Genome-wide identification of the WRKY gene family in Angraecum sesquipedale and exploration of its role in cold stress response

Genome-wide identification of the WRKY gene family in Angraecum sesquipedale and exploration of its role in cold stress response | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide identification of the WRKY gene family in Angraecum sesquipedale and exploration of its role in cold stress response Chun Gu, Chao Hu, Mengfan Wang, Shenshen Pang, Ziyi Ni, Maohong Zhang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7505680/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 Background Angraecum sesquipedale , commonly known as Darwin’s orchid, is a distinctive ornamental species uniquely susceptible to biotic and abiotic stresses, primarily owing to its confinement to specialized habitats. WRKY transcription factors play pivotal roles in plant stress responses, growth and development regulation, and secondary metabolism. However, research on this gene family in A. sesquipedale remains unreported. Results In this study, 70 WRKY genes ( AsWRKYs ) were identified in A. sesquipedale through genome-wide analysis. Phylogenetic analysis, integrating WRKY genes from four model plants ( Arabidopsis thaliana , Solanum lycopersicum , Oryza sativa , and Glycine max ), classified these 70 AsWRKYs into three major groups, with Group Ⅱ further subdivided into five subgroups. Genes within the same group exhibited high conservation in gene structure and motif composition. Tissue expression profiling revealed that several AsWRKYs ( AsWRKY21 and AsWRKY49 ) exhibit root-preferential expression, suggesting their potential involvement in regulating root growth and development in A. sesquipedale . The expression patterns under cold stress showed that 7 genes in roots and 4 genes in leaves exhibited early rapid responses, while 15 genes in leaves and 4 genes in roots exhibited long-term sustained response features. Integrating expression patterns with phylogenetic relationships, key candidate genes potentially implicated in cold stress regulation, including AsWRKY17 , AsWRKY23 , AsWRKY30 , AsWRKY56 , and AsWRKY58 , were identified. Conclusions This study identified 70 WRKY genes in A. sesquipedale , elucidated their classification, evolutionary characteristics, and expression patterns, and unveiled the potential mechanisms of AsWRKYs in cold stress responses. The findings establish a foundation for understanding the evolutionary adaptability of AsWRKYs and the cold regulatory network in A. sesquipedale , and lay a foundation for molecular breeding and genetic improvement of stress resistance in orchids. Angraecum sesquipedale WRKY gene family transcription factor cold tolerance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background The WRKY gene family represents a pivotal family of transcription factors (TFs) in plants, playing crucial roles in regulating plant development and orchestrating responses to diverse stresses. Structurally, WRKY proteins are characterized by a conserved domain architecture that includes one or two WRKY domains at the N-terminus and specific zinc-finger motifs at the C-terminus. Each WRKY domain encompasses the highly conserved amino acid sequence WRKYGQK, while the C-terminal region contains functionally essential zinc-finger structures that exhibit distinct structural variations. These variations are exemplified by the C2H2-type motifs (with the consensus sequence CX4-5CX22-23HXH) and C2HC-type motifs (with the consensus sequence CX7CX23HXC) [ 1 , 2 ] . Based on these structural variations, the WRKY family is classified into three major groups (Group I, Group II, and Group III), with Group II further subdivided into five subgroups [ 3 ] . WRKY TFs act as key regulators in plant growth and development, participating in a range of biological processes, including seed dormancy [ 4 ] and embryogenesis [ 5 ] to plant growth, senescence [ 6 ] , and metabolism [ 7 ] . For instance, in rice ( Oryza sativa ), OsWRKY29 acts as a