Identification and Expression Analysis of the MADS-box Gene Family in Camellia sinensis

Identification and Expression Analysis of the MADS-box Gene Family in Camellia sinensis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification and Expression Analysis of the MADS-box Gene Family in Camellia sinensis Jing Yang, Hanxue Wei, Guangzhen Zhou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7855824/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A woody crop that has been important to the economy is Camellia sinensis . Tea plants may experience physiological and metabolic changes as a result of drought and high salinity. The MADS-box gene family is a significant of transcription factors essential for various facets of plant growth and development. However, comprehensive research on MADS-box genes in C.sinensis is still sparse. In this study, based on the genomic datas of C. sinensis , a total of 70 MADS-box genes were identified and phylogenetically categorized into two types Type I (which includes Mα, Mβ, Mγ, and Mγ, consisting of 26 members) and Type II (comprising MIKCC and MIKC*, with 44 members). Members of the same subfamily have very conserved exon-intron patterns and protein motifs, according to gene structure analysis. The initial detailed study of the MADS-box gene family expression profiles and evolutionary features of this gene in C.sinensis is presented in this paper. Moreover, qRT-PCR tests showed that these genes are frequently engaged in the reaction to salt stress and drought. These results not only offer valuable insights into the genetic mechanisms that contribute to tea plant diversity but also lay the groundwork for molecular breeding efforts focused on enhancing tea quality and aesthetic characteristics. C.sinensis MADS-box gene family Gene expression pattern Abiotic stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Camellia sinensis belongs to the family Theaceae, and it is a significant economic crop of the genus Camellia. This plant is extensively cultivated throughout tropical and subtropical zones and is prized for the intense fragrance of the tea derived from its leaves [ 1 ] . Higher plants are frequently exposed to environmental stressors during growth and development. Drought, low temperature, and high salinity are some of the most prevalent abiotic stresses that can elicit various physiological and metabolic responses in plants, hinder their metabolism and growth, and, in extreme cases, result in irreversible damage or even death. [ 2 ] . The MADS-box gene family is widely found in plants, animals, and fungi, and plays a central role in multiple stages of plant development [ 3 – 5 ] . This gene family includes members that control different parts of the plant growth and development process, such as the stress response, the length of the flowering period, the maintenance of the floral meristem, the formation of thefloral organ, and the development of fruit [ 6 – 7 ] . The MADS-box gene family has a core function in plant responses to abiotic stress, highlighting its role as a key integrator of environmental signals and developmental regulation. By modulating processes such as hormone signaling, ROS homeostasis, nutrient response, and flowering time, MADS-box genes help plants achieve survival and reproductive success in changing environments [ 8 ] . The name of the MADS-box gene family derives from its conserved DNA-binding domain(the MADS domain), formed from the initials of four founder genes:M(MCM1, yeast), A(AGAMOUS, Arabidopsis), D(DEFICIENS, snapdragon), and S(SRF, human) [ 9 ] . The MADS-box genes may be separated into the two main classes according to protein domain architecture and phylogenetic relationships:Type I Mα、Mβ、Mγand Type II.(C)e II gene are specific to plants and contain, besides the MADS domain, three additional domains:I(intervening), K-box(keratin-like), and a variable C-terminal domain(C). They are additionally categorized into MIKCC and MIKC*subtypes [ 10 ] . With known expression patterns or mutant characteristics, the majority are made up of the MIKCC kind, which also includes all of the plant MADS-box genes. MIKCC-type genes can be divided into 12 different clades according to phylogeny: AG-like, AGL12-like, AGL15-like, AGL17-like, AGL6-like, AP1-like, AP3/PI-like, Bsister, FLC-like, SEP-like, SOC1-like, and SVP-like. Type I genes evolve more rapidly and have relatively diverse functions, whereas Type II genes are more conserved in floral organ development and form the genetic basis of the classical ABC(D)E model [ 11 – 12 ] . Yifei Mou et al. were the first to comprehensively identify the MIKC-type MADS-box gene family in peanuts, revealing its phylogeny, structural characteristics, expression patterns, and potential functions in abiotic stress responses. Their results indicated that peanut MADS-box genes may be involved in various stress adaptation processes through hormone signaling pathways and stress-responsive elements [ 13 ] . Xue Y et al. first identified a MIKCC-class MADS transcription factor gene, GhFYF, in cotton. They discovered that it is concentrated in the nucleus and is highly expressed in flowers and seeds.This gene is activated by drought and salt stress;its overexpression improves salt tolerance in Arabidopsis, boosts seed germination and proline accumulation, and interacts with the stress-responsive protein GhGPP2.These data show that GhFYF may be important in abiotic stress responses, especially salt stress, in cotton [ 14 ] . Lu J et al. systematically identified the MADS-box gene family in flax and clarified the key functions of these genes in floral organ development and stress response. Their study further revealed that Type II genes are primarily involved in floral organ development, whereas Type I genes are more associated with embryonic development. In addition, multiple LuMADS genes showed significant expression changes under cold, drought, and salt stress conditions, among which LuMADS27 exhibited regulatory roles under multiple stresses [ 15 ] . Although abiotic stresses significantly impact C. sinensis , the role of the MADS-box gene family remains poorly characterized. To fill this knowledge gap, this study provides a genome-wide systematic analysis. Firstly, we identified all MADS-box genes and analyzed their phylogenetic evolution, gene structure, and conserved motifs. Additionally, RNA-seq and qRT-PCR were employed to determine their expression patterns under different abiotic stresses.The findings will contribute to elucidating the molecular regulatory mechanisms of C. sinensis under stress and provide a theoretical basis and candidate genes for molecular breeding and genetic improvement of tea plants. 2. Results 2.1 Identification of MADS-box gene family members in C. sinensis . To ensure a comprehensive screening of MADS-box gene family members, putative MADS-box genes were identified by combining the results of Hidden Markov Model (HMM) and BLAST search. Our genome-wide analysis identified 70 members of the MADS-box gene family in C. sinensis. These proteins exhibited different lengths, with the longest being 1358 aa ( CaS14G024890.1 ) and the shortest being 100 aa ( CaS05G005620.1 )(Fig. 1 ). Correspondingly, CaS14G024890.1 had the heaviest molecular weight (MW) at 154041.95 Da, while CaS05G005620.1 had the lightest protein at 11721.28 Da. The isoelectric point (PI) of the protein varied from 4.76 ( CaS07G000990.1 ) to 10.99 ( CaS05G005610.1 ), with 26 members of the MADS-box family exhibiting an acidic isoelectric point below 7, while the remaining 44 proteins displayed a PI greater than 7, classifying them as basic ( Fig. 1 ) . According to the instability index, only three members, CaS13G020990.1 , CaS03G014820.1 , and CaS05G002930.1 , were considered stable proteins (value less than 40), and the remaining 67 were unstable. The variation in the aliphatic amino acid index (A.I.), from 66.32 in CaS13G020990.1 to 102.16 in CaS13G013420.1 , demonstrates significant differences in protein thermal stability ( Fig. 1 ) . The total average hydrophobicity scores (GRAVY) of the proteins were all negative, implying that the detected MADS-box family members all encode hydrophilic proteins ( Fig. 1 ) . Based on the results of subcellular localization prediction, the primary location of the proteins was found to be the nucleus, which was followed by the cytoplasm and chloroplasts. Five members CaS14G013270.1 , CaS06G034490.1 , CaS08G023160.1 , CaS08G023340.1 , and CaS05G008260.1 are located in the mitochondria, and two members CaS06G011830.1 and CaS06G014230.1 are located in the plasma membrane(Fig. 1 ). 2.2 Phylogenetic analysis of MADS-box genes of C. sinensis var. assamica var. A phylogenetic analysis was performed to determine the relationships and subfamilies of the C. sinensis MADS-box proteins. The tree was constructed in MEGA 11 using the neighbor-joining method ( Fig. 2 A ) . The tree was based on the results of the comparison of MADS-box gene proteins, i.e., 108 sequences from Arabidopsis thaliana and 70 sequences from C. sinensis . Based on earlier research on the phylogenetic tree, the MADS-box gene family from C. sinensis includes 70 members were categorized into two groups, namely: type I, consisting of 26 members (8 Mα, 16 Mβ, and 2 Mγ), whereas the remaining 44 members were classified as type II(Fig. 2 B). Within the type II group, only one MADS-box gene protein belonged to the MIKC* category, whereas the remaining members were categorized as MIKC C , and the MIKC C proteins were further classified into 12 branches, in all of which sequences could be identified in both C. sinensis and Arabidopsis thaliana (Fig. 2 C). Phylogenetic analysis revealed that the majority of type II MADS-box genes in C. sinensis were located within the SOC1-like branch (9 members), while the AGL17-like and AGL15-like branches contained only one member each. 2.3 Examination of the structure, conserved structural domains and cis-acting elements of the MADS gene of C. sinensis var. assamica . An analysis of conserved motifs and exon-intron structures was conducted to explore the structural diversity and evolutionary relationships of the MADS-box genes in C. sinensis .