification of LcC2DPs gene family in Lotus corniculatus provides insights into regulatory network in response to abiotic stresses","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlobal climate change is increasing the frequency and severity of extreme weather, like droughts and cold spells\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, which threaten ecosystems, agriculture, and forage production\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. These conditions disrupt water supply, hinder forage growth, thus reduce yield and quality, sometimes leading to plant death\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. For instance, summer droughts with high temperatures can cut forage yield by about 30%\u003csup\u003e6\u003c/sup\u003e. In plant cells, genes and their products manage signal perception and transduction, activating downstream genes to protect against stress\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Calcium-dependent protein kinases (CDPKs) are vital in forage grasses\u0026rsquo; response to abiotic stress\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, acting as key signaling effectors. They detect environmental changes and trigger intracellular pathways\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Under drought and low temperature stress, \u003cem\u003eCDPKs\u003c/em\u003e enhance forage grass adaptability by influencing stomatal movement, antioxidant enzymes, osmoprotectants synthesis, and gene expression\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eProtein kinases, through phosphorylation and dephosphorylation, are crucial for plant response to abiotic stresses such as drought and low temperature\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Phosphorylation of protein kinases amplifies intracellular signal cascades by altering its charge, conformation, and function, while dephosphorylation removes foreign signals in plant pathways\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Most protein kinases are calcium-dependent, playing a role in calcium signal responses\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Calcium-dependent (CD) factors, such CDPK, are crucial in plant stress responses, growth, and development\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Studies indicate that cells detect signals via Receptor-like kinase (RLK) and membrane hardening, which then activate Ca\u003csup\u003e2+\u003c/sup\u003e channels to raise cytoplasm Ca\u003csup\u003e2+\u003c/sup\u003e levels\u003csup\u003e14\u003c/sup\u003e. Ca\u003csup\u003e2+\u003c/sup\u003e receptors like Ca\u003csup\u003e2+\u003c/sup\u003e-dependent protein kinase (CDPK), Calmodulin (CaM), Calmodulin-like proteins (CMLs), and Calcineurin B-like proteins (CBLs), facilitate signal transduction\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. CDPK with a C2 domain, crucial for binding Ca\u003csup\u003e2+\u003c/sup\u003e, are extensively involved in regulating cell signaling, perception, conduction, gene expression, growth, and development\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Additionally, hormones, particularly Abscisic acid (ABA), are vital in plant responses to various stresses, including low temperature\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Research indicates that ABA boosts salt and drought resistance in \u003cem\u003eSuaeda liaotungensis\u003c/em\u003e through working with jasmonic acid (JA) under cold stress\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. MAPK protein kinase is key in the signal transduction of indole-3-acetic acid (IAA)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, where G protein activates intracellular signals after IAA is detected by cell membrane receptors\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, can be to induce the stransduction of IAA.\u003c/p\u003e \u003cp\u003eTranscription factors (TFs) play a pivotal role in helping plants respond to harsh environments by binding to specific promoter and adjusting gene expression\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. TFs generally have two domains: the DNA-binding domain (DBD) and the transcriptional activation or inhibition domain\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Based on DBD structure, plant TFs are categorized into families like \u003cem\u003eMYB\u003c/em\u003e, \u003cem\u003eWRKY\u003c/em\u003e, \u003cem\u003eGARP\u003c/em\u003e, \u003cem\u003eTCP\u003c/em\u003e, \u003cem\u003ebZIP\u003c/em\u003e, \u003cem\u003ebHLH\u003c/em\u003e, and \u003cem\u003eHSF\u003c/em\u003e, etc.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Among them, \u003cem\u003ebHLH\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eNAC\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eMYB_related\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eMYB\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eC3H\u003c/em\u003e, and \u003cem\u003eC2H2\u003c/em\u003e\u003csup\u003e27\u003c/sup\u003e are crucial in regulating plant stress responses to drought, low temperature, and salt.\u003c/p\u003e \u003cp\u003eC2 domain protein (C2DP), a class of calcium-dependent binding proteins in eukaryotes, is involved in stress response and growth regulation\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30 CR31 CR32\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. CDPK proteins with C2 domain play a crucial role in plant growth, development, and stress responses like drought\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. While their functions have been studied in model plants like Arabidopsis (\u003cem\u003eArabidopsis thaliana\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, tobacco (\u003cem\u003eNicotiana tabacum\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, and rice (\u003cem\u003eOryza sativa\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, they are under-researched in forage crops \u003cem\u003eLotus corniculatus\u003c/em\u003e, also known as five-leaf grass or cowhorn, is a high-quality perennial legume with strong stress resistance, widely cultivated for forage and soil conservation\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. This study identified and characterized the LcC2DPs protein kinase gene family, analyzing their expression in response to ABA and IAA hormone treatments and stress conditions like drought and low temperature. The findings offer insights and potential gene candidates for enhancing stress resistance in \u003cem\u003eLotus corniculatus\u003c/em\u003e breeding.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003ePlant materials\u003c/p\u003e \u003cp\u003e \u003cem\u003eLotus corniculatus\u003c/em\u003e was planted at a key laboratory's experimental base of Education Ministry at Guizhou University (Guiyang, Guizhou, 106\u0026deg; 675 'E; 26\u0026deg; 427 ' N).\u003c/p\u003e \u003cp\u003eFamily members identification\u003c/p\u003e \u003cp\u003eThe research utilized genome and protein sequences, and annotation data from a \u003cem\u003eLotus corniculatus\u003c/em\u003e database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.kazusa.or.jp/lotus/\u003c/span\u003e\u003cspan address=\"http://www.kazusa.or.jp/lotus/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) The Hidden Markov Model (HMM) profile (version 3.3) (PF00168) was used to identify C2DPs proteins by searching the Pfam database\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. An initial domain search with an E-value\u0026thinsp;\u0026lt;\u0026thinsp;1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e was conducted to create a species-specific model for the \u003cem\u003eLotus corniculatus\u003c/em\u003e genome protein sequence. C2 domain protein sequences of \u003cem\u003eLotus corniculatus\u003c/em\u003e were then submitted to the Conserved Domain Database (CDD) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and screened with SMART (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl-heidelberg.de\u003c/span\u003e\u003cspan address=\"http://smart.embl-heidelberg.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Pfam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org/\u003c/span\u003e\u003cspan address=\"http://pfam.xfam.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to exclude sequences without C2 domain\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. This process identified high-cofidence C2 domain family proteins in \u003cem\u003eLotus corniculatus\u003c/em\u003e from all non-redundant genes.\u003c/p\u003e \u003cp\u003ePhysicochemical and molecular analysis\u003c/p\u003e \u003cp\u003eExpasy (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and WoLF PSORT (\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) software were used to analyze theoretical molecular weight (MW)\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, isoelectric point (pI), and subcellular localization. MEME Suite 5.5.