repressor of seed dormancy by attenuating abscisic acid (ABA)-mediated responses [ 8 ] . OsWRKY53 modulates anther development through the transcriptional regulation of gibberellin (GA) metabolic genes, and its knockout mutants exhibit significantly enhanced cold tolerance under low-temperature stress [ 9 ] . In addition, overexpression of LcWRKY17 promotes monoterpene biosynthesis [ 10 ] . In Arabidopsis , AtWRKY53 serves as a convergence node for jasmonic acid (JA) signaling and age-dependent pathways; it directly upregulates the expression of senescence-associated genes, thereby functioning as a positive regulator of leaf senescence [ 11 ] . Members of the WRKY family act as critical mediators in plant responses to both biotic and abiotic stresses, enabling plants to cope with bacterial infections [ 12 ] , mechanical damage [ 13 ] , and environmental stresses such as heat, drought, salinity, and cold [ 14 ] . For instance, transgenic Arabidopsis overexpressing Vitis vinifera VvWRKY28 exhibits significantly enhanced tolerance to both low-temperature and high-salinity stresses, highlighting its role in cross-stress adaptation [ 15 ] . Overexpression of wheat ( Triticum aestivum ) TaWRKY19 confers broad-spectrum tolerance to salt, drought, and freezing stresses in transgenic plants [ 16 ] . In Arabidopsis , AtWRKY25 , AtWRKY26 , and AtWRKY33 cooperatively improve thermotolerance by regulating ethylene-activated signaling pathways [ 17 ] . Overexpression of maize ( Zea mays ) ZmWRKY106 enhances drought and heat resistance, linking its function to multiple abiotic stress pathways [ 18 ] . Cucumber ( Cucumis sativus ) CsWRKY46 confers cold tolerance to transgenic plants and positively regulates the cold signaling pathway in an ABA-dependent manner [ 19 ] . These WRKY TFs regulate downstream gene expression by specifically binding to W-box elements in the promoters of target genes, thereby activating or repressing transcription to enhance plant adaptation to adverse environments [ 1 , 2 ] . Although the WRKY gene family has been extensively identified and functionally characterized in various plant species, research on orchids remains relatively scarce [ 20 , 21 , 22 ] . Existing studies have identified WRKY family members in Dendrobium officinale and analyzed their expression profiles under abiotic stress [ 23 ] . In Cymbidium sinense , CsWRKY18 regulates abiotic stress responses via an ABA-dependent pathway [ 24 ] . Angraecum sesquipedale (Angraecinae, Orchidaceae), endemic to Madagascar, is renowned for its unique co-evolutionary relationship with pollinators, attributed to its extraordinarily long nectar spur. As a rare endemic orchid dependent on specific microhabitats, A. sesquipedale is vulnerable to abiotic stresses such as low temperatures, making the WRKY family a pivotal target for investigating stress adaptation mechanisms. However, research on the WRKY gene family in A. sesquipedale is currently lacking. Here, we performed genome-wide identification of WRKY genes in A. sesquipedale (designated AsWRKYs ) and analyzed their conserved domains, phylogenetic relationships, gene structure, motif composition, cis-acting elements, gene duplication events, and collinearity. Additionally, transcriptome sequencing was used to investigate the role of AsWRKYs in cold stress responses, aiming to provide valuable insights into the molecular mechanisms underlying cold tolerance. Methods Identification of the AsWRKY Gene Family The genome of A. sesquipedale previously assembled by our research group was employed in this study [ 25 ] . A. thaliana WRKY ( AtWRKYs ) protein sequences were retrieved from The Arabidopsis Information Resource (TAIR) [ 26 ] . Sequences of SlWRKY33 ( Solyc09g014990 ), SlWRKY45 ( Solyc08g067360 ), SlWRKY46 ( Solyc08g067340 ), and SlWRKY50 ( Solyc06g068460 ) were obtained from the Phytozome database [ 27 ] . Sequences of OsWRKY71 ( Os02g0181300 ), OsWRKY76 ( Os09g0417600 ), and GmWRKY21 (NP_001237327.2) were acquired from the National Center for Biotechnology