(Fig. 3 ). From the results, it can be observed that all 70 members of the C. sinensis MADS-box gene family contain coding sequences (CDS); however, untranslated regions (UTRs) are not present in all members. Furthermore, an investigation into the conserved motifs was conducted using the MEME search tool. This analysis revealed a total of ten conserved motifs across the 70 MADS-box proteins. It was found that members within the same phylogenetic group generally exhibited similar motif structures, a pattern that was particularly evident in the SOC1-like and B-sister groups. Nevertheless, the number and distribution of these motifs varied significantly among different C. sinensis MADS-box proteins, ranging from a maximum of seven to a minimum of only one. In addition to motifs, a total of 15 distinct types of structural domains were predicted among the MADS-box family members. Analysis showed that no single domain was universally present in all members. Among these domains, the MADS_MEF2_like and the K-box were the most abundant in terms of their occurrence, which may suggest that these two domains are more highly conserved within the C. sinensis MADS-box gene family. The cis-element species and distribution may partially imply the transcriptional regulation and expression patterns of the genes involved. To elucidate the biological pathways in which the members of the C. sinensis MADS-box gene family may be involved, a prediction and analysis of the promoter cis-acting elements of these gene family members were performed.(Fig. 5 ). A total of 34 cis-elements were identified using the PlantCARE database and classified into four categories ( Fig. 6 ) : light-responsive (17), phytohormone-responsive (9), stress-responsive (5), and plant growth-regulated (3). Most of the genes exhibited high proportions of light-responsive progenitors, followed by phytohormone-responsive elements, whereas elements related to plant growth regulation were the most sparse. The distribution of cis-acting elements suggests that the expression of MADS-box genes in C. sinensis is significantly influenced by light. No single element type was found in all members. The most prevalent light-responsive elements were Box 4 (171), G-box (112), and GT1-motif (74). A substantial fraction of elements were phytohormone-related, with ABRE (abscisic acid; 112), CGTCA-motif (methyl jasmonate; 65), TGACG-motif (methyl jasmonate; 64), and TCA-element (salicylic acid; 52) being the most abundant. Notably, certain elements were concentrated on specific genes; for instance, a high number of ABRE elements in CaS12G015340.1 and CaS03G004360.1 implies their potential importance in corresponding hormone regulation. Elements associated with abiotic stress responses, including ARE (anaerobic induction; 166), MBS (drought stress; 51), TC-rich repeats (defense and stress; 28), and LTR (low temperature; 21), were also identified. Among the 70 members, CaS04G016390.1 possessed the greatest variety and number of elements (18 types, 36 total), whereas CaS07G002960.1 and CaS08G023160.1 had the fewest (5 and 6, respectively). Based on these results, it is hypothesized that C. sinensis MADS-box genes are involved in light and stress responses, potentially influencing flowering and stress tolerance pathways. 2.4 Chromosomal localization and covariance analysis of the MADS gene in Camellia Chromosomal localization and gene covariance analysis of MADS-box genes in C. sinensis was performed based on the genome annotation files ( Fig. 6 ) . The results indicated that the 70 MADS-box gene family members were unevenly distributed across 15 chromosomes, with no genes located on chromosome 9. The distribution was concentrated on chromosomes 4, 5, 6, 12, 13, and 14, with chromosome 5 harboring the highest number. In contrast, chromosomes 1, 2, 3, 7, 8, 10, 11, and 15 contained relatively few members. Specifically, only one gene was found on chromosomes 1 and 11, while two genes were located on each of chromosomes 7 and 10. To investigate the evolutionary relationships, covariance analysis was conducted using MCScanX in TBtools. The analysis identified six tandem duplicated gene pairs, indicated by red curves, on chromosomes 2, 5, 6, 13, and 14. These colinear genes may regulate core biological processes in C. sinensis , such as flowering time, floral organ development, or secondary metabolism. Their selective retention during evolution suggests that these genes possess redundant or complementary functions, potentially enhancing the plant's adaptability to environmental stresses. Fewer covariant genes in the MADS-box of C. sinensis may reflect the tendency of the family to replicate in tandem or to experience stronger selective pressures in C. sinensis . Collinearity analysis between C. sinensis and other species revealed 18, 36, 2, and 7 collinear gene pairs with Arabidopsis thaliana, grape, maize, and rice, respectively ( Fig. 7 ) . These results indicate that C. sinensis shares the highest degree of collinearity with grape (36 pairs), substantially more than with A. thaliana (18 pairs), rice (7 pairs), or maize (2 pairs). A plausible explanation is that tea plant and grape share a closer phylogenetic relationship as core eudicots. The grape genome, which has experienced fewer ancient polyploidization events, likely retains a gene complement more similar to that of tea plant. In addition to this, both grape and C. sinensis are perennial woody plants that may share certain MADS-box gene-regulated developmental pathways. As a model plant, Arabidopsis thaliana , which has a more well-studied MADS-box gene function, has a lower number of covariates than grapes, probably because Arabidopsis thaliana has experienced more frequent genome rearrangements or lineage-specific gene loss. The very low number of collinear genes between C. sinensis and rice/maize can be attributed to their evolutionary divergence. As monocotyledons, rice and maize diverged from dicotyledonous plants like C. sinensis. This separation was followed by independent genome duplication events in their respective lineages, which weakened the detectable signals of collinear genes. Consequently, some MADS-box genes in monocots may have undergone functional divergence or been lost. 2.5 Analysis of Quantitative qRT- PCR Results To elucidate the expression patterns of the MADS-box gene family under abiotic stress in tea plants ( C. sinensis ), seven selected genes were analyzed following six-day drought and salt stress treatments. The results showed that under drought stress, the expression levels of CaS14G031860.1 , CaS01G012140.1 , CaS13G024900.1 , and CaS04G016390.1 changed significantly over time. Among them, CaS14G031860.1 , CaS01G012140.1 , and CaS13G024900.1 were significantly upregulated on day 3, with expression levels 3–4 times higher than those of the control group. In contrast, the expression of CaS04G016390.1 increased continuously with prolonged drought treatment, reaching a level 4 times higher than the control by day 6(Fig. 8 A). Under salt stress, CaS05G010120.1 and CaS10G020060.1 exhibited the most pronounced upregulation on day 3, with expression levels 3 times that of the control. Meanwhile, the expression of CaS14G031860.1 , CaS01G012140.1 , CaS10G020070.1 , and CaS04G016390.1 increased steadily over time and peaked on day 6. A notable observation was the significant downregulation of CaS13G024900.1 in response to salt stress ( Fig. 8 B ) . 3. Discussion In this study, a genome-wide analysis of C. sinensis identified 70 MADS-box gene family members. Based on the classification system established for Arabidopsis thaliana , these members were categorized into 26 type I (including Mα, Mβ, and Mγ) and 44 type II (including MIKCC and MIKC*) genes. This number is similar to that found in closely related species, such as oil tea, which possesses 68 MADS-box genes [ 16 ] . The significant numerical disparity between types likely reflects greater functional complexity and evolutionary constraints in Type II genes. Currently, 103, 87, 117, 81 and 95 members of the MADS-box gene family have been identified in Arabidopsis thaliana , dove, yellow early four, quinoa, vermilion rhododendron and chilli pepper, respectively, and the differences in the number of MADS-box genes among the species may be due to their genome sizes and whole-genome replication levels resulting from the differences in their genome size and genome-wide replication levels [ 17 ] . Among Type II subfamilies, SOC1-like contained the most members (9), consistent with its conserved role in flowering time regulation, suggesting its critical function in tea phenology [ 18 ] . Conversely, AGL17-like and AGL15-like each contained only one member, aligning with Oryza sativa AGL17-like involvement in root development, possibly indicating functional specialization within C. sinensis [ 19 ] . Physicochemical property analysis revealed substantial variation in protein sequence length, isoelectric point (pI), and hydrophilicity among members, potentially reflecting functional diversification. Gene structure analysis revealed that members within the same subfamily exhibit highly conserved exon-intron patterns and protein motifs. Notably, the MADS_MEF2_like and K-box domains were ubiquitous among the family members, leading to the hypothesis that they are functionally crucial. As one of the most extensively studied plant transcription factor families, the MADS-box genes are known to play vital roles in growth, development, stress response, and secondary metabolism. Consistent with this, our analysis of promoter cis-acting elements in C. sinensis indicates that the expression of its MADS-box genes is likely regulated by light, phytohormones, and abiotic stresses. The MADS-box genes exhibited an uneven dispersion across the 15 chromosomes of C. sinensis , mainly concentrated on chromosomes 4, 5, 6, 12, 13 and 14, and the covariance analysis showed that C. sinensis had the most covariant gene pairs with grapes (36 gene pairs), which was much higher than that with Arabidopsis thaliana (18 gene pairs), which was probably due to the fact that C. sinensis and grapes are dicotyledonous woody plants and have a similar retention pattern of the genome. This work provides the first systematic analysis of the MADS-box gene family in C. sinensis , elucidating its evolutionary features and expression patterns, and thereby offering a theoretical foundation for understanding the genetic mechanisms underlying the plant's morphological diversity. Compared to model plants, the MADS-box family in C. sinensis exhibits both functional conservation (e.g., the organ-identity functions of B- and C-class genes) and divergence (e.g., the reduction of AGL17-like members). These evolutionary dynamics may be associated with its perennial woody growth habit and specific environmental adaptations. Future research should employ CRISPR-Cas9 editing or transgenic approaches to functionally validate key genes, providing candidate targets for tea molecular breeding. Additionally, promoter cis-element predictions suggest MADS-box involvement in abiotic stress responses, opening new avenues for studying tea plant resilience mechanisms. 4. materials and methods 4.1 Studying species genomic data The whole genome sequence, protein sequence, and annotation files of C. sinensis were obtained from the Tea Plant Genome Database (https://eplant.njau.edu.cn/tea/index.html). The corresponding files for the Arabidopsis thaliana MADS-box gene family were acquired from the TAIR database (https://www.arabidopsis.org/). 4.2 Identification of the MADS -box gene family members in C.sinensis To identify the MADS-box genes in C. elegans , two different strategies were used: BLAST search versus Hidden Markov Model (HMM) search. First, the gene IDs of known Arabidopsis thaliana MADS-box genes were obtained from previous studies. Subsequently, their corresponding protein sequences were extracted using TBtools as queries for a BLASTP search against the C. sinensis genome. Finally, only homologous sequences with E-values less than 1e-5 were retained as candidate genes for subsequent analysis.To identify sequences containing typical MADS-box or K-box domains, the corresponding Hidden Markov model (HMM) profiles (PF00319, PF01486) were downloaded from the PFAM database (https://pfam-legacy.xfam.org/) and employed in a domain search. The final MADS-box genes were obtained by merging the sequences identified by the BLAST search with the HMM search and removing redundant members. The subcellular localization of MADS-box proteins was predicted using the online tool WoLF PSORT (https://wolfpsort.hgc.jp/). Meanwhile, the physicochemical properties of these proteins were analyzed with the ProtParam-based “Protein Parameter Calc” function in TBtools. [20] . 4.3 Phylogenetic analysis of the MADS gene of C. sinensis A multiple sequence alignment of all identified MADS-box protein sequences from C. sinensis was conducted using MUSCLE v3.8. [21] . First, the protein sequences of the C. sinensis MADS-box gene family were merged with those from Arabidopsis thaliana . Subsequently, the optimal model for phylogenetic construction was determined using the Model Selection tool built into MEGA11. Finally, a phylogenetic tree was constructed with the Neighbor-Joining (NJ) method, applying the Gamma-distributed rate variation model (+G) [22] . After the evolutionary tree construction was completed the tree files generated by MEGA were beautified using Evolview v3(https://www.evolgenius.info/evolview/#/treeview) by annotating the type I (Mα, Mβ, Mγ) and type II (MIKCC, MIKC*, etc.) genes with different colours [23] . 4.4 Analysis of the structure, conserved structural domains and cis-acting elements of the MADS gene of C. sinensis Identification of conserved motifs was performed using the MEME online tool (https://meme-suite.org/meme/index.html), configured to search for 10 motifs, with subsequent visualization conducted using the relevant function in TBtools [24] . For structural domain prediction, protein sequences were submitted to NCBI CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi), and the resulting data were visualized [25] . Investigation of potential regulatory elements involved the extraction of a 2000 bp putative promoter region upstream of the translation initiation codon for each gene from the genomic files using TBtools. Cis-acting elements within these promoter sequences were identified via the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and visualized using TBtools [26] . 4.5 Genome-Wide Identification of MADS-box Genes: Chromosome Distribution and Synteny in C. sinensis The chromosomal locations of the MADS-box genes were determined from the C. sinensis genome annotation file and visualized using the "Gene Location Visualize" function in TBtools. The One Step MCScan X-Super Fast function of the TBtools software was then used to analyse the covariance of the MADS-box gene family members within the C. sinensis species.A similar procedure was used to demonstrate the distribution and covariance of MADS-box homologous genes on different chromosomes by going to the Ensembl Plantsdatabase to retrieve the genomic data of grapevine, Arabidopsis thaliana , rice, and maize for inter-species covariance with the MADS-box genes of C. sinensis [27] . 4.6 RNA Extraction, Reverse Transcription, and Quantitative RT-PCR Analysis in Response to Simulated Drought and Salt Stress Four potted large-leaf tea plants ( C. sinensis ) from Wuzhishan, Hainan, with uniform growth status were selected and divided into two groups. One group was irrigated with 20% PEG6000 to simulate drought stress, while the other was treated with 200 mmol/L NaCl for salt stress. Leaves were collected on day 0, day 3, and day 6 to form the following groups: CK, PEG-3Day, PEG-6Day, NaCl-3Day, and NaCl-6Day. Upon harvest, all leaf samples were promptly wrapped in aluminum foil, flash-frozen in liquid nitrogen, and stored at -80°C. Extraction of total RNA from all collected samples was performed using an RNA extraction kit (Tiangen Biotech, Beijing, China) in accordance with the manufacturer’s instructions. Following an assessment of RNA purity, reverse transcription was conducted using the FastKing One-Step cDNA Synthesis PreMix (KR118). Quantitative real-time PCR amplification was executed on a LightCycler® 480 II system, and the relative expression levels of seven selected Type II MADS-box genes were determined by the 2–∆∆Ct method. The corresponding quantitative primers are provided in Supplementary Table 1. Declarations Acknowledgements This work was supported by the Hainan Province Science and Technology Special Fund：ZDYF2024KJTPY026. Author contributions J conducted the majority of the experimental work and drafted the manuscript. J and HX contributed to the experimental work. GZ supervised this study. Data availability The authors confirm that the data supporting the findings of this study are available within the Supplementary Materials. Ethics declarations Competing interests The authors declare no competing interests. Ethical approval and consent to participate Not applicable. Conflicts of Interes t: The authors declare no conflict of interest. References Sánchez M, González-Burgos E, Iglesias I et al (2020) The Pharmacological Activity of Camellia sinensis (‎L.‎) ‎Kuntze‎ on Metabolic and Endocrine Disorders: A Systematic Review. Biomolecules 10(4):603 Wan S, Liang B, Yang L et al (2023) The MADS-box family gene PtrANR1 encodes a transcription activator promoting root growth and enhancing plant tolerance to drought stress. Plant Cell Rep 43(1):16 Ng M, Yanofsky M (2001) Function and evolution of the plant MADS -box gene family. Nat Rev 2(3):186–195 Saedler H, Becker A, Winter KU, Kirchner C, Theissen G (2001) MADS -box genes are involved in floral development and evolution. Acta Biochim Pol 48(2):351–358 Smaczniak C, Immink RG, Angenent GC, Kaufmann K (2012) Developmental and evolutionary diversity of plant MADS -domain factors: insights from recent studies. Development 139(17):3081–3098. https://doi.org/10.1242/dev.074674 Theissen G (2001) Development of floral organ identity:stories from the MADS house. Curr Opin Plant Biol 4(1):75–85 Becker A, Theissen G (2003) The major clades of MADS-box genes and their role in the development and evolution of flowering plants. Mol Phylogenet Evol 29(3):464–489 Castelán-Muñoz N, Herrera J, Cajero-Sánchez W et al (2019) MADS-Box Genes Are Key Components of Genetic Regulatory Networks Involved in Abiotic Stress and Plastic Developmental Responses in Plants. Front Plant Sci 10:853 de PARENICOVÁL S, KIEFFER M et al (2003) Molecular and phylogeneticanalyses of the complete MADS -box transcriptionfactor family in Arabidopsis: new openings to the MADS world. Plant Cell 15(7):1538–1551 RIECHMANN JL, KRIZEK BA, MEYEROWITZ EM (1996) Dimerization specificity of Arabidopsis MADS domainhomeotic proteins APETALA1, APETALA3,PISTILLATA, and AGAMOUS. Proceedings of theNational Academy of Sciences of the United States ofAmerica 93(10): 4793–4798 Gramzow L, Theissen G (2010) A hitchhiker’s guide tothe MADS world of plants. Genome Biol 11(6):1–11 Becker A, Theissen G (2003) The major clades of MADS -box genes and their role in the development andevolution of flowering plants. Mol Phylogenet Evol 29(3):464–489 Mou Y, Yuan C, Sun Q, Yan C et al (2022) MIKC-type MADS-box transcription factor gene family in peanut: Genome-wide characterization and expression analysis under abiotic stress. Front Plant Sci 13:980933 Xue Y, Ma L, Wang H et al (2022) The MADS transcription factor GhFYF is involved in abiotic stress responses in upland cotton (Gossypium hirsutum L). Gene 815:146138 Lu J, Wu H, Pitt DM et al (2024) Identification and characterization of MADS-box gene family in flax, Linum usitatissimum L. and its role under abiotic stress. iScience 27(12):111092 Zhang Y, Li X, Yang H et al (2022) Genome-wide identification and expression analysis of MADS-box genes in Camellia oleifera. Horticulturae 8:102 Chen J, Yang Y, Li C et al (2023) Genome-Wide Identification of MADS -Box Genes in Taraxacum kok-saghyz and Taraxacum mongolicum: Evolutionary Mechanisms, Conserved Functions and New Functions Related to Natural Rubber Yield Formation. Int J Mol Sci 24(13):10997 Lee H, Suh SS et al (2000) The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis. Genes Dev 14(18):2366–2376 Yu LH, Miao et al (2014) MADS-box transcription factor AGL21 regulates lateral root development and responds to multiple external and physiological signals. Mol Plant 7(11):1653–1669 Chen C, Chen H, Zhang Y et al (2020) TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant 13:1194–1202 Edgar RC (2004) MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792–1797 Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis version 11. Mol Biol Evol 38(7):3022–3027 Subramanian B, Gao S, Lercher et al (2019) Evolview v3: A webserver for visualization, annotation, and management of phylogenetic trees. Nucleic Acids Res 47(W1):W270–W275 Bailey TL, Johnson J, Grant CE et al (2015) The MEME Suite. Nucleic Acids Res 43:W39–W49 Marchler-Bauer A, Lu S, Anderson JB et al (2011) CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res 39:D225–D229 Lescot M, Déhais P, Thijs G et al (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30:325–327 Andrew D, Yates JA et al (2022) Ensembl Genomes 2022: an expanding genome resource for non-vertebrates. Nucleic Acids Res 50(1):D996–D100 Additional Declarations No competing interests reported. Supplementary Files Schedule.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7855824","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":544439579,"identity":"4ef7a5f2-23d9-408f-9881-537fd9e142ac","order_by":0,"name":"Jing Yang","email":"","orcid":"","institution":"Hainan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Yang","suffix":""},{"id":544439581,"identity":"48fbca0e-8c24-4a66-b0ed-3616adb7d701","order_by":1,"name":"Hanxue Wei","email":"","orcid":"","institution":"Hainan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Hanxue","middleName":"","lastName":"Wei","suffix":""},{"id":544439594,"identity":"b8651821-3adf-4f06-9929-9a871e875689","order_by":2,"name":"Guangzhen Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYHACNiC2SWBghvEPEKcljXQthxMQfEJa5P2PP3vwse18Hn8778HPhW0Mcnw3Ehg/F+DRYnjgQLrhzLbbxRKH+ZKlZ7YxGEveSGCWnoFPS2PDMWnettuJDYd5DIAMhsQNNxLYmHnwaWlmbAOqPJc4/zCP8W+glnqCWuTZmNmAWg4kbjjMYwayJcGAkBYDHjY2yRnnkhM3ArVY85yTMJx55mGzNF5b+o8/k/hQZpc47/wZ49s8ZTbyfMeTD37Ga8sBVL4EEDM24NEAtAW/9CgYBaNgFIwCIAAAzF5H0UPspnwAAAAASUVORK5CYII=","orcid":"","institution":"Hainan Normal University","correspondingAuthor":true,"prefix":"","firstName":"Guangzhen","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2025-10-14 08:08:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7855824/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7855824/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97666599,"identity":"950266e6-a6b7-4061-950f-9e615a784258","added_by":"auto","created_at":"2025-12-08 09:21:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":210550,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhysicochemical properties of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMADS-box\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene family members of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. sinensis var. assamica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e var.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7855824/v1/b9b4c6775f62b81decc1387a.png"},{"id":97425920,"identity":"418b63a4-5919-41fb-a142-73e20b82b2af","added_by":"auto","created_at":"2025-12-04 09:13:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":439964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMADS-box\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. sinensis var. assamica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thaliana. \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(A)Phylogenetic tree of Type I \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMADS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-box genes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. sinensis var. assamica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and Arabidopsis thaliana. (B) Phylogenetic tree of Type II.\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMADS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-box genes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. sinensis var. assamica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thaliana. \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(C) Heatmap of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMADS-box \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene holdings in\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e C. sinensis var. assamica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thaliana\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7855824/v1/7e2d80766e6bfa396dcfcba8.png"},{"id":97667496,"identity":"354ed018-02bb-470e-ad69-361161eed8c9","added_by":"auto","created_at":"2025-12-08 09:23:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":334378,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructure and conserved motif analysis of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMADS-box\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. sinensis var. Assamica. \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(A) Distribution of conserved motifs in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMADS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-box gene proteins. (B) \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMADS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e domain, K-box domain, and other domains in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMADS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-box gene.(C) The gene structure of MADS-box includes untranslated regions (UTRs) and coding sequence (CDS).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7855824/v1/24c460ec5b5fc67c6dbab629.png"},{"id":97667018,"identity":"35eb92e1-ef85-4f14-8b45-aae128968376","added_by":"auto","created_at":"2025-12-08 09:22:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":274400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConserved structural domains of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMADS-box\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. sinensis var. assamica\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7855824/v1/9575fd2f75d246f1e421ceb3.png"},{"id":97666699,"identity":"c8b72c16-42c7-4030-9596-938a6c4f47ab","added_by":"auto","created_at":"2025-12-08 09:21:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1033563,"visible":true,"origin":"","legend":"\u003cp\u003ePredictive analysis of cis-acting elements of the promoter of the \u003cem\u003eMADS-box\u003c/em\u003egene of \u003cem\u003eC. sinensis var. Assamica. (\u003c/em\u003eA) Analysis of cis-regulatory elements in the promoter regions of \u003cem\u003eCamellia sinensis\u003c/em\u003e var. assamica \u003cem\u003eMADS\u003c/em\u003e-box genes. (B) Heatmap of cis-regulatory element counts. (C) Bar chart showing aggregate counts of cis-regulatory elements by category\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7855824/v1/221d699b225f9695ebe1da48.png"},{"id":97666083,"identity":"108e078f-a8be-485c-a8d0-fdc69ba4886c","added_by":"auto","created_at":"2025-12-08 09:20:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":529188,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution and covariance of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMADS-box\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. sinensis var. assamica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genome\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7855824/v1/859964e3dd797fba232da386.png"},{"id":97666769,"identity":"20d8b92d-c0c9-4da1-926e-494633a83f65","added_by":"auto","created_at":"2025-12-08 09:22:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":835169,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of covariance between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. sinensis var. assamica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and other species. (A) Analysis of covariance between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. sinensis var. assamica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and Grape. (B) Analysis of covariance between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. sinensis var. assamica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (C) Analysis of covariance between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. sinensis var. assamica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and Rice. (D) Analysis of covariance between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. sinensis var. assamica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and Maize\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7855824/v1/08efccd2552ba4a7b01922fa.png"},{"id":97667525,"identity":"96543241-3e0e-497d-9e8e-fa6c67860ee3","added_by":"auto","created_at":"2025-12-08 09:23:42","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":159867,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression levels of Type II genes at different time points under drought and salt stress treatments. (A) Drought stress. (B) Salt stress.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7855824/v1/47874db760c01c68449ddff0.png"},{"id":100036623,"identity":"bea280a7-5d5a-465e-84f3-e0a7def9d1c9","added_by":"auto","created_at":"2026-01-12 10:25:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4675539,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7855824/v1/99e03167-a655-4fdb-a645-de51f989b0c6.pdf"},{"id":97425924,"identity":"2076787b-9493-46f7-9173-40cd8a4d0aa3","added_by":"auto","created_at":"2025-12-04 09:13:22","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":10209,"visible":true,"origin":"","legend":"","description":"","filename":"Schedule.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7855824/v1/0bb6ab8803cb386b965b29ef.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification and Expression Analysis of the MADS-box Gene Family in Camellia sinensis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cem\u003eCamellia sinensis\u003c/em\u003e belongs to the family Theaceae, and it is a significant economic crop of the genus Camellia. This plant is extensively cultivated throughout tropical and subtropical zones and is prized for the intense fragrance of the tea derived from its leaves\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Higher plants are frequently exposed to environmental stressors during growth and development. Drought, low temperature, and high salinity are some of the most prevalent abiotic stresses that can elicit various physiological and metabolic responses in plants, hinder their metabolism and growth, and, in extreme cases, result in irreversible damage or even death.\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eMADS-box\u003c/em\u003e gene family is widely found in plants, animals, and fungi, and plays a central role in multiple stages of plant development\u003csup\u003e[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. This gene family includes members that control different parts of the plant growth and development process, such as the stress response, the length of the flowering period, the maintenance of the floral meristem, the formation of thefloral organ, and the development of fruit\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. The \u003cem\u003eMADS-box\u003c/em\u003e gene family has a core function in plant responses to abiotic stress, highlighting its role as a key integrator of environmental signals and developmental regulation. By modulating processes such as hormone signaling, ROS homeostasis, nutrient response, and flowering time, \u003cem\u003eMADS-box\u003c/em\u003e genes help plants achieve survival and reproductive success in changing environments\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. The name of the \u003cem\u003eMADS-box\u003c/em\u003e gene family derives from its conserved DNA-binding domain(the MADS domain), formed from the initials of four founder genes:M(MCM1, yeast), A(AGAMOUS, Arabidopsis), D(DEFICIENS, snapdragon), and S(SRF, human) \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. The \u003cem\u003eMADS-box\u003c/em\u003e genes may be separated into the two main classes according to protein domain architecture and phylogenetic relationships:Type I Mα、Mβ、Mγand Type II.(C)e II gene are specific to plants and contain, besides the MADS domain, three additional domains:I(intervening), K-box(keratin-like), and a variable C-terminal domain(C). They are additionally categorized into MIKCC and MIKC*subtypes \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. With known expression patterns or mutant characteristics, the majority are made up of the MIKCC kind, which also includes all of the plant \u003cem\u003eMADS-box\u003c/em\u003e genes. \u003cem\u003eMIKCC-type\u003c/em\u003e genes can be divided into 12 different clades according to phylogeny: AG-like, AGL12-like, AGL15-like, AGL17-like, AGL6-like, AP1-like, AP3/PI-like, Bsister, FLC-like, SEP-like, SOC1-like, and SVP-like. Type I genes evolve more rapidly and have relatively diverse functions, whereas Type II genes are more conserved in floral organ development and form the genetic basis of the classical ABC(D)E model\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eYifei Mou et al. were the first to comprehensively identify the MIKC-type \u003cem\u003eMADS-box\u003c/em\u003e gene family in peanuts, revealing its phylogeny, structural characteristics, expression patterns, and potential functions in abiotic stress responses. Their results indicated that peanut \u003cem\u003eMADS-box\u003c/em\u003e genes may be involved in various stress adaptation processes through hormone signaling pathways and stress-responsive elements\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Xue Y et al. first identified a MIKCC-class MADS transcription factor gene, GhFYF, in cotton. They discovered that it is concentrated in the nucleus and is highly expressed in flowers and seeds.This gene is activated by drought and salt stress;its overexpression improves salt tolerance in Arabidopsis, boosts seed germination and proline accumulation, and interacts with the stress-responsive protein GhGPP2.These data show that GhFYF may be important in abiotic stress responses, especially salt stress, in cotton\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Lu J et al. systematically identified the \u003cem\u003eMADS-box\u003c/em\u003e gene family in flax and clarified the key functions of these genes in floral organ development and stress response. Their study further revealed that Type II genes are primarily involved in floral organ development, whereas Type I genes are more associated with embryonic development. In addition, multiple LuMADS genes showed significant expression changes under cold, drought, and salt stress conditions, among which LuMADS27 exhibited regulatory roles under multiple stresses\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Although abiotic stresses significantly impact \u003cem\u003eC. sinensis\u003c/em\u003e, the role of the \u003cem\u003eMADS-box\u003c/em\u003e gene family remains poorly characterized. To fill this knowledge gap, this study provides a genome-wide systematic analysis. Firstly, we identified all \u003cem\u003eMADS-box\u003c/em\u003e genes and analyzed their phylogenetic evolution, gene structure, and conserved motifs. Additionally, RNA-seq and qRT-PCR were employed to determine their expression patterns under different abiotic stresses.The findings will contribute to elucidating the molecular regulatory mechanisms of \u003cem\u003eC. sinensis\u003c/em\u003e under stress and provide a theoretical basis and candidate genes for molecular breeding and genetic improvement of tea plants.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Identification of \u003cem\u003eMADS-box\u003c/em\u003e gene family members in \u003cem\u003eC. sinensis .\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTo ensure a comprehensive screening of \u003cem\u003eMADS-box\u003c/em\u003e gene family members, putative \u003cem\u003eMADS-box\u003c/em\u003e genes were identified by combining the results of Hidden Markov Model (HMM) and BLAST search. Our genome-wide analysis identified 70 members of the \u003cem\u003eMADS-box\u003c/em\u003e gene family in \u003cem\u003eC. sinensis.\u003c/em\u003eThese proteins exhibited different lengths, with the longest being 1358 aa (\u003cem\u003eCaS14G024890.1\u003c/em\u003e) and the shortest being 100 aa (\u003cem\u003eCaS05G005620.1\u003c/em\u003e)(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Correspondingly, \u003cem\u003eCaS14G024890.1\u003c/em\u003e had the heaviest molecular weight (MW) at 154041.95 Da, while \u003cem\u003eCaS05G005620.1\u003c/em\u003e had the lightest protein at 11721.28 Da. The isoelectric point (PI) of the protein varied from 4.76 (\u003cem\u003eCaS07G000990.1\u003c/em\u003e) to 10.99 (\u003cem\u003eCaS05G005610.1\u003c/em\u003e), with 26 members of the \u003cem\u003eMADS-box\u003c/em\u003e family exhibiting an acidic isoelectric point below 7, while the remaining 44 proteins displayed a PI greater than 7, classifying them as basic \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. According to the instability index, only three members, \u003cem\u003eCaS13G020990.1\u003c/em\u003e, \u003cem\u003eCaS03G014820.1\u003c/em\u003e, and \u003cem\u003eCaS05G002930.1\u003c/em\u003e, were considered stable proteins (value less than 40), and the remaining 67 were unstable. The variation in the aliphatic amino acid index (A.I.), from 66.32 in \u003cem\u003eCaS13G020990.1\u003c/em\u003e to 102.16 in \u003cem\u003eCaS13G013420.1\u003c/em\u003e, demonstrates significant differences in protein thermal stability \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. The total average hydrophobicity scores (GRAVY) of the proteins were all negative, implying that the detected \u003cem\u003eMADS-box\u003c/em\u003e family members all encode hydrophilic proteins \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Based on the results of subcellular localization prediction, the primary location of the proteins was found to be the nucleus, which was followed by the cytoplasm and chloroplasts. Five members \u003cem\u003eCaS14G013270.1\u003c/em\u003e, \u003cem\u003eCaS06G034490.1\u003c/em\u003e, \u003cem\u003eCaS08G023160.1\u003c/em\u003e, \u003cem\u003eCaS08G023340.1\u003c/em\u003e, and \u003cem\u003eCaS05G008260.1\u003c/em\u003e are located in the mitochondria, and two members \u003cem\u003eCaS06G011830.1\u003c/em\u003e and \u003cem\u003eCaS06G014230.1\u003c/em\u003e are located in the plasma membrane(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Phylogenetic analysis of \u003cem\u003eMADS-box\u003c/em\u003e genes of \u003cem\u003eC. sinensis var. assamica\u003c/em\u003e var.\u003c/h2\u003e\u003cp\u003eA phylogenetic analysis was performed to determine the relationships and subfamilies of the \u003cem\u003eC. sinensis MADS-box\u003c/em\u003e proteins. The tree was constructed in MEGA 11 using the neighbor-joining method \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The tree was based on the results of the comparison of \u003cem\u003eMADS-box\u003c/em\u003e gene proteins, i.e., 108 sequences from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and 70 sequences from \u003cem\u003eC. sinensis\u003c/em\u003e. Based on earlier research on the phylogenetic tree, the \u003cem\u003eMADS-box\u003c/em\u003e gene family from \u003cem\u003eC. sinensis\u003c/em\u003e includes 70 members were categorized into two groups, namely: type I, consisting of 26 members (8 Mα, 16 Mβ, and 2 Mγ), whereas the remaining 44 members were classified as type II(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Within the type II group, only one \u003cem\u003eMADS-box\u003c/em\u003e gene protein belonged to the MIKC* category, whereas the remaining members were categorized as MIKC\u003csup\u003eC\u003c/sup\u003e, and the MIKC\u003csup\u003eC\u003c/sup\u003e proteins were further classified into 12 branches, in all of which sequences could be identified in both \u003cem\u003eC. sinensis\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Phylogenetic analysis revealed that the majority of type II \u003cem\u003eMADS-box\u003c/em\u003e genes in \u003cem\u003eC. sinensis\u003c/em\u003e were located within the SOC1-like branch (9 members), while the AGL17-like and AGL15-like branches contained only one member each.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.3 Examination of the structure, conserved structural domains and cis-acting elements of the\u003c/b\u003e \u003cb\u003eMADS\u003c/b\u003e \u003cb\u003egene of\u003c/b\u003e \u003cb\u003eC. sinensis var. assamica\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eAn analysis of conserved motifs and exon-intron structures was conducted to explore the structural diversity and evolutionary relationships of the \u003cem\u003eMADS-box\u003c/em\u003e genes in \u003cem\u003eC. sinensis\u003c/em\u003e.(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). From the results, it can be observed that all 70 members of the \u003cem\u003eC. sinensis MADS-box\u003c/em\u003e gene family contain coding sequences (CDS); however, untranslated regions (UTRs) are not present in all members. Furthermore, an investigation into the conserved motifs was conducted using the MEME search tool. This analysis revealed a total of ten conserved motifs across the 70 \u003cem\u003eMADS-box\u003c/em\u003e proteins. It was found that members within the same phylogenetic group generally exhibited similar motif structures, a pattern that was particularly evident in the SOC1-like and B-sister groups. Nevertheless, the number and distribution of these motifs varied significantly among different \u003cem\u003eC. sinensis MADS-box\u003c/em\u003e proteins, ranging from a maximum of seven to a minimum of only one. In addition to motifs, a total of 15 distinct types of structural domains were predicted among the \u003cem\u003eMADS-box\u003c/em\u003e family members. Analysis showed that no single domain was universally present in all members. Among these domains, the \u003cem\u003eMADS_MEF2_like\u003c/em\u003e and the \u003cem\u003eK-box\u003c/em\u003e were the most abundant in terms of their occurrence, which may suggest that these two domains are more highly conserved within the \u003cem\u003eC. sinensis MADS-box\u003c/em\u003e gene family.\u003c/p\u003e\u003cp\u003eThe cis-element species and distribution may partially imply the transcriptional regulation and expression patterns of the genes involved. To elucidate the biological pathways in which the members of the \u003cem\u003eC. sinensis MADS-box\u003c/em\u003e gene family may be involved, a prediction and analysis of the promoter cis-acting elements of these gene family members were performed.(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). A total of 34 cis-elements were identified using the PlantCARE database and classified into four categories\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e: light-responsive (17), phytohormone-responsive (9), stress-responsive (5), and plant growth-regulated (3). Most of the genes exhibited high proportions of light-responsive progenitors, followed by phytohormone-responsive elements, whereas elements related to plant growth regulation were the most sparse. The distribution of cis-acting elements suggests that the expression of \u003cem\u003eMADS-box\u003c/em\u003e genes in \u003cem\u003eC. sinensis\u003c/em\u003e is significantly influenced by light. No single element type was found in all members. The most prevalent light-responsive elements were Box 4 (171), G-box (112), and GT1-motif (74). A substantial fraction of elements were phytohormone-related, with ABRE (abscisic acid; 112), CGTCA-motif (methyl jasmonate; 65), TGACG-motif (methyl jasmonate; 64), and TCA-element (salicylic acid; 52) being the most abundant. Notably, certain elements were concentrated on specific genes; for instance, a high number of ABRE elements in \u003cem\u003eCaS12G015340.1\u003c/em\u003e and \u003cem\u003eCaS03G004360.1\u003c/em\u003e implies their potential importance in corresponding hormone regulation. Elements associated with abiotic stress responses, including ARE (anaerobic induction; 166), MBS (drought stress; 51), TC-rich repeats (defense and stress; 28), and LTR (low temperature; 21), were also identified. Among the 70 members, \u003cem\u003eCaS04G016390.1\u003c/em\u003e possessed the greatest variety and number of elements (18 types, 36 total), whereas \u003cem\u003eCaS07G002960.1\u003c/em\u003e and \u003cem\u003eCaS08G023160.1\u003c/em\u003e had the fewest (5 and 6, respectively). Based on these results, it is hypothesized that \u003cem\u003eC. sinensis MADS-box\u003c/em\u003e genes are involved in light and stress responses, potentially influencing flowering and stress tolerance pathways.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Chromosomal localization and covariance analysis of the \u003cem\u003eMADS\u003c/em\u003e gene in Camellia\u003c/h2\u003e\u003cp\u003eChromosomal localization and gene covariance analysis of \u003cem\u003eMADS-box\u003c/em\u003e genes in \u003cem\u003eC. sinensis\u003c/em\u003e was performed based on the genome annotation files \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. The results indicated that the 70 \u003cem\u003eMADS-box\u003c/em\u003e gene family members were unevenly distributed across 15 chromosomes, with no genes located on chromosome 9. The distribution was concentrated on chromosomes 4, 5, 6, 12, 13, and 14, with chromosome 5 harboring the highest number. In contrast, chromosomes 1, 2, 3, 7, 8, 10, 11, and 15 contained relatively few members. Specifically, only one gene was found on chromosomes 1 and 11, while two genes were located on each of chromosomes 7 and 10. To investigate the evolutionary relationships, covariance analysis was conducted using MCScanX in TBtools. The analysis identified six tandem duplicated gene pairs, indicated by red curves, on chromosomes 2, 5, 6, 13, and 14. These colinear genes may regulate core biological processes in \u003cem\u003eC. sinensis\u003c/em\u003e, such as flowering time, floral organ development, or secondary metabolism. Their selective retention during evolution suggests that these genes possess redundant or complementary functions, potentially enhancing the plant's adaptability to environmental stresses. Fewer covariant genes in the \u003cem\u003eMADS-box\u003c/em\u003e of \u003cem\u003eC. sinensis\u003c/em\u003e may reflect the tendency of the family to replicate in tandem or to experience stronger selective pressures in \u003cem\u003eC. sinensis\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eCollinearity analysis between \u003cem\u003eC. sinensis\u003c/em\u003e and other species revealed 18, 36, 2, and 7 collinear gene pairs with Arabidopsis thaliana, grape, maize, and rice, respectively \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. These results indicate that \u003cem\u003eC. sinensis\u003c/em\u003e shares the highest degree of collinearity with grape (36 pairs), substantially more than with A. thaliana (18 pairs), rice (7 pairs), or maize (2 pairs). A plausible explanation is that tea plant and grape share a closer phylogenetic relationship as core eudicots. The grape genome, which has experienced fewer ancient polyploidization events, likely retains a gene complement more similar to that of tea plant. In addition to this, both grape and \u003cem\u003eC. sinensis\u003c/em\u003e are perennial woody plants that may share certain \u003cem\u003eMADS-box\u003c/em\u003e gene-regulated developmental pathways. As a model plant, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, which has a more well-studied \u003cem\u003eMADS-box\u003c/em\u003e gene function, has a lower number of covariates than grapes, probably because \u003cem\u003eArabidopsis thaliana\u003c/em\u003e has experienced more frequent genome rearrangements or lineage-specific gene loss. The very low number of collinear genes between \u003cem\u003eC. sinensis\u003c/em\u003e and rice/maize can be attributed to their evolutionary divergence. As monocotyledons, rice and maize diverged from dicotyledonous plants like \u003cem\u003eC. sinensis.\u003c/em\u003e This separation was followed by independent genome duplication events in their respective lineages, which weakened the detectable signals of collinear genes. Consequently, some \u003cem\u003eMADS-box\u003c/em\u003e genes in monocots may have undergone functional divergence or been lost.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Analysis of Quantitative qRT- PCR Results\u003c/h2\u003e\u003cp\u003eTo elucidate the expression patterns of the \u003cem\u003eMADS-box\u003c/em\u003e gene family under abiotic stress in tea plants (\u003cem\u003eC. sinensis\u003c/em\u003e), seven selected genes were analyzed following six-day drought and salt stress treatments. The results showed that under drought stress, the expression levels of \u003cem\u003eCaS14G031860.1\u003c/em\u003e, \u003cem\u003eCaS01G012140.1\u003c/em\u003e, \u003cem\u003eCaS13G024900.1\u003c/em\u003e, and \u003cem\u003eCaS04G016390.1\u003c/em\u003e changed significantly over time. Among them, \u003cem\u003eCaS14G031860.1\u003c/em\u003e, \u003cem\u003eCaS01G012140.1\u003c/em\u003e, and \u003cem\u003eCaS13G024900.1\u003c/em\u003e were significantly upregulated on day 3, with expression levels 3\u0026ndash;4 times higher than those of the control group. In contrast, the expression of \u003cem\u003eCaS04G016390.1\u003c/em\u003e increased continuously with prolonged drought treatment, reaching a level 4 times higher than the control by day 6(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eUnder salt stress, \u003cem\u003eCaS05G010120.1\u003c/em\u003e and \u003cem\u003eCaS10G020060.1\u003c/em\u003e exhibited the most pronounced upregulation on day 3, with expression levels 3 times that of the control. Meanwhile, the expression of \u003cem\u003eCaS14G031860.1\u003c/em\u003e, \u003cem\u003eCaS01G012140.1\u003c/em\u003e, \u003cem\u003eCaS10G020070.1\u003c/em\u003e, and \u003cem\u003eCaS04G016390.1\u003c/em\u003e increased steadily over time and peaked on day 6. A notable observation was the significant downregulation of \u003cem\u003eCaS13G024900.1\u003c/em\u003e in response to salt stress \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eIn this study, a genome-wide analysis of \u003cem\u003eC. sinensis\u003c/em\u003e identified 70 \u003cem\u003eMADS-box\u003c/em\u003e gene family members. Based on the classification system established for \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, these members were categorized into 26 type I (including Mα, Mβ, and Mγ) and 44 type II (including MIKCC and MIKC*) genes. This number is similar to that found in closely related species, such as oil tea, which possesses 68 \u003cem\u003eMADS-box\u003c/em\u003e genes\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. The significant numerical disparity between types likely reflects greater functional complexity and evolutionary constraints in Type II genes. Currently, 103, 87, 117, 81 and 95 members of the \u003cem\u003eMADS-box\u003c/em\u003e gene family have been identified in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, dove, yellow early four, quinoa, vermilion rhododendron and chilli pepper, respectively, and the differences in the number of \u003cem\u003eMADS-box\u003c/em\u003e genes among the species may be due to their genome sizes and whole-genome replication levels resulting from the differences in their genome size and genome-wide replication levels\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAmong Type II subfamilies, SOC1-like contained the most members (9), consistent with its conserved role in flowering time regulation, suggesting its critical function in tea phenology\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Conversely, AGL17-like and AGL15-like each contained only one member, aligning with Oryza sativa AGL17-like involvement in root development, possibly indicating functional specialization within C. sinensis \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Physicochemical property analysis revealed substantial variation in protein sequence length, isoelectric point (pI), and hydrophilicity among members, potentially reflecting functional diversification.\u003c/p\u003e\u003cp\u003eGene structure analysis revealed that members within the same subfamily exhibit highly conserved exon-intron patterns and protein motifs. Notably, the \u003cem\u003eMADS_MEF2_like\u003c/em\u003e and \u003cem\u003eK-box\u003c/em\u003e domains were ubiquitous among the family members, leading to the hypothesis that they are functionally crucial. As one of the most extensively studied plant transcription factor families, the \u003cem\u003eMADS-box\u003c/em\u003e genes are known to play vital roles in growth, development, stress response, and secondary metabolism. Consistent with this, our analysis of promoter cis-acting elements in \u003cem\u003eC. sinensis\u003c/em\u003e indicates that the expression of its \u003cem\u003eMADS-box\u003c/em\u003e genes is likely regulated by light, phytohormones, and abiotic stresses.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eMADS-box\u003c/em\u003e genes exhibited an uneven dispersion across the 15 chromosomes of \u003cem\u003eC. sinensis\u003c/em\u003e, mainly concentrated on chromosomes 4, 5, 6, 12, 13 and 14, and the covariance analysis showed that \u003cem\u003eC. sinensis\u003c/em\u003e had the most covariant gene pairs with grapes (36 gene pairs), which was much higher than that with \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (18 gene pairs), which was probably due to the fact that \u003cem\u003eC. sinensis\u003c/em\u003e and grapes are dicotyledonous woody plants and have a similar retention pattern of the genome.\u003c/p\u003e\u003cp\u003eThis work provides the first systematic analysis of the \u003cem\u003eMADS-box\u003c/em\u003e gene family in \u003cem\u003eC. sinensis\u003c/em\u003e, elucidating its evolutionary features and expression patterns, and thereby offering a theoretical foundation for understanding the genetic mechanisms underlying the plant's morphological diversity. Compared to model plants, the \u003cem\u003eMADS-box\u003c/em\u003e family in \u003cem\u003eC. sinensis\u003c/em\u003e exhibits both functional conservation (e.g., the organ-identity functions of B- and C-class genes) and divergence (e.g., the reduction of AGL17-like members). These evolutionary dynamics may be associated with its perennial woody growth habit and specific environmental adaptations. Future research should employ CRISPR-Cas9 editing or transgenic approaches to functionally validate key genes, providing candidate targets for tea molecular breeding. Additionally, promoter cis-element predictions suggest \u003cem\u003eMADS-box\u003c/em\u003e involvement in abiotic stress responses, opening new avenues for studying tea plant resilience mechanisms.\u003c/p\u003e"},{"header":"4. materials and methods","content":"\u003cp\u003e\u003cstrong\u003e4.1 Studying species genomic data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe whole genome sequence, protein sequence, and annotation files of \u003cem\u003eC. sinensis\u003c/em\u003e were obtained from the Tea Plant Genome Database (https://eplant.njau.edu.cn/tea/index.html). The corresponding files for the Arabidopsis thaliana \u003cem\u003eMADS-box\u003c/em\u003e gene family were acquired from the TAIR database (https://www.arabidopsis.org/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eIdentification of the \u003cem\u003eMADS\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e-box\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;gene family members in \u003cem\u003eC.sinensis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the \u003cem\u003eMADS-box\u003c/em\u003e genes in \u003cem\u003eC. elegans\u003c/em\u003e, two different strategies were used: BLAST search versus Hidden Markov Model (HMM) search. First, the gene IDs of known Arabidopsis thaliana \u003cem\u003eMADS-box\u003c/em\u003e genes were obtained from previous studies. Subsequently, their corresponding protein sequences were extracted using TBtools as queries for a BLASTP search against the \u003cem\u003eC. sinensis\u003c/em\u003e genome. Finally, only homologous sequences with E-values less than 1e-5 were retained as candidate genes for subsequent analysis.To identify sequences containing typical \u003cem\u003eMADS-box\u003c/em\u003e or \u003cem\u003eK-box\u003c/em\u003e domains, the corresponding Hidden Markov model (HMM) profiles (PF00319, PF01486) were downloaded from the PFAM database (https://pfam-legacy.xfam.org/) and employed in a domain search. The final \u003cem\u003eMADS-box\u003c/em\u003e genes were obtained by merging the sequences identified by the BLAST search with the HMM search and removing redundant members. The subcellular localization of \u003cem\u003eMADS-box\u003c/em\u003e proteins was predicted using the online tool WoLF PSORT (https://wolfpsort.hgc.jp/). Meanwhile, the physicochemical properties of these proteins were analyzed with the ProtParam-based \u0026ldquo;Protein Parameter Calc\u0026rdquo; function in TBtools.\u003csup\u003e[20]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3 Phylogenetic analysis of the \u003cem\u003eMADS\u003c/em\u003e gene of \u003cem\u003eC. sinensis\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA multiple sequence alignment of all identified \u003cem\u003eMADS-box\u003c/em\u003e protein sequences from \u003cem\u003eC. sinensis\u003c/em\u003e was conducted using MUSCLE v3.8.\u003csup\u003e[21]\u003c/sup\u003e. First, the protein sequences of the \u003cem\u003eC. sinensis MADS-box\u003c/em\u003e gene family were merged with those from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Subsequently, the optimal model for phylogenetic construction was determined using the Model Selection tool built into MEGA11. Finally, a phylogenetic tree was constructed with the Neighbor-Joining (NJ) method, applying the Gamma-distributed rate variation model (+G)\u003csup\u003e[22]\u003c/sup\u003e. After the evolutionary tree construction was completed the tree files generated by MEGA were beautified using Evolview v3(https://www.evolgenius.info/evolview/#/treeview) by annotating the type I (M\u0026alpha;, M\u0026beta;, M\u0026gamma;) and type II (MIKCC, MIKC*, etc.) genes with different colours\u003csup\u003e[23]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4 Analysis of the structure, conserved structural domains and cis-acting elements of the \u003cem\u003eMADS\u003c/em\u003e gene of \u003cem\u003eC. sinensis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIdentification of conserved motifs was performed using the MEME online tool (https://meme-suite.org/meme/index.html), configured to search for 10 motifs, with subsequent visualization conducted using the relevant function in TBtools \u003csup\u003e[24]\u003c/sup\u003e. For structural domain prediction, protein sequences were submitted to NCBI CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi), and the resulting data were visualized \u003csup\u003e[25]\u003c/sup\u003e. Investigation of potential regulatory elements involved the extraction of a 2000 bp putative promoter region upstream of the translation initiation codon for each gene from the genomic files using TBtools. Cis-acting elements within these promoter sequences were identified via the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and visualized using TBtools \u003csup\u003e[26]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5 Genome-Wide Identification of \u003cem\u003eMADS-box\u003c/em\u003e Genes: Chromosome Distribution and Synteny in \u003cem\u003eC. sinensis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chromosomal locations of the \u003cem\u003eMADS-box\u003c/em\u003e genes were determined from the \u003cem\u003eC. sinensis\u003c/em\u003e genome annotation file and visualized using the \u0026quot;Gene Location Visualize\u0026quot; function in TBtools. The One Step MCScan X-Super Fast function of the TBtools software was then used to analyse the covariance of the \u003cem\u003eMADS-box\u003c/em\u003e gene family members within the \u003cem\u003eC. sinensis\u003c/em\u003e species.A similar procedure was used to demonstrate the distribution and covariance of \u003cem\u003eMADS-box\u0026nbsp;\u003c/em\u003ehomologous genes on different chromosomes by going to the Ensembl Plantsdatabase to retrieve the genomic data of grapevine, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, rice, and maize for inter-species covariance with the \u003cem\u003eMADS-box\u003c/em\u003e genes of \u003cem\u003eC. sinensis\u003c/em\u003e\u003csup\u003e[27]\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.6 RNA Extraction, Reverse Transcription, and Quantitative RT-PCR Analysis in Response to Simulated Drought and Salt Stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour potted large-leaf tea plants (\u003cem\u003eC. sinensis\u003c/em\u003e) from Wuzhishan, Hainan, with uniform growth status were selected and divided into two groups. One group was irrigated with 20% PEG6000 to simulate drought stress, while the other was treated with 200 mmol/L NaCl for salt stress. Leaves were collected on day 0, day 3, and day 6 to form the following groups: CK, PEG-3Day, PEG-6Day, NaCl-3Day, and NaCl-6Day. Upon harvest, all leaf samples were promptly wrapped in aluminum foil, flash-frozen in liquid nitrogen, and stored at -80\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eExtraction of total RNA from all collected samples was performed using an RNA extraction kit (Tiangen Biotech, Beijing, China) in accordance with the manufacturer\u0026rsquo;s instructions. Following an assessment of RNA purity, reverse transcription was conducted using the FastKing One-Step cDNA Synthesis PreMix (KR118). Quantitative real-time PCR amplification was executed on a LightCycler\u0026reg; 480 II system, and the relative expression levels of seven selected Type II MADS-box genes were determined by the 2\u0026ndash;∆∆Ct method. The corresponding quantitative primers are provided in Supplementary Table 1.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Hainan Province Science and Technology Special Fund：ZDYF2024KJTPY026.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ conducted the majority of the experimental work and drafted the manuscript. J and HX contributed to the experimental work. GZ supervised this study. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the Supplementary Materials.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interes\u003c/strong\u003et: The authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eS\u0026aacute;nchez M, Gonz\u0026aacute;lez-Burgos E, Iglesias I et al (2020) The Pharmacological Activity of Camellia sinensis (\u0026lrm;L.\u0026lrm;) \u0026lrm;Kuntze\u0026lrm; on Metabolic and Endocrine Disorders: A Systematic Review. Biomolecules 10(4):603\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWan S, Liang B, Yang L et al (2023) The MADS-box family gene PtrANR1 encodes a transcription activator promoting root growth and enhancing plant tolerance to drought stress. Plant Cell Rep 43(1):16\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNg M, Yanofsky M (2001) Function and evolution of the plant \u003cem\u003eMADS\u003c/em\u003e-box gene family. Nat Rev 2(3):186\u0026ndash;195\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaedler H, Becker A, Winter KU, Kirchner C, Theissen G (2001) \u003cem\u003eMADS\u003c/em\u003e-box genes are involved in floral development and evolution. Acta Biochim Pol 48(2):351\u0026ndash;358\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSmaczniak C, Immink RG, Angenent GC, Kaufmann K (2012) Developmental and evolutionary diversity of plant \u003cem\u003eMADS\u003c/em\u003e-domain factors: insights from recent studies. Development 139(17):3081\u0026ndash;3098. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/dev.074674\u003c/span\u003e\u003cspan address=\"10.1242/dev.074674\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTheissen G (2001) Development of floral organ identity:stories from the MADS house. Curr Opin Plant Biol 4(1):75\u0026ndash;85\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBecker A, Theissen G (2003) The major clades of MADS-box genes and their role in the development and evolution of flowering plants. Mol Phylogenet Evol 29(3):464\u0026ndash;489\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCastel\u0026aacute;n-Mu\u0026ntilde;oz N, Herrera J, Cajero-S\u0026aacute;nchez W et al (2019) MADS-Box Genes Are Key Components of Genetic Regulatory Networks Involved in Abiotic Stress and Plastic Developmental Responses in Plants. Front Plant Sci 10:853\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede PARENICOV\u0026Aacute;L S, KIEFFER M et al (2003) Molecular and phylogeneticanalyses of the complete \u003cem\u003eMADS\u003c/em\u003e-box transcriptionfactor family in Arabidopsis: new openings to the\u003cem\u003eMADS\u003c/em\u003e world. Plant Cell 15(7):1538\u0026ndash;1551\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRIECHMANN JL, KRIZEK BA, MEYEROWITZ EM (1996) Dimerization specificity of Arabidopsis \u003cem\u003eMADS\u003c/em\u003e domainhomeotic proteins APETALA1, APETALA3,PISTILLATA, and AGAMOUS. Proceedings of theNational Academy of Sciences of the United States ofAmerica 93(10): 4793\u0026ndash;4798\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGramzow L, Theissen G (2010) A hitchhiker\u0026rsquo;s guide tothe \u003cem\u003eMADS\u003c/em\u003e world of plants. Genome Biol 11(6):1\u0026ndash;11\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBecker A, Theissen G (2003) The major clades of \u003cem\u003eMADS\u003c/em\u003e-box genes and their role in the development andevolution of flowering plants. Mol Phylogenet Evol 29(3):464\u0026ndash;489\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMou Y, Yuan C, Sun Q, Yan C et al (2022) MIKC-type MADS-box transcription factor gene family in peanut: Genome-wide characterization and expression analysis under abiotic stress. Front Plant Sci 13:980933\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXue Y, Ma L, Wang H et al (2022) The MADS transcription factor GhFYF is involved in abiotic stress responses in upland cotton (Gossypium hirsutum L). Gene 815:146138\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLu J, Wu H, Pitt DM et al (2024) Identification and characterization of MADS-box gene family in flax, Linum usitatissimum L. and its role under abiotic stress. iScience 27(12):111092\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Y, Li X, Yang H et al (2022) Genome-wide identification and expression analysis of \u003cem\u003eMADS-box\u003c/em\u003e genes in Camellia oleifera. Horticulturae 8:102\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen J, Yang Y, Li C et al (2023) Genome-Wide Identification of \u003cem\u003eMADS\u003c/em\u003e-Box Genes in Taraxacum kok-saghyz and Taraxacum mongolicum: Evolutionary Mechanisms, Conserved Functions and New Functions Related to Natural Rubber Yield Formation. Int J Mol Sci 24(13):10997\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee H, Suh SS et al (2000) The AGAMOUS-LIKE 20 \u003cem\u003eMADS\u003c/em\u003e domain protein integrates floral inductive pathways in Arabidopsis. Genes Dev 14(18):2366\u0026ndash;2376\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu LH, Miao et al (2014) \u003cem\u003eMADS-box\u003c/em\u003e transcription factor AGL21 regulates lateral root development and responds to multiple external and physiological signals. Mol Plant 7(11):1653\u0026ndash;1669\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen C, Chen H, Zhang Y et al (2020) TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant 13:1194\u0026ndash;1202\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEdgar RC (2004) MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792\u0026ndash;1797\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis version 11. Mol Biol Evol 38(7):3022\u0026ndash;3027\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSubramanian B, Gao S, Lercher et al (2019) Evolview v3: A webserver for visualization, annotation, and management of phylogenetic trees. Nucleic Acids Res 47(W1):W270\u0026ndash;W275\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBailey TL, Johnson J, Grant CE et al (2015) The MEME Suite. Nucleic Acids Res 43:W39\u0026ndash;W49\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarchler-Bauer A, Lu S, Anderson JB et al (2011) CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res 39:D225\u0026ndash;D229\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLescot M, D\u0026eacute;hais P, Thijs G et al (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30:325\u0026ndash;327\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAndrew D, Yates JA et al (2022) Ensembl Genomes 2022: an expanding genome resource for non-vertebrates. Nucleic Acids Res 50(1):D996\u0026ndash;D100\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"C.sinensis, MADS-box gene family, Gene expression pattern, Abiotic stress","lastPublishedDoi":"10.21203/rs.3.rs-7855824/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7855824/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA woody crop that has been important to the economy is \u003cem\u003eCamellia sinensis\u003c/em\u003e. Tea plants may experience physiological and metabolic changes as a result of drought and high salinity. The \u003cem\u003eMADS-box\u003c/em\u003e gene family is a significant of transcription factors essential for various facets of plant growth and development. However, comprehensive research on \u003cem\u003eMADS-box\u003c/em\u003e genes in \u003cem\u003eC.sinensis\u003c/em\u003e is still sparse. In this study, based on the genomic datas of \u003cem\u003eC. sinensis\u003c/em\u003e, a total of 70 \u003cem\u003eMADS-box\u003c/em\u003e genes were identified and phylogenetically categorized into two types Type I (which includes Mα, Mβ, Mγ, and Mγ, consisting of 26 members) and Type II (comprising MIKCC and MIKC*, with 44 members). Members of the same subfamily have very conserved exon-intron patterns and protein motifs, according to gene structure analysis. The initial detailed study of the \u003cem\u003eMADS-box\u003c/em\u003e gene family expression profiles and evolutionary features of this gene in \u003cem\u003eC.sinensis\u003c/em\u003e is presented in this paper. Moreover, qRT-PCR tests showed that these genes are frequently engaged in the reaction to salt stress and drought. These results not only offer valuable insights into the genetic mechanisms that contribute to tea plant diversity but also lay the groundwork for molecular breeding efforts focused on enhancing tea quality and aesthetic characteristics.\u003c/p\u003e","manuscriptTitle":"Identification and Expression Analysis of the MADS-box Gene Family in Camellia sinensis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 09:13:17","doi":"10.21203/rs.3.rs-7855824/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d3a47ef0-2006-4a29-928d-1038d7964775","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T10:24:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-04 09:13:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7855824","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7855824","identity":"rs-7855824","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}