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/tools/meme\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) identified conserved motifs in \u003cem\u003eLotus corniculatus C2DP\u003c/em\u003e genes, with a maximum of 10 motifs\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. TBtools' Visualize Gene Structure examined intron and exon regions of \u003cem\u003eLcC2DPs\u003c/em\u003e, TBtools' Visualize Gene Structure examined intron and exon regions, and Gene Location Visualize from GFF mapped chromosome localization. A phylogenetic tree was created using the Neighbor-Joining (NJ) algorithm in MEGA 7.0, and final plot was visualized with iTOL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e5\u003c/sup\u003e. Collinearity analysis was performed with MCScanX. TBtools aided in visualizing collinearity analysis. A 2000 bp upstream DNA sequence from \u003cem\u003eLcC2DPs\u003c/em\u003e was used for promoter analysis, with \u003cem\u003ecis\u003c/em\u003e-acting elements predicted via the PlantCARE database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e40\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFunction prediction\u003c/p\u003e \u003cp\u003eGO and KEGG analyses were conducted using the DAVID tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov/\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and figures were created with Microscopic Letter (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinformatics.com.cn/\u003c/span\u003e\u003cspan address=\"https://bioinformatics.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). A \u003cem\u003eLotus corniculatus\u003c/em\u003e protein-protein interaction network was constructed by importing \u003cem\u003eLotus corniculatus\u003c/em\u003e C2DPs sequences into the STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), saved as a TSV file, and visualized the PPI network with Cytoscape software. Using the Japonicus plant database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://planttfdb.gao-lab.org/prediction.php\u003c/span\u003e\u003cspan address=\"https://planttfdb.gao-lab.org/prediction.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e41\u003c/sup\u003e. A Python script analyzed gene transcriptome FPKM values, and a coexpression network was built with DEGs and TF data Cytoscape v3.7.10 software was used for visual analysis.\u003c/p\u003e \u003cp\u003eGene expression analysis\u003c/p\u003e \u003cp\u003eTo study \u003cem\u003eLcC2DP\u003c/em\u003e gene expression, 12-day-old hydroponic seedlings with healthy rhizomes were transplanted into soil for 2 weeks culture. During this time, they underwent drought, low temperature, and hormone treatment. Drought stress was simulated by applying respectively carried out during this period, 500 ml 20% PEG solution daily for 15 days. The \u003cem\u003eLotus corniculatus\u003c/em\u003e seedlings were placed in a growth incubator (Ningbo Southeast Instruments Co., Ltd.) at 4\u0026deg;C for 72 hours. For 15 days, 100 umol IAA and ABA were applied to the leaves and soil twice daily. Except for the low temperature group, the culture conditions were maintained at 25 ℃ with a 16-hour light and 8-hour dark cycle, 60% humidity, and 20000 Lk light intensity. Total RNA was extracted from each sample (drought and hormone, 0, 3, 6, 9,12, and 15 days; low temperature, 0, 6, 12, 24, 48, and 72 hours) using the OMEGA Plant RNA Kit (OMEGA, Guangzhou, China), and approximately 2 \u0026micro;g of RNA was used for reverse transcription with a StarScript II First-strand cDNA Synthesis Mix (CenStar, Beijing, China). \u003cem\u003eLcC2DPs\u003c/em\u003e and \u003cem\u003eLcUBI\u003c/em\u003e detection primers were designed with Primer Premier 5.0 software. The cDNA served as templates for quantitative real-time PCR (qRT-PCR) using these primers (Table S2), with cycling conditions of 94 C for 3 min, then 35 cycles of 94\u0026deg;C for 30 s, 58\u0026deg;C for 30 s, and 72 C for 30 s on a qTower3G System (Analytik Jena AG, Jena city, Germany). \u003cem\u003eLcUBI\u003c/em\u003e gene was the endogenous control for data normalization\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, and gene expression was quantified using the comparative CT method\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. All experiments included at least three independent biological replicates.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData processing was performed using Microsoft Excel 2013, statistical analysis was carried out using SPSS24.0, and figures were generated using GraphPad Prism 8.4.3. In this research, all the experiments were carried out in three independent biological replicates. In each graph, error bars indicate the mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE) of three replicates. To test the statistical significance between measurements of different treatments, a multiple test selection with analysis of variance (ANOVA) and Student\u0026rsquo;s t-test were used. The significance level between the various time points has been placed (*P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eLcC2DPs\u003c/em\u003e family members\u003c/p\u003e \u003cp\u003eUsing HMMER, InterPro (PF00168), and SMART software, 90 \u003cem\u003eLcC2DPs\u003c/em\u003e genes were identified after searching for sequences with typical C2 structural domains in \u003cem\u003eLotus corniculatus\u003c/em\u003e genome. Protein sequences revealed significant variation in the lengths and molecular weights of LcC2DP members. Their amino acids ranged from 60 (LcC2DP24) to 1,056 (LcC2DP3, LcC2DP27), and molecular weights varied from 6,681.80 (LcC2DP24) to 78,9062.1 (LcC2DP9) kDa. The \u003cem\u003eLcC2DPs\u003c/em\u003e family included 34 basic proteins and 56 acidic members with isoelectric points below 7. Fat coefficients of the family ranged from 60.13 (LcC2DP49) to113.15 (LcC2DP61), with 87 proteins being hydrophilic. The \u003cem\u003eLcC2DPs\u003c/em\u003e family consists of 42 stable and 48 unstable proteins, with LcC2DP62 having the highest instability index of 60.07. These proteins were found in various cellular locations, including the nucleus, cytoplasm, cytoplasmic membrane, chloroplast, endoplasmic reticulum, cytoskeleton, peroxisome, vesicles, and mitochondria. Specifically, 28 proteins were in the nucleus, 23 in the cytoplasm, and 14 in the plasma membrane (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This indicates that \u003cem\u003eLcC2DPs\u003c/em\u003e family members are widely involved in various life processes.\u003c/p\u003e \u003cp\u003eMotif, intron and exon of LcC2DPs\u003c/p\u003e \u003cp\u003eTo explore the diversity of \u003cem\u003eLcC2DPs\u003c/em\u003e family members in \u003cem\u003eLotus corniculatus\u003c/em\u003e, the conserved motifs of LcC2DP proteins were analyzed using online MEME software. The analysis identified five subfamilies within \u003cem\u003eLcC2DPs\u003c/em\u003e family (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Group I had at least 6 conserved motifs (motif 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) in 13 proteins. Group II primarily featured motif1 (77.8%) or motif1\u0026thinsp;+\u0026thinsp;motif6 (22.2%). Group III mainly included motif1 (50%) or motif1\u0026thinsp;+\u0026thinsp;motif6 (50%). Group IV, with 8 members, consisted of motif1, 4, 6, 9 (67%) and motif 1, 6 (33%). The co-occurrence of motif 1 and motif6 is crucial for Group IV members. In group V, the combination included motif1 (7.1%), motif6 (7.1%), motif1\u0026thinsp;+\u0026thinsp;motif6 (75%), and motif1\u0026thinsp;+\u0026thinsp;motif6\u0026thinsp;+\u0026thinsp;motif7 (10.71%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This suggests that motif1 and 6 are the most conserved in \u003cem\u003eLcC2DPs\u003c/em\u003e family, indicating the diverse and special roles in plant cell. According to the method\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, the \u003cem\u003eLcC2DPs\u003c/em\u003e gene structure was mapped using genome annotation, showed 1\u0026ndash;18 exons and 1\u0026ndash;15 introns (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). An exon appeared in 38 genes across Groups I-IV, while an intron was found in 7 genes from Groups I-III and V. Exons were more frequent than introns, and the size of coding sequences (CDS) after intron removal varied significantly, reflecting differences in protein molecular weight and amino acid composition among \u003cem\u003eLcC2DPs\u003c/em\u003e family members.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eChromosome localization\u003c/p\u003e \u003cp\u003eTBtools analysis showed that \u003cem\u003eLcC2DPs\u003c/em\u003e genes in \u003cem\u003eLotus corniculatus\u003c/em\u003e are distributed across chromosomes chr0 to chr6. Chr0 contains the most genes, with 34. Chr1, 2, and 4 each have 13 genes. Chr5 has the fewest, with just 1 gene, while chr6 has 9 genes. The tandem gene distribution is common, with genes \u003cem\u003eLcC2DP\u003c/em\u003e39, 40, 41, and 65 on chr1; \u003cem\u003eLcC2DP44\u003c/em\u003e and \u003cem\u003eLcC2DP51\u003c/em\u003e on chr2; \u003cem\u003eLcC2DP82\u003c/em\u003e and \u003cem\u003eLcC2DP84\u003c/em\u003e on chr3; \u003cem\u003eLcC2DP71\u003c/em\u003e, 72, \u003cem\u003e74\u003c/em\u003e, \u003cem\u003e75\u003c/em\u003e, \u003cem\u003e76\u003c/em\u003e, \u003cem\u003e77\u003c/em\u003e and \u003cem\u003e78\u003c/em\u003e on chr4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePhyletic evolution\u003c/p\u003e \u003cp\u003eA phylogenetic tree was created using amino acid sequence alignment of C2DPs proteins from \u003cem\u003eLotus corniculatus\u003c/em\u003e and Arabidopsis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), dividing them into five groups (I-V). Both species showed similarities: Group I includes 26 LcC2DPs and 11 AtC2DPs, some with only the C2 domain and others with an additional phospholipase D domain, PLDc, which produces phosphatidic acid from phosphatidylcholine, may play a role in forming transport vesicles or act as a component in signaling pathway trafficking. Group II comprises 20 LcC2DPs and 21 AtC2DPs members, some with a C2 domain and others with a C2_ArfGAP domain related to glycosylation. Group III includes 8 LcC2DPs and 16 AtC2DPs members, featuring either a C2 domain or a C2B_MCTP_PRT domain. The PRT-c domain, often found with a calcium-dependent C2 domain, is located at the C-terminus of phosphoribosyl transferases and similar proteins, and typically includes a potential transmembrane region. Group IV consists of 8 LcC2DPs and 5 AtC2DPs, with some members having only a C2 domain and others having an additional VWA domain. Group V includes 27 LcC2DPs and 22 AtC2DPs, with some members having only a C2 domain, while others have an additional GRAM domain. The GRAM domain is mainly found in membrane-associated proteins and may play a role in protein and lipid binding.\u003c/p\u003e \u003cp\u003eCollinearity genes\u003c/p\u003e \u003cp\u003eGene duplication and divergence contribute to gene family expansion and the evolution of new functions\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. A collinear gene analysis was conducted within \u003cem\u003eLotus corniculatus\u003c/em\u003e to explore the origin and evolutionary history of the \u003cem\u003eLcC2DPs\u003c/em\u003e family. The \u003cem\u003eLcC2DPs\u003c/em\u003e family contains 10 pairs of homologous genes on chromosomes 1 to 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Collinearity analysis between \u003cem\u003eLotus corniculatus\u003c/em\u003e and Arabidopsis revealed 34 homologous relationships among 24 \u003cem\u003eLotus corniculatus\u003c/em\u003e and 25 genes Arabidopsis genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Gene pairings vary, with some Arabidopsis genes, like \u003cem\u003eAtC2DP\u003c/em\u003e22, corresponding to multiple \u003cem\u003eLotus corniculatus\u003c/em\u003e genes (\u003cem\u003eLcC2DP57\u003c/em\u003e, \u003cem\u003e79\u003c/em\u003e, \u003cem\u003e87)\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eCis-acting elements of promoter\u003c/p\u003e \u003cp\u003eCis-acting elements in gene promoter affect the expression patterns. To understand the function of \u003cem\u003eLcC2DPs\u003c/em\u003e gene family promoters, 2000 bp upstream sequences were analyzed by bioinformatics. These promoters are mostly enriched with elements responsive to light (GT1-motif), auxin (AuxRR-core), and abscisic acid (ABRE), related to the growth and development. They also contain elements for stress regulation, including responses to cold (MYB), drought (MYC or MBS), hypoxia (ARE), and defense (TC-rich repeats) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGO and KEGG enrichment pathways\u003c/p\u003e \u003cp\u003eGO analysis revealed that 90 \u003cem\u003eLcC2DP\u003c/em\u003e genes were categorized into 30 groups, spanning cellular components, molecular functions, and biological processes. The cellular components primarily involved endosome, cytoplasm, endoplasmic reticulum, membrane, lysosomal membrane, plasma desmosis, vesicles, and membranes. Molecular functions of the endoplasmic reticulum mainly included calcium ion binding, protein binding, calmodulin binding, enzyme regulator activity, GTPase activation, kinase activity, protein serine/threonine kinase activity, catalytic activity, organic acid and carboxylic acid binding. Biological processes covered abscisic acid response, hormone signaling pathways, cell signal transduction, growth and developmental regulation, abiotic stimulus response, oxidative stress and defense response regulation. GO analysis revealed that the pathways of hormone response, protein binding activation, and kinase activity are crucial for the biologically active of \u003cem\u003eC2DPs\u003c/em\u003e family members, including responses to environmental stress, growth regulation and other functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKEGG enrichment analysis identified 90 \u003cem\u003eLcC2DPs\u003c/em\u003e genes across 13 pathways, primarily involving material metabolism: substance metabolism and transportation, ubiquitin system, coenzyme and protein metabolism, genetic information processing, pantothenic acid biosynthesis, gene transcription. This indicates that the metabolism, ubiquitin system, and gene transcription mechanism are key pathways enriched for \u003cem\u003eLcC2DPs\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eGene clustering group\u003c/p\u003e \u003cp\u003eUnder abiotic stress, 90 \u003cem\u003eLcC2DP\u003c/em\u003e genes were grouped into different clusters\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In stem node tissue, gene expression showed three patterns: 53 genes were up-regulated (red), 25 were down-regulated (blue), and 12 showed no change (yellow), representing 58.89%, 27.8%, and 13.33% of the total, respectively. Over 0, 3, 6, 9, and 12 days, up-regulated genes numbered 36, 28, 43, 39, and 19, while down-regulated genes were 42, 49, 39, 35, and 39. Twelve genes showed no significant expression change over 12 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The \u003cem\u003eLcC2DPs\u003c/em\u003e family, expressed in various plant tissues, plays diverse roles in cellular stress response, mainly by regulating pathways.\u003c/p\u003e \u003cp\u003eGene expression pattern\u003c/p\u003e \u003cp\u003eqRT-PCR analysis of 10 \u003cem\u003eLcC2DPs\u003c/em\u003e genes screened in the root, stem, and leaf of \u003cem\u003eLotus corniculatus\u003c/em\u003e confirmed their expression in all these organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The expression levels of certain genes, like \u003cem\u003eLcC2DP1\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e, \u003cem\u003e24\u003c/em\u003e, \u003cem\u003e43\u003c/em\u003e, \u003cem\u003e60\u003c/em\u003e, \u003cem\u003e61\u003c/em\u003e, and \u003cem\u003e62\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), were notably higher in stems and leaves compared to roots. \u003cem\u003eLcC2DP8\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and \u003cem\u003eLcC2DP\u003c/em\u003e31, 36 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were primarily expressed in stems and leaves, respectively. This suggests that \u003cem\u003eLcC2DPs\u003c/em\u003e genes play a crucial roles across root, stem, and leaf.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of ABA-responsive (ABRE) and IAA-responsive (AuxRR-core) elements in \u003cem\u003eLotus corniculatus C2DPs\u003c/em\u003e family showed that treating plants with 100 \u0026micro;M ABA and IAA improved the growth over 15 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Samples were collected at various growth stages for qRT-PCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Results showed that \u003cem\u003eLcC2DPs\u003c/em\u003e family members respond to ABA and IAA hormones, with expression patterns varying over 0 to 15 days, forming four groups. The first group (\u003cem\u003eLcC2DP\u003c/em\u003e1, 24, and 31) had higher early expression with ABA (0\u0026ndash;9 or 0\u0026ndash;6 days) than IAA. The second group (\u003cem\u003eLcC2DP\u003c/em\u003e2, 8, 36, 43, and 61) showed opposite, higher expression trends for IAA and ABA throughout. The third group, \u003cem\u003eLcC2DP\u003c/em\u003e60, exhibited notable transcription fluctuations in response to both hormones across all stages (0\u0026ndash;6, 6\u0026ndash;12, and 12\u0026ndash;15 days). The fourth group, \u003cem\u003eLcC2DP\u003c/em\u003e62, showed higher gene expression under ABA treatment than IAA throughout the 0\u0026ndash;15 period.\u003c/p\u003e \u003cp\u003eCis-acting elements in gene promoters can help analyze gene expression patterns and functions. To investigate the \u003cem\u003eLcC2DPs\u003c/em\u003e gene family's functions, \u003cem\u003eLotus corniculatus\u003c/em\u003e was exposed to PEG6000 for drought simulation (0, 3, 6, 9, 12, and 15 days) and 4\u0026deg;C for cold stress (0, 6, 12, 24, 48, and 72 hours). Under drought, plant showed leaf curling, poor growth, and slow height increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-a). In cold conditions, wilting began after 12 hours, worsening in the upper leaves, and by 72 hours, the \u003cem\u003eLotus corniculatus\u003c/em\u003e drooped (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-b). qRT-PCR analysis of \u003cem\u003eLcC2DP\u003c/em\u003e genes in young leaves under drought and low-temperature conditions revealed differences in the expression of 10 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Compared to the initial expression, the \u003cem\u003eLcC2DP1\u003c/em\u003e gene showed fluctuating expression, being up-regulated in the early stages (3\u0026ndash;6 days, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and down-regulated later (12\u0026ndash;15 days, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 or p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). \u003cem\u003eLcC2DP8\u003c/em\u003e was up-regulated during the middle to later phase (6\u0026ndash;15 days, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while \u003cem\u003eLcC2DP24, 31, 43\u003c/em\u003e, and \u003cem\u003e60\u003c/em\u003e consistently showed increased expression throughout (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). \u003cem\u003eLcC2DP60\u003c/em\u003e and \u003cem\u003eLcC2DP61\u003c/em\u003e were up-regulated in the early to middle stages (3\u0026ndash;12 days, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnder the 4 ℃ environment, the expression of \u003cem\u003eLcC2DPs\u003c/em\u003e family genes in \u003cem\u003eLotus corniculatus\u003c/em\u003e varied (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). \u003cem\u003eLcC2DP1\u003c/em\u003e was significantly up-regulated between 12 and 48 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while \u003cem\u003eLcC2DP24\u003c/em\u003e showed up-regulation only at 72 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). \u003cem\u003eLcC2DP8\u003c/em\u003e and \u003cem\u003eLcC2DP36\u003c/em\u003e exhibited an \u0026lsquo;increase-decrease-rise\u0026rsquo; pattern. \u003cem\u003eLcC2DP43\u003c/em\u003e and \u003cem\u003eLcC2DP60\u003c/em\u003e were continuously down-regulated from 6 to 72 and 12 to 72 hours, respectively. Notably, \u003cem\u003eLcC2DP61\u003c/em\u003e showed no significant expression changes, indicating that \u003cem\u003eLcC2DP61\u003c/em\u003e is not induced by low temperature for transcription. \u003cem\u003eLcC2DP62\u003c/em\u003e in plant cell was up-regulated at 6\u0026ndash;72 hours. Results showed that in addition to \u003cem\u003eLcC2DP61\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e other genes are involved in cold response in \u003cem\u003eLotus corniculatus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eRegulatory pathways\u003c/p\u003e \u003cp\u003eThe C2DPs protein kinase family creates complex networks for cellular adaptation to stress\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. To explore the cooperative or division of labor among \u003cem\u003eC2DPs\u003c/em\u003e family members, they were analyzed using STRING (STRING://cn.string-db.org) for PCC statistics. Results showed that five core proteins, LcC2DP5, 11, 15, 38 and 58, primarily control the regulatory pathways among 90 family members, with 27 members interacting to varying extents with these core proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Results indicates that LcC2DP1, 20, and 67 proteins interact with XLG2 protein from the G-alpha superfamily, which acts as an energy-releasing engine in signal transduction\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The activation and conformational changes in \u003cem\u003eLcC2DPs\u003c/em\u003e depend on XLG2 activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm the response of five core genes to drought and low-temperature stress, qRT-PCR was used to measure the expression of \u003cem\u003eLcC2DP5\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e, \u003cem\u003e38\u003c/em\u003e, and \u003cem\u003e58\u003c/em\u003e. The study found that all five core genes responded to drought stress, initially increasing and then decreasing. Gene expression for \u003cem\u003eLcC2DP5\u003c/em\u003e and \u003cem\u003e15\u003c/em\u003e increased continuously from 0 to 9 days, \u003cem\u003eLcC2DP11\u003c/em\u003e and \u003cem\u003e38\u003c/em\u003e from 0 to 6 days, and \u003cem\u003eLcC2DP58\u003c/em\u003e from 0 to 3 days. \u003cem\u003eLcC2DP11\u003c/em\u003e showed a significant decrease at 12 days compared to day 0 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eLcC2DP11\u003c/em\u003e, \u003cem\u003e38\u003c/em\u003e, and \u003cem\u003e58\u003c/em\u003e had notable expression changes from 3 to 6 days (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, or P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eLcC2DP5\u003c/em\u003e showed the most significant expression from 9 to 15 days (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while \u003cem\u003eLcC2DP15\u003c/em\u003e was significantly expressed from 6\u0026ndash;12 days (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Five core genes also responded to low-temperature. \u003cem\u003eLcC2DP5\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e, and \u003cem\u003e38\u003c/em\u003e continuously increased from 0 to 72 hours at 4 ℃, while \u003cem\u003eLcC2DP15\u003c/em\u003e and \u003cem\u003eLcC2DP58\u003c/em\u003e peaked at 72 and 24 hours, respectively. \u003cem\u003eLcC2DP5\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e, and \u003cem\u003e38\u003c/em\u003e showed differential expression from 6 to 48 hours (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and decreased after 72 hours. \u003cem\u003eLcC2DP11\u003c/em\u003e and \u003cem\u003eLcC2DP15\u003c/em\u003e were significantly expressed from 48 to 72 hours (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). \u003cem\u003eLcC2DP58\u003c/em\u003e was significantly expressed only at 24 hours (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eCo-expression transcription factor\u003c/p\u003e \u003cp\u003eCell regulates its physiological process by TFs acting as mediators\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. To explore the abiotic stress response mechanism of \u003cem\u003eLcC2DPs\u003c/em\u003e family members, a Python script was used to identify co-expressed TFs for five core proteins. Only LcC2DP5 and LcC2DP15 provided co-expressed data, suggesting they are key in regulating abiotic stress responses within the \u003cem\u003eLcC2DPs\u003c/em\u003e family (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). This analysis was based on FPKM data from our existing transcriptome using a Python script\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, we conducted co-expression analysis of transcription factors (r\u0026thinsp;\u0026gt;\u0026thinsp;0.9). The study identified 49 types TFs, totaling 134, co-expressed with \u003cem\u003eLcC2DP5\u003c/em\u003e gene. The top eight TF types by member count include \u003cem\u003ebHLH\u003c/em\u003e (8), \u003cem\u003eWRKY\u003c/em\u003e (7), \u003cem\u003eC3H\u003c/em\u003e (7), \u003cem\u003ebZIP\u003c/em\u003e (7), \u003cem\u003eMYB\u003c/em\u003e (5), \u003cem\u003eMYB_related\u003c/em\u003e (5), \u003cem\u003eC2H2\u003c/em\u003e (5), and \u003cem\u003eNAC\u003c/em\u003e (5). Analysis showed the \u003cem\u003eLcC2DP15\u003c/em\u003e gene is co-expressed with 38 types, totaling 125, including \u003cem\u003ebHLH\u003c/em\u003e (15), \u003cem\u003eWRKY\u003c/em\u003e (14), \u003cem\u003eMYB_related\u003c/em\u003e (10), \u003cem\u003eNAC\u003c/em\u003e (9), \u003cem\u003ebZIP\u003c/em\u003e (9), \u003cem\u003eC2H2\u003c/em\u003e (7), and \u003cem\u003eC3H\u003c/em\u003e (7) (Table S3). These TF families are known to play roles in abiotic stress responses\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan additionalcitationids=\"CR51 CR52 CR53 CR54 CR55\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, suggesting that \u003cem\u003ebHLH\u003c/em\u003e, \u003cem\u003eWRKY\u003c/em\u003e, \u003cem\u003eC3H\u003c/em\u003e, \u003cem\u003ebZIP\u003c/em\u003e, \u003cem\u003eMYB\u003c/em\u003e, \u003cem\u003eNAC\u003c/em\u003e, \u003cem\u003eC2H2\u003c/em\u003e, and \u003cem\u003eMYB_related\u003c/em\u003e are key pathways for \u003cem\u003eLcC2DPs\u003c/em\u003e family in mediating abiotic stress regulation in plants.\u003c/p\u003e \u003cp\u003eExpression of transcription factors\u003c/p\u003e \u003cp\u003eDifferential expression analysis of key abiotic stress-related TFs (\u003cem\u003ebZIP\u003c/em\u003e, \u003cem\u003ebHLH\u003c/em\u003e, \u003cem\u003eWRKY\u003c/em\u003e, \u003cem\u003eNAC\u003c/em\u003e, \u003cem\u003eMYB_related\u003c/em\u003e, \u003cem\u003eMYB\u003c/em\u003e, \u003cem\u003eC3H\u003c/em\u003e, and \u003cem\u003eC2H2\u003c/em\u003e) in \u003cem\u003eLotus corniculatus\u003c/em\u003e under PEG-induced drought showed that all eight TFs were up-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The \u003cem\u003ebHLH\u003c/em\u003e was significantly expressed only at the initial stage (3 days) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) before decreasing. \u003cem\u003eMYB_related\u003c/em\u003e was significantly expressed during both the initial and middle stages (3\u0026ndash;9 days) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with reduced expression after 9 days (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast, other TFs like \u003cem\u003ebZIP\u003c/em\u003e, \u003cem\u003eWRKY\u003c/em\u003e, \u003cem\u003eNAC\u003c/em\u003e, \u003cem\u003eMYB\u003c/em\u003e, \u003cem\u003eC3H\u003c/em\u003e, and \u003cem\u003eC2H2\u003c/em\u003e were up-regulated throughout the stress period (3\u0026ndash;15 days) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, or P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with \u003cem\u003ebZIP\u003c/em\u003e, \u003cem\u003eNAC\u003c/em\u003e, and \u003cem\u003eC2H2\u003c/em\u003e TFs showing the most significant differential expression in response to drought (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In \u003cem\u003eLotus corniculatus\u003c/em\u003e exposed to low temperature (4\u0026deg;C), all eight TFs (\u003cem\u003ebZIP\u003c/em\u003e, \u003cem\u003ebHLH\u003c/em\u003e, \u003cem\u003eWRKY\u003c/em\u003e, \u003cem\u003eNAC\u003c/em\u003e, \u003cem\u003eMYB_related\u003c/em\u003e, \u003cem\u003eMYB\u003c/em\u003e, \u003cem\u003eC3H\u003c/em\u003e, and \u003cem\u003eC2H2\u003c/em\u003e) co-expressed with \u003cem\u003eLcC2DPs\u003c/em\u003e were induced by cold stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). \u003cem\u003eMYB_related\u003c/em\u003e showed differential expression in early and middle stages (6\u0026ndash;24 hours) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and \u003cem\u003eMYB\u003c/em\u003e in initial stage (6\u0026ndash;12 hours), both peaking at 6 hours (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). \u003cem\u003eNAC\u003c/em\u003e and \u003cem\u003eC3H\u003c/em\u003e were active during early and middle phases (6\u0026ndash;48 hours) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of core TFs linked to \u003cem\u003eLcC2DPs\u003c/em\u003e under drought and low temperature stress showed that \u003cem\u003ebZIP\u003c/em\u003e was consistently up-regulated throughout both conditions (3\u0026ndash;12 days for drought and 6\u0026ndash;48 hours for low temperature) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). \u003cem\u003ebHLH\u003c/em\u003e was up-regulated early in drought (3 days) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and entire low temperature exposure (6\u0026ndash;72 hours) but down-regulated later in both stresses (15 days for drought and 72 hours for low temperature) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eWRKY\u003c/em\u003e was up-regulated during 3\u0026ndash;6 and 12\u0026ndash;15 days of drought, and at 6 and 24\u0026ndash;72 hours of low temperature treatment (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eNAC\u003c/em\u003e was consistently up-regulated during drought period (3\u0026ndash;15 days) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but down-regulated in later stage (72 hours) under low temperature, showing different regulatory patterns for each stress. \u003cem\u003eMYB_related\u003c/em\u003e had varied expression early on, then gradually decreased. The transcription of \u003cem\u003eMYB\u003c/em\u003e differed between stresses, increasing under drought and decreasing under low temperature (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, or P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eC3H\u003c/em\u003e and \u003cem\u003eC2H2\u003c/em\u003e showed differential expression patterns under drought and low temperature stress. \u003cem\u003eC3H\u003c/em\u003e was up-regulated early and mid-stress (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while \u003cem\u003eC2H2\u003c/em\u003e was consistently up-regulated (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Under drought, TFs expression ranked: \u003cem\u003eNAC\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eC2H2\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eWRKY\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003ebZIP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eC3H\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eMYB\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eMYB_related\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003ebHLH.\u003c/em\u003e In low temperatures, it was: \u003cem\u003ebZIP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eMYB_related\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eC2H2\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eC3H\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eNAC\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003ebHLH\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eMYB\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eWRKY\u003c/em\u003e. \u003cem\u003eNAC\u003c/em\u003e and \u003cem\u003eC2H2\u003c/em\u003e were key under drought, while \u003cem\u003ebZIP\u003c/em\u003e and \u003cem\u003eMYB_related\u003c/em\u003e were crucial under low temperature.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe continuing warming of global climate has severely affected grass growth and development. Research into genes involved in stress regulation will be essential for grass molecular breeding. This paper identifies and analyzes 90 \u003cem\u003eC2DP\u003c/em\u003e genes in \u003cem\u003eLotus corniculatus\u003c/em\u003e genome, which are linked to stress responses in rice (\u003cem\u003eOryza sativa\u003c/em\u003e L)\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, cotton (\u003cem\u003eGossypium hirsutum\u003c/em\u003e L)\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eArabidopsis\u003c/em\u003e (\u003cem\u003eArabidopsis thaliana\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e were found in of \u003cem\u003eLotus corniculatus\u003c/em\u003e (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The \u003cem\u003eLcC2DP\u003c/em\u003e gene family is larger than in rice, cotton and \u003cem\u003eArabidopsis\u003c/em\u003e, which have 82, 31, and 16 \u003cem\u003eC2DP\u003c/em\u003e genes, respectively. Rich functionality in a gene family is contingent upon its member numbers\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. The complexity and diversity of \u003cem\u003eC2DPs\u003c/em\u003e gene family suggest that variations among plant species may result from gene loss and duplication, key factors in gene family evolution. These processes can lead to the emergence of new genes with unique functions\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAfter identifying \u003cem\u003eLcC2DPs\u003c/em\u003e family, their physicochemical properties were examined (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Subcellular localization showed that LcC2DPs proteins in the cell nucleus and cytoplasm, similar to CDPK in other plant\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. This suggests that LcC2DP proteins activate transcription factors and regulate gene expression in response to signals, like other protein kinases. The division of labor family members is linked to their nuclear and cytoplasmic localization, affecting their role in signal transduction.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLcC2DP\u003c/em\u003e genes primarily cluster on chromosomes 1, 2, 3, and 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), indicating possible tandem duplications in these regions of the \u003cem\u003eLotus corniculatus\u003c/em\u003e genome\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Such duplications are key in stress resistance, membrane function in rice and \u003cem\u003eArabidopsis\u003c/em\u003e, and signal transduction in legumes\u003csup\u003e\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. This implies that many \u003cem\u003eLcC2DP\u003c/em\u003e genes play roles in stress responses and cellular signal transduction, potentially explaining the large number of family members in plants like rice and sweet potato, and suggesting interactions between chromosomes. Phylogenetic analysis based on gene structure classified 90 family members into five groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The \u003cem\u003eC2DP\u003c/em\u003e genes of \u003cem\u003eArabidopsis\u003c/em\u003e were similarly divided into five groups\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, confirming our analysis. \u003cem\u003eC2DPs\u003c/em\u003e genes in \u003cem\u003eLotus corniculatus\u003c/em\u003e outnumber those in cotton and rice\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eC2DPs\u003c/em\u003e were grouped into 5 in \u003cem\u003eArabidopsis\u003c/em\u003e and 7 in rice, with rice having fewer members than \u003cem\u003eLotus corniculatus\u003c/em\u003e. Gene functional redundancy has emerged during evolution. Gene duplication is a key driver of new gene functions but can cause redundancy. This redundancy helps maintain metabolic balance across environmental changes and development\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. The study indicates that under purifying selection, duplicated genes varied expression or specialization supports plant adaptation to diverse environments\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGenome analysis shows that tandem duplication plays a crucial role in gene family expansion by generating new genes and clusters\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e, In this study, tandem duplication analysis identified 10 pairs of collinear homologous genes within \u003cem\u003eLotus corniculatus\u003c/em\u003e and 34 pairs between \u003cem\u003eLotus japonicas\u003c/em\u003e and Arabidopsis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). This suggests that tandem duplication has contributed to gene expansion in the \u003cem\u003eLotus corniculatus\u003c/em\u003e genome, potentially aiding its adaptation to complex environmental conditions and physiological functions compared to plant like potato\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Collinearity analysis revealed only 20 homologous gene pairs between \u003cem\u003eCucumis melo\u003c/em\u003e, \u003cem\u003eVitis vinifera\u003c/em\u003e, and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Our findings indicate a strong evolutionary relationship between the \u003cem\u003eLcC2DPs\u003c/em\u003e gene family and Arabidopsis, suggesting similar functions. Overall, \u003cem\u003eLotus corniculatus\u003c/em\u003e shares a similar evolutionary process and genetic trait variation with Arabidopsis and other organisms.\u003c/p\u003e \u003cp\u003eCis-acting elements in promoters can elucidate gene expression regulation, cellular signaling network, and gene-environment interactions\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. The prediction of cis-acting elements in \u003cem\u003eLcC2DP\u003c/em\u003e promoter regions identified multiple hormone (AuxRR-core and ABRE) and stress response elements (MYC, MBS, ARE, TC-rich, and MYB) were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Evidence suggests that \u003cem\u003eAtCDPKs\u003c/em\u003e is involved in external stimuli responses with ABA\u003csup\u003e71\u0026ndash; \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eCDPKs\u003c/em\u003e have been linked to abiotic stresses like drought, cold, heat, and salinity\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. This indicates \u003cem\u003eLcC2DP\u003c/em\u003e family genes may play a complex role in stress responses related to plant hormone regulation. Changes in \u003cem\u003eLcC2DPs\u003c/em\u003e gene expression under hormone and abiotic stress were analyzed using qRT-PCR. This study confirms that \u003cem\u003eLcC2DPs\u003c/em\u003e is involved in plant hormone and abiotic stress responses, including ABA, IAA, drought, and low temperature.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCDPK\u003c/em\u003e regulate ABA-mediated signaling and influence phytohormones responses, such as IAA and cytokinins (CTK)\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. qRT-PCR results showed \u003cem\u003eLcC2DP\u003c/em\u003e's sensitivity to 100 \u0026micro;M ABA and IAA in \u003cem\u003eLotus corniculatus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), leading to its classification into four groups based on the hormones response differences over time. Specifically, \u003cem\u003eLcC2DP1\u003c/em\u003e, \u003cem\u003e24\u003c/em\u003e, \u003cem\u003e31\u003c/em\u003e, \u003cem\u003eLcC2DP2\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e, \u003cem\u003e36\u003c/em\u003e, \u003cem\u003e43\u003c/em\u003e, \u003cem\u003e61\u003c/em\u003e, \u003cem\u003eLcC2DP60\u003c/em\u003e, and \u003cem\u003eLcC2DP62\u003c/em\u003e belong to groups 1, 2, 3, 4, respectively, according to RNA-seq data\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. CDPK proteins are crucial for signal transduction, gene expression, growth, and development by binding to Ca\u003csup\u003e2+\u003c/sup\u003e, and they enhance plant resilience to various stresses\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Our study highlights the diverse biological roles of protein kinases. In grape and rice\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eVpCDPK16\u003c/em\u003e and \u003cem\u003eOsCPK21\u003c/em\u003e respond to ABA signaling, while IAA affect CDPK expression and activity, with \u003cem\u003eNtCDPK1\u003c/em\u003e transcripts increasing upon IAA treatment\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. The expression level of different \u003cem\u003eLcC2DP\u003c/em\u003e genes in \u003cem\u003eLotus corniculatus\u003c/em\u003e, treated with ABA and IAA, suggest a potential role in hormone signaling regulation.\u003c/p\u003e \u003cp\u003eThe cis-acting elements analysis and qRT-PCR assays on tender leaves exposed to PEG6000 and low temperature (4 ℃) revealed that 10 gene showed expression differences under drought and cold conditions. These genes exhibited varying expression patterns during drought treatment, categorized into four models: \u003cem\u003eLcC2DP1\u003c/em\u003e, \u003cem\u003e24, 31\u003c/em\u003e; \u003cem\u003eLcC2DP2, 8, 36, 43\u003c/em\u003e, and \u003cem\u003e61\u003c/em\u003e; \u003cem\u003eLcC2DP60\u003c/em\u003e; \u003cem\u003eLcC2DP62\u003c/em\u003e groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). CDPK are crucial for \u003cem\u003eArabidopsis\u003c/em\u003e under drought and other stresses\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. For example, \u003cem\u003eGhCDPK60\u003c/em\u003e enhances drought resistance\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e, while \u003cem\u003eGhCDPK4\u003c/em\u003e boosts tobacco's drought tolerance\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. In conclusion, \u003cem\u003eLcC2DPs\u003c/em\u003e family is closely linked to drought response and exhibits complex regulation in \u003cem\u003eLotus corniculatus.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eCDPK proteins have diverse roles in responding to abiotic stresses like cold, salinity, and heat in plants\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eLcC2DP1, 2, 24\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e, \u003cem\u003e31, 36\u003c/em\u003e, \u003cem\u003e60\u003c/em\u003e, and \u003cem\u003e62\u003c/em\u003e genes show varied expression at different stages under low-temperatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). \u003cem\u003eLcC2DP61\u003c/em\u003e showed no significant changes throughout the process and was unaffected by low temperature. \u003cem\u003eLcC2DP61\u003c/em\u003e's expression did not align with its promoter's \u0026lsquo;defense and stress responsiveness\u0026rsquo; elements, possibly due to other regulatory like trans-acting factor\u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. Besides, \u003cem\u003eLcC2DP43\u003c/em\u003e expression significantly decreased at each stage in 4 ℃ environment, indicating a unique transcription model pattern. \u003cem\u003eShCDPK12\u003c/em\u003e and \u003cem\u003eShCDPK30\u003c/em\u003e function differently in response to cold stress in \u003cem\u003eSolanum habrochaites\u003c/em\u003e and Arabidopsis\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eCDPKs\u003c/em\u003e respond to abiotic stresses like drought, heat, salinity, and cold\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. This study reveals that \u003cem\u003eLcC2DPs\u003c/em\u003e reacts differently to low-temperature, possibly due to their abundance of stress-related cis-elements and family members.\u003c/p\u003e \u003cp\u003eProtein-protein interactions are crucial in biological pathways\u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. Using the online tool STRING (available at //cn.string-db.org) with Cytoscape for predicting and visualizing the interactions among \u003cem\u003eLcC2DPs\u003c/em\u003e family members. Analysis revealed that this family has five core proteins (\u003cem\u003eLcC2DP5\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e, \u003cem\u003e38\u003c/em\u003e, and \u003cem\u003e58\u003c/em\u003e). \u003cem\u003eLcC2DP1\u003c/em\u003e, \u003cem\u003eLcC2DP20\u003c/em\u003e, and \u003cem\u003eLcC2DP67\u003c/em\u003e interact with XLG2, a G-alpha superfamily member (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). XLG2 and G-alpha protein positively influence hormone signal transduction like IAA, ABA, and gibberellin (GA), affecting cell division, developmental, and stress response in plants\u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e. Thus, \u003cem\u003eLcC2DP\u003c/em\u003e family genes are crucial in plant hormone signaling and stress response pathways.\u003c/p\u003e \u003cp\u003eThe TFs bind to cis-regulatory elements in target gene regions to regulate plant growth, development, and stress responses\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. A Python script identified co-expressed TFs for five core proteins, revealing that only LcC2DP5 and LcC2DP15 provided insights with co-expressed TFs. This suggests they are the central genes in the \u003cem\u003eLcC2DPs\u003c/em\u003e family involved in plant abiotic stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Co-expression analysis identified eight TFs (\u003cem\u003ebZIP\u003c/em\u003e, \u003cem\u003ebHLH\u003c/em\u003e, \u003cem\u003eWRKY\u003c/em\u003e, \u003cem\u003eNAC\u003c/em\u003e, \u003cem\u003eMYB_related\u003c/em\u003e, \u003cem\u003eMYB\u003c/em\u003e, \u003cem\u003eC3H\u003c/em\u003e, and \u003cem\u003eC2H2\u003c/em\u003e) in the regulation network of \u003cem\u003eLcC2DP5\u003c/em\u003e and \u003cem\u003eLcC2DP15\u003c/em\u003e for drought and 4 ℃ responses. Among them, \u003cem\u003ebHLH\u003c/em\u003e and \u003cem\u003eMYB\u003c/em\u003e regulate drought, \u003cem\u003eNAC\u003c/em\u003e and \u003cem\u003eMYB_related\u003c/em\u003e regulate low temperature, while \u003cem\u003ebZIP\u003c/em\u003e, \u003cem\u003eC2H2\u003c/em\u003e, \u003cem\u003eWRKY\u003c/em\u003e, \u003cem\u003eand C3H\u003c/em\u003e form a common regulatory network under these stresses. The qRT-PCR analysis confirmed these findings after PEG6000 and 4 ℃ treatments.\u003c/p\u003e \u003cp\u003eNAC (NAM, ATAF1/2, and CUC2) and MYB proteins are important factors in the stress response\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e. Our study showed that \u003cem\u003eNAC\u003c/em\u003e and \u003cem\u003eMYB\u003c/em\u003e expression increased after 20% PEG treatment for 15 days in \u003cem\u003eLotus corniculatus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA), highlighting their role as key stress response regulators. As a crucial regulator in heat and drought stress responses, \u003cem\u003eSNAC3\u003c/em\u003e, in particular, enhances plant tolerance to heat and drought\u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e. Overexpressing \u003cem\u003eTaMYB33\u003c/em\u003e and \u003cem\u003eGbMYB5\u003c/em\u003e genes enhances the drought tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e and tobacco\u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, indicating that \u003cem\u003eNAC\u003c/em\u003e and \u003cem\u003eMYB\u003c/em\u003e transcription factors are vital for drought resistance in \u003cem\u003eLotus corniculatus\u003c/em\u003e. Low temperature pose significant environmental challenges, impacting plant physiology and yield\u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e. This study shows that \u003cem\u003ebHLH\u003c/em\u003e and \u003cem\u003eMYB-related\u003c/em\u003e are highly expressed at 4\u0026deg;C in \u003cem\u003eLotus corniculatus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), with \u003cem\u003ebHLH\u003c/em\u003e family members playing a key role in responding to cold stress by regulating cold -responsive genes like \u003cem\u003eCOR\u003c/em\u003e (cold-responsive) and \u003cem\u003eCBF\u003c/em\u003e (cold-regulated binding factor)\u003csup\u003e\u003cspan additionalcitationids=\"CR89\" citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u003c/sup\u003e. Simultaneously, the \u003cem\u003eMYB_related\u003c/em\u003e family is crucial in responding to low temperature stress. Research shows that \u003cem\u003eAhMYB30\u003c/em\u003e from peanut \u003cem\u003eMYB_related\u003c/em\u003e family enhances the freezing tolerance in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e via DREB/CBF and ABA pathways\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Our findings indicate that \u003cem\u003ebHLH\u003c/em\u003e and \u003cem\u003eMYB_related\u003c/em\u003e transcription factors are key in regulating \u003cem\u003eLotus\u003c/em\u003e's response to low temperature.\u003c/p\u003e \u003cp\u003eNotably, high expression of \u003cem\u003ebZIP\u003c/em\u003e, \u003cem\u003eWRKY, C3H\u003c/em\u003e, and \u003cem\u003eC2H2\u003c/em\u003e was detected in both drought and cold conditions, suggesting their role in relating these stresses in \u003cem\u003eLotus corniculatus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, B). Various TF families, such as \u003cem\u003ebZIP\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eWRKY\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eC3H\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eC2H2\u003c/em\u003e\u003csup\u003e27\u003c/sup\u003e, are crucial in regulating plant responses to stress, particularly cold and drought, by enhancing tolerance. Our findings support these roles. \u003cem\u003ebZIP\u003c/em\u003e, \u003cem\u003eWRKY, C3H\u003c/em\u003e, and \u003cem\u003eC2H2\u003c/em\u003e genes are activated by cold and drought stress. While \u003cem\u003ebHLH\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003ebZIP\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eNAC\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eMYB_related\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eMYB\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eC2H2\u003c/em\u003e\u003csup\u003e95\u003c/sup\u003egenes are found in both the nucleus and cytoplasm, \u003cem\u003eWRKY\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eC3H\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e genes are localized in the nucleus. This study implicates that \u003cem\u003eNAC\u003c/em\u003e, \u003cem\u003eC2H2\u003c/em\u003e, \u003cem\u003ebHLH\u003c/em\u003e, \u003cem\u003eMYB\u003c/em\u003e, \u003cem\u003ebZIP\u003c/em\u003e, and \u003cem\u003eMYB_related\u003c/em\u003e TFs move to the nucleus after cytoplasmic activation, whereas \u003cem\u003eWRKY\u003c/em\u003e and \u003cem\u003eC3H\u003c/em\u003e are activated in the nucleus to regulate gene expression.\u003c/p\u003e \u003cp\u003e \u003cem\u003eC2DP\u003c/em\u003e, a calcium-dependent protein kinase, is activated by Ca\u003csup\u003e2+\u003c/sup\u003e during stress signal transduction, leading to phosphorylation and related TFs expression for stress response\u003csup\u003e\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eZmNAC84\u003c/em\u003e phosphorylation activates downstream genes by CDPK to participate in ABA-induced antioxidant defense\u003csup\u003e\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e\u003c/sup\u003e. Additionally, \u003cem\u003eMYB\u003c/em\u003e is involved in the ABA signaling pathway for drought tolerance, regulated by PYR/PYL/RCARs, protein phosphate 2C (PP2Cs), and SNF1-related protein kinase 2s (nRK2s) in \u003cem\u003eArabidopsis\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ebHLH\u003c/em\u003e plays a role in plant's response to low temperatures through CDPK phosphorylation pathways\u003csup\u003e\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e\u003c/sup\u003e. MAPK (mitogen-activated protein kinase) activation enhances the cold resistance via \u003cem\u003eMYB-related\u003c/em\u003e under low temperature stress\u003csup\u003e\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e\u003c/sup\u003e. It is suggested that \u003cem\u003eLcC2PDs\u003c/em\u003e phosphorylate activates \u003cem\u003eNAC\u003c/em\u003e, \u003cem\u003eMYB\u003c/em\u003e, and \u003cem\u003ebHLH\u003c/em\u003e, \u003cem\u003eMYB-related\u003c/em\u003e to improve drought and cold stress tolerance in \u003cem\u003eLotus\u003c/em\u003e plants. Additionally, \u003cem\u003ebZIP\u003c/em\u003e, \u003cem\u003eWRKY\u003c/em\u003e, \u003cem\u003eC3H\u003c/em\u003e, and \u003cem\u003eC2H2\u003c/em\u003e are involved in both drought and cold stress responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Understanding these TFs can provide insights into plant abiotic regulation, aiding in the functional identification and analysis of stress regulation genes in forage crops.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study used bioinformatics and experiments to identify and analyze the LcC2DP protein kinase family in \u003cem\u003eLotus corniculatus\u003c/em\u003e, finding 90 \u003cem\u003eLcC2DP\u003c/em\u003e genes divided into five subgroups. Homologous genes pairs were present in both in \u003cem\u003eLotus corniculatus\u003c/em\u003e and Arabidopsis chromosomes. The \u003cem\u003eLcC2DPs\u003c/em\u003e promoters contained hormone response elements (AuxRR-core, ABRE) and stress response elements (MYB, MYC, MBS) for cold and drought. GO enrichment revealed hormone response and kinase activity functions, and 13 KEGG pathways were identified. RNA-seq analysis showed 78 \u003cem\u003eLcC2DPs\u003c/em\u003e related to stress response, with 10 genes linked to ABA, IAA, drought, and low temperature through qRT-PCR. Protein-protein interaction analysis identified five core proteins, with LcCDP5 and LcC2DP15 co-expressed with various TFs, including \u003cem\u003ebZIP\u003c/em\u003e, \u003cem\u003ebHLH\u003c/em\u003e, \u003cem\u003eWRKY\u003c/em\u003e, \u003cem\u003eNAC\u003c/em\u003e, \u003cem\u003eMYB_related\u003c/em\u003e, \u003cem\u003eMYB\u003c/em\u003e, \u003cem\u003eC3H\u003c/em\u003e, and \u003cem\u003eC2H2\u003c/em\u003e, under stress conditions. \u003cem\u003eNAC\u003c/em\u003e and \u003cem\u003eC2H2\u003c/em\u003e primarily respond to drought stress, while \u003cem\u003ebZIP\u003c/em\u003e and \u003cem\u003eMYB_related\u003c/em\u003e respond to low temperature. These findings form a basis for exploring \u003cem\u003eLcC2DP\u003c/em\u003e family gene mechanisms in drought and cold stress responses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by the National Natural Science Foundation of China (No.32260338) and (No.31660685), and the Guizhou Province Science and Technology Project (2023ZK119).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor contribution LS conceived and designed the experiments. GY performed the experiments. GY carried out the bioinformatics analysis. GY wrote the manuscript. YL and SC gave insightful suggestions. LS improved the manuscript errors and English language. All authors read and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank master student Zouxian Gong (Clinical Medical College of Guizhou Medical University, Guiyang 550004, Guizhou Province, China.) for participating in the English revision.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, Beijing Institute of Genomics (China National Center for Bioinformation), Chinese Academy of Sciences, under accession number CRA002426 that are publicly accessible at https://bigd.big.ac.cn/gsa.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePizzorni, M., Innocent, A \u0026amp; Tollin, N. 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Research advances of \u003cem\u003eMYB\u003c/em\u003e transcription factors in plant stress resistance and breeding\u003cem\u003e.\u003c/em\u003e \u003cem\u003ePlant Signal Behav\u003c/em\u003e \u003cstrong\u003e14, \u003c/strong\u003e1613131. https://10.1080/15592324.2019.1613131 (2019).\u003c/li\u003e\n\u003cli\u003ePraat, M., De Smet, I. \u0026amp; van Zanten, M. Protein kinase and phosphatase control of plant temperature responses\u003cem\u003e.\u003c/em\u003e \u003cem\u003eJ Exp Bot \u003c/em\u003e72. https://10.1093/jxb/erab345 (2021).\u003c/li\u003e\n\u003cli\u003eAbdullah, S.N.A., Azzeme, A.M. \u0026amp; Yousefi, K. Fine-Tuning Cold Stress Response Through Regulated Cellular Abundance and Mechanistic Actions of Transcription Factors\u003cem\u003e.\u003c/em\u003e \u003cem\u003eFront Plant Sci\u003c/em\u003e \u003cstrong\u003e13, \u003c/strong\u003e850216. https://10.3389/fpls.2022.850216 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"calcium-dependent protein kinase, C2 domain, Lotus corniculatus, Signal transduction, Drought response, Cold stress","lastPublishedDoi":"10.21203/rs.3.rs-5763738/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5763738/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLow temperatures and drought reduce forage yield and quality, with protein kinases crucial for plant stress response. This study examines the LcC2DPs protein kinase family in \u003cem\u003eLotus corniculatus\u003c/em\u003e, identifying 90 members, with some tandemly distributed on chromosomes 2\u0026ndash;6, and grouped into 5 subfamilies (I-V). 34 homologous gene pairs were found in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. \u003cem\u003eLcC2DP\u003c/em\u003e genes promoters contain hormone and stress response elements. GO analysis highlights enrichment in hormone response and kinase activity. Transcriptomic analysis links 78 genes to environmental response and stress growth, with 10 validated by qRT-PCR after treatment with 100 \u0026micro;M ABA and IAA, 20% PEG6000, and 4\u0026deg;C. Protein interaction analysis identifies 5 core proteins (LcC2DP5, 11, 15, 38, and 58) activated by drought and cold stress. Gene analysis revealed that only LcC2DP5 and LcC2DP15 share co-expression transcription factors, with \u003cem\u003ebZIP\u003c/em\u003e, \u003cem\u003ebHLH\u003c/em\u003e, \u003cem\u003eWRKY\u003c/em\u003e, \u003cem\u003eNAC\u003c/em\u003e, \u003cem\u003eMYB_related\u003c/em\u003e, \u003cem\u003eMYB\u003c/em\u003e, \u003cem\u003eC3H\u003c/em\u003e, and \u003cem\u003eC2H2\u003c/em\u003e being prominent. These proteins are expressed under drought and cold conditions, highlighting \u003cem\u003eLcC2DP5\u003c/em\u003e and \u003cem\u003eLcC2DP15\u003c/em\u003e activity. \u003cem\u003eNAC\u003c/em\u003e and \u003cem\u003eC2H2\u003c/em\u003e are vital for drought response, while \u003cem\u003ebZIP\u003c/em\u003e and \u003cem\u003eMYB_related\u003c/em\u003e are important for cold response. This suggests that various \u003cem\u003eLcC2DPs\u003c/em\u003e in \u003cem\u003eLotus corniculatus\u003c/em\u003e respond to hormones and stress via a TF regulatory network.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification of LcC2DPs gene family in Lotus corniculatus provides insights into regulatory network in response to abiotic stresses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-13 07:12:47","doi":"10.21203/rs.3.rs-5763738/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-07T11:24:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-05T15:31:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54507313729436835900843200087630805685","date":"2025-02-20T15:00:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"265628273026427874383152766026767477407","date":"2025-02-18T03:16:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-17T13:04:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"130322016541606774353240591769675518050","date":"2025-02-16T08:00:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-15T14:10:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-10T04:34:38+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-01-09T12:42:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-09T09:55:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-01-04T13:12:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9d756adf-ffd5-4efa-ba69-ae343ed7b58f","owner":[],"postedDate":"January 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":42658640,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":42658641,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-04-21T16:00:18+00:00","versionOfRecord":{"articleIdentity":"rs-5763738","link":"https://doi.org/10.1038/s41598-025-97896-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-04-18 15:57:27","publishedOnDateReadable":"April 18th, 2025"},"versionCreatedAt":"2025-01-13 07:12:47","video":"","vorDoi":"10.1038/s41598-025-97896-2","vorDoiUrl":"https://doi.org/10.1038/s41598-025-97896-2","workflowStages":[]},"version":"v1","identity":"rs-5763738","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5763738","identity":"rs-5763738","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}