Information (NCBI) Protein database [ 28 ] . The hidden Markov model (HMM) profile of the WRKY domain (PF03106) was retrieved from the Pfam database [ 29 ] . Candidate WRKY genes were identified by searching the local A. sesquipedale protein database using HMMER 3.3.2 software with the parameter setting: E-value < 1e-5. To ensure accuracy, the integrity of the WRKY domain (core sequence: WRKYGQK) and zinc-finger motifs within the sequences was further verified using SMART [ 30 ] , NCBI CDD [ 31 ] , and Pfam [ 29 ] , leading to the final determination of the AsWRKY genes set. Based on the physical location information in the A. sesquipedale genome annotation file, 70 WRKY genes were mapped to their respective chromosomes. Analysis of Physicochemical Properties of the AsWRKY Gene Family The Protein Parameter Calculator tool in TBtools software with default parameters was used to analyze the number of amino acids, molecular weight (MW), isoelectric point (pI), and instability index of A. sesquipedale WRKY proteins (with an instability index > 40 indicating unstable proteins). Subcellular localization was predicted using the WoLFPSORT tool [ 32 ] with default parameters, which infers subcellular compartment distribution based on amino acid composition and sorting signals (e.g., nuclear localization signals, chloroplast transit peptides). Phylogenetic Analysis and Sequence Alignment Multiple sequence alignment of A. sesquipedale WRKY ( AsWRKY ) protein sequences was performed using the MUSCLE algorithm in MEGA 11.0 software with the following parameters: gap opening penalty = -400, gap extension penalty = 0, and default matrix. The alignment results, which highlight conserved amino acid residues, were visualized using GeneDoc software. A maximum likelihood (ML) phylogenetic tree was constructed in MEGA 11.0, with branch reliability evaluated via 1000 bootstrap replicates; other parameters were set to default. The phylogenetic tree was annotated (e.g., group labeling, branch coloring) and refined using the Interactive Tree of Life (iTOL) platform [ 33 ] . Based on topological congruence with the established phylogenetic framework of AtWRKYs , AsWRKYs were classified into major groups (I, II, III) and subgroups (IIa–IIe). Gene Structure and Conserved Motif Analysis The exon-intron structures of AsWRKYs were visualized using the "Gene Structure View" tool in TBtools software, based on genome annotation files containing exon and intron position information. Conserved motifs within AsWRKY protein sequences were predicted using the MEME suite [ 34 ] with the following parameter settings: maximum number of motifs = 10, motif width range = 20 amino acids, motif E-value threshold < 1e-5, and other parameters set to default. Cis-Acting Element Analysis Promoter regions (1500 bp upstream of the transcription start site, with the transcription start site determined based on genome annotation files) were extracted using a custom Perl script. Cis-regulatory elements were identified using the PlantCARE database [ 35 ] , which provides comprehensive support for the annotation of plant cis-acting regulatory elements. Elements associated with stress responses and hormone regulation (e.g., ABRE, MYB binding sites) were filtered, and the identified cis-elements were visualized (colored by element type) using the "Visualize Cis-elements" tool in TBtools software. Gene Duplication and Synteny Analysis Gene duplication events were analyzed using the MCScanX algorithm integrated in TBtools with the following parameter settings: sequence similarity ≥ 70% and alignment length coverage ≥ 80%. Paralogous gene pairs and segmental duplication blocks were identified based on protein sequence similarity and genomic synteny, with a threshold for excluding tandem duplications set as gene spacing < 5 genes. Genomic data of A. thaliana , Phalaenopsis aphrodite , and O. sativa were retrieved from the NCBI Genome database [ 36 ] . Interspecific synteny maps, with connecting lines representing homologous gene pairs, were generated using Advanced Circos software with default parameters [ 37 ] . Plant Materials Angraecum sesquipedale seedlings were obtained from the orchid nursery of Shanghai Chenshan Botanical Garden, China. For cold stress treatment, nine seedlings with uniform growth status (plant height: 10 ± 1 cm; 3–4 leaves) were transferred to a 4°C growth chamber, maintained under the same light, photoperiod, and humidity conditions as the control environment. Leaf and root samples were collected at 0 h (control), 6 h, and 12 h post-treatment, with three seedlings allocated per time point and each seedling serving as an independent biological replicate. Immediately after collection, tissue samples were flash-frozen in liquid nitrogen for 5 min and stored at -80°C in an ultra-low temperature refrigerator to preserve RNA integrity. RNA extraction and transcriptome sequencing were conducted by MetWare Biotechnology Co., Ltd. (Wuhan, China) using the Illumina sequencing platform. Transcriptome Analysis Raw sequencing reads were filtered using FASTP software [ 38 ] with the following parameter settings: removal of reads containing Illumina universal adapter sequences, reads with N content exceeding 10%, and low-quality reads (reads in which bases with a Qphred value ≤ 20 accounted for more than 50% of the total read length) to obtain clean data. Clean reads were aligned to the reference genome of A. sesquipedale from our laboratory's previous study [ 25 ] using HISAT software [ 39 ] . The expression level of each gene was quantified as its TPM (Transcripts Per Million) value, with differentially expressed genes (DEGs) identified using the criteria: |log₂Fold Change| ≥ 1 and FDR < 0.05. Inter-sample clustering and visualization were performed using the "pheatmap" package in R software. Results Identification of the WRKY Gene in A. sesquipedale A total of 70 WRKY genes ( AsWRKY1 – AsWRKY70 ) were identified in the A. sesquipedale genome and systematically named based on their chromosomal locations (Fig. S1 ). To lay a foundation for functional analysis, a comprehensive physicochemical characterization of these genes was conducted, with key parameters determined, including amino acid (aa) length, molecular weight (MW), isoelectric point (pI), instability index, aliphatic index, hydropathy (Grand Average of Hydropathy, GRAVY), and predicted subcellular localization (Table S1 ). The amino acid lengths of AsWRKYs ranged from 73 residues ( AsWRKY50 ) to 665 residues ( AsWRKY65 ), with corresponding molecular weights of 8.48 kDa and 72.97 kDa, respectively. The pI values spanned from 4.76 ( AsWRKY54 ) to 10.17 ( AsWRKY25 ), with a mean of 7.51, indicating a slight bias toward alkalinity. Instability index analysis classified most proteins as unstable, except for AsWRKY10 , AsWRKY13 , AsWRKY47 , AsWRKY50 , AsWRKY63 , AsWRKY65 , AsWRKY67 , and AsWRKY70 . The aliphatic index varied between 46.93 ( AsWRKY5 ) and 106.95 ( AsWRKY45 ). All AsWRKYs exhibited negative GRAVY values, consistent with hydrophilic properties. Subcellular localization predictions indicated nuclear localization for the majority of AsWRKY proteins. Phylogenetic and Multiple Sequence Alignment Analysis of the AsWRKY Gene Family To explore the evolutionary relationships among members of the AsWRKY gene family and identify potential cold-responsive genes, a phylogenetic tree was constructed using MEGA 11.0 software, incorporating WRKY gene sequences from A. sesquipedale , A. thaliana (72 genes), S. lycopersicum (4 genes), O. sativa (2 genes), and G. max (1 gene) (Fig. 1 ). Notably, the selected WRKY genes from tomato, rice, and soybean have been previously reported to be associated with plant cold stress responses. Analysis results showed that the 70 AsWRKYs could be classified into three major groups based on the established grouping criteria for AtWRKYs : Group I (14 genes), Group II (45 genes), and Group III (11 genes). Group II was further subdivided into five subgroups: IIa (4 genes), IIb (3 genes), IIc (25 genes), IId (3 genes), and IIe (10 genes). Phylogenetic tree analysis not only clarified the evolutionary relationships among family members but also provided potential evidence for functional conservation. Notably, WRKY proteins with analogous functions typically exhibit a clustered distribution pattern. Specifically, the cold-responsive WRKY genes from rice and tomato clustered on the same branch as AsWRKY23 , AsWRKY45 , AsWRKY46 , and AsWRKY48 , all of which belong to Group IIa. Additionally, AsWRKY1 and AsWRKY55 showed a close phylogenetic relationship with G. max GmWRKY21 , while AsWRKY56 and AsWRKY58 clustered with A. thaliana AtWRKY41 on a separate branch. To further investigate the evolutionary relationships among the structural domains of AsWRKY proteins across the seven subgroups, multiple sequence alignment of the highly conserved 60-residue WRKY domains was performed to assess structural conservation (Fig. 2 ). The WRKYGQK heptapeptide was highly conserved across most AsWRKY proteins, although notable substitutions were observed in specific members. For instance, AsWRKY1 , AsWRKY10 , and AsWRKY37 contained a WRKYGKK substitution, while AsWRKY40 exhibited an Arg6-to-Asn (R6N) substitution, resulting in a modified WNKYGQK sequence within its DNA-binding domain. Additionally, three Group IIc members ( AsWRKY17 , AsWRKY55 , and AsWRKY63 ) possessed truncated WRKY domains, whereas three Group I genes ( AsWRKY50 , AsWRKY65 , and AsWRKY70 ) displayed a single WRKY domain architecture. Furthermore, 8 AsWRKYs (e.g., AsWRKY18 , AsWRKY59 , AsWRKY60 ) contained non-canonical zinc-finger motifs. Conserved Motif and Gene Structure Analysis of the AsWRKY Gene Family To further explore the conservation and divergence of protein motif composition, the online tool MEME was used to analyze the conserved motifs of AsWRKYs . Ten distinct motifs were identified, designated as Motif 1 to Motif 10, each consisting of 20 amino acids (Fig. 3 ). Across all AsWRKYs , the number of motifs per protein ranges from 2 to 8, with Motif 1 and Motif 3 collectively constituting the conserved domains that harbor the WRKYGQK sequence and zinc-finger structures (Fig. 4 ). Motif 1, Motif 2, and Motif 3 are highly conserved, present in 79% of all AsWRKY proteins. As noted earlier, these motifs form the structural foundation of the WRKY domain, underscoring their importance for core family functionality. Motif 5, Motif 6, and Motif 9 are uniquely present in Group I AsWRKY proteins. Notably, AsWRKY50 , AsWRKY55 , AsWRKY63 , AsWRKY65 , AsWRKY66 , and AsWRKY70 all lack Motif 1, which typically contains the "WRKYGQK" signature sequence. This absence may indicate functional divergence or alternative modes of action for these members, necessitating further investigation into their DNA-binding capacity or regulatory roles. To further explore the structural diversity and evolutionary conservation of AsWRKY proteins, an analysis of their exon-intron organizations was conducted (Fig. S2 ). The 70 AsWRKYs exhibited significant structural heterogeneity, with the number of exons ranging from 1 to 10 and introns from 0 to 9. Notably, genes clustered within the same phylogenetic group tended to share consistent numbers of coding exons, reflecting evolutionary conservation. Among these, AsWRKY65 displayed the most complex structure, containing 10 exons and 9 introns, while AsWRKY50 and AsWRKY70 were intronless. Cis-Acting Element Analysis Analysis of the 1500 bp promoter regions upstream of AsWRKY genes identified a diverse array of cis-acting elements, which were functionally categorized into hormone-responsive, stress-responsive, light-responsive, and growth/development-related elements (Fig. S3). Hormone-associated elements included ABRE (abscisic acid response element), AuxRR-core (auxin signaling element), CGTCA-motif and TGACG-motif (methyl jasmonate/MeJA responsiveness elements), GARE-motif and TATC-box (gibberellin regulatory elements), TCA-element (salicylic acid response element), and TGA-element (auxin signaling element). Stress-related elements encompassed MBS (drought response element), LTR (low-temperature response element), GC-motif (anoxia tolerance element), WUN-motif (wound signaling element), and ARE (antioxidant defense element). Light-responsive elements comprised G-Box, ACE, Sp1, and GT1-motif. Notably, 20 AsWRKYs contained LTR cis-elements. Gene Duplication Events and Synteny Analysis A total of 68 AsWRKYs are unevenly distributed across 17 chromosomes of A. sesquipedale , while the remaining two genes ( AsWRKY69 , AsWRKY70 ) are located on unanchored scaffolds (Fig. 5 ). Chromosome 1 contained the highest number of AsWRKYs (9 members), making it a hotspot for family localization. In contrast, Chromosomes 8 and 15 each harbored only one AsWRKY gene, representing the lowest gene density (excluding unlocalized scaffolds). Notably, no AsWRKY genes were detected on chromosomes 10 and 19. Gene duplication is a major driver of gene family expansion; analysis of AsWRKY genes identified 21 homologous gene pairs among the 70 members. AsWRKY26 and AsWRKY31 , located on chromosome 5, form a pair of tandemly duplicated genes, while the remaining 20 homologous gene pairs are derived from segmental duplication. To elucidate evolutionary mechanisms, syntenic relationships were analyzed between A. sesquipedale and three reference species: A. thaliana (Fig. 6 A), P. aphrodite (Fig. 6 B), and O. sativa (Fig. 6 C). Comparative synteny mapping identified 14 collinear gene pairs with A. thaliana , 40 with P. aphrodite , and 48 with O. sativa . Compared to the dicotyledonous plant A. thaliana , A. sesquipedale exhibits higher genomic homology with P. aphrodite (a fellow Orchidaceae member) and O. sativa (another monocotyledonous species). Expression patterns of AsWRKY gene family in response to cold stress Low temperature is a critical environmental stress factor constraining plant growth, development, and geographical distribution. To investigate the potential roles of AsWRKYs in the cold stress response of A. sesquipedale , we analyzed the expression patterns of 70 AsWRKYs in leaves and roots based on seedling transcriptome data (Fig. S3) and examined their expression profiles under 4°C cold stress (Fig. 7 ). The results showed that 42.8% (30/70) of AsWRKYs were weakly expressed or not expressed in both tissues. Meanwhile, seven genes ( AsWRKY2 , AsWRKY6 , AsWRKY21 , AsWRKY26 , AsWRKY30 , AsWRKY33 , and AsWRKY40 ) exhibited root-preferential expression, whereas AsWRKY1 , AsWRKY7 , AsWRKY10 , and AsWRKY66 displayed leaf-preferential expression. Additionally, AsWRKY44 and AsWRKY49 showed high expression levels in both tissues (log₂(TPM + 1) > 1.5). Under low-temperature stress, most AsWRKYs responded in both roots and leaves. Among these, 45% (32/70) of AsWRKYs were downregulated to varying degrees in both tissues as the duration of low-temperature treatment increased (Fig. 7 ). Notably, in leaves, the expression levels of 19 AsWRKYs ( AsWRKY4 , AsWRKY9 , AsWRKY16 , AsWRKY17 , AsWRKY21 , AsWRKY25 , AsWRKY23 , AsWRKY30 , AsWRKY32 , AsWRKY36 , AsWRKY37 , AsWRKY41 , AsWRKY44 , AsWRKY49 , AsWRKY56 , AsWRKY58 , AsWRKY59 , AsWRKY61 , and AsWRKY67 ) increased rapidly over time or showed a sustained increase to varying degrees, with peak expression observed at 6 h or 12 h (Fig. 7 A). Similarly, in roots, 11 AsWRKYs ( AsWRKY7 , AsWRKY13 , AsWRKY21 , AsWRKY30 , AsWRKY31 , AsWRKY32 , AsWRKY43 , AsWRKY44 , AsWRKY49 , AsWRKY52 , and AsWRKY61 ) exhibited similar dynamic expression patterns under cold stress (Fig. 7 B). Discussion Under natural conditions, plant growth, development, and reproduction are frequently challenged by both biotic and abiotic stresses. To cope with these adverse factors, plants have evolved elaborate regulatory mechanisms, with the WRKY gene family serving as a particularly representative example [ 40 ] . Its members are extensively involved in diverse physiological processes, including the regulation of plant growth and development, secondary metabolite synthesis, and stress signal transduction [ 41 , 42 ] . Although the functions of the WRKY family in model angiosperms have been relatively well characterized, systematic research on their molecular mechanisms in orchids remains scarce, especially in A. sesquipedale , a species with unique evolutionary significance. Structural conservation and divergence of AsWRKY genes Genomic screening identified 70 putative AsWRKY proteins, with physicochemical characterization revealing substantial heterogeneity in encoded protein attributes, including molecular mass (8.48–72.97 kDa) and isoelectric point (pI 4.76–10.17) (Table S1 ). Such biophysical divergence suggests adaptive specialization for context-dependent stress responses. Subcellular localization predictions indicated nuclear enrichment for the majority of AsWRKY proteins, congruent with their canonical roles as transcriptional regulators and consistent with orthologous studies in other vascular plants [ 16 ] . The evolutionary relationship of AsWRKY genes To explore the evolutionary relationships of the AsWRKY gene family, a phylogenetic tree was constructed alongside the A. thaliana WRKY gene family, classifying the 70 AsWRKYs into three major groups (I, II, III), with Group II further subdivided into five subgroups (IIa–IIe) (Fig. 1 ). This classification is consistent with observations of the WRKY gene family in other plant species [ 43 , 44 ] . Members within the same group exhibit high similarity in conserved domains and gene structures, reflecting the relative evolutionary stability of WRKY family genes in A. sesquipedale . Notably, several AsWRKYs cluster closely with cold stress-associated WRKY genes from four other plant species, suggesting they may participate in cold stress responses through analogous mechanisms. Additionally, several Group I AsWRKYs (e.g., AsWRKY50 , AsWRKY65 , AsWRKY70 ) possess only one WRKY domain (Fig. 2 ), a phenomenon also reported in cassava [ 45 ] and maize [ 46 ] . The study further revealed that the highly conserved WRKYGQK motif in specific AsWRKYs (e.g., AsWRKY1 , AsWRKY10 , AsWRKY37 ) has mutated to WRKYGKK, mirroring WRKY domain mutations in soybean GmWRKY6 and GmWRKY21 . Such mutations impair normal binding to the W-box element (TTGACC/T), significantly reducing or even eliminating the ability to interact with target gene promoters [ 47 ] . Interestingly, mutations in the WRKY domain and zinc finger structures are primarily concentrated in subgroup IIc, consistent with previous reports of greater sequence diversity in plant subgroup IIc [ 48 ] . This pattern suggests a potential association with functional divergence during plant adaptation to diverse ecological niches. Promoter cis-acting elements provide insights into the potential regulatory mechanisms and biological functions of genes. Analysis of AsWRKYs identified numerous hormone-responsive motifs (e.g., ABRE, TGA elements) and stress-responsive motifs (e.g., MBS, LTR) (Fig. S3). These findings suggest that AsWRKYs may be involved in mediating plant hormone crosstalk and environmental stress adaptation processes. Regarding gene family expansion mechanisms, both tandem duplication and segmental duplication contribute to the expansion of the WRKY gene family. In A. sesquipedale , segmental duplication events (20 pairs) were far more frequent than tandem duplication events (1 pair) (Fig. 5 ), consistent with macroevolutionary patterns reported for WRKY families in other plant species [ 49 , 50 , 51 ] . This indicates that segmental duplication is the primary driver of WRKY gene family expansion during the evolution of A. sesquipedale . Interspecific synteny reflects evolutionary relationships between species. The phylogenetic relationship between A. sesquipedale and P. aphrodite , as well as with O. sativa , is significantly closer than that with A. thaliana (Fig. 6 ), resulting in greater genomic structural conservation and more abundant collinear gene pairs among the former three. Notably, previous studies have shown that the Cymbidium goeringii CgWRKY57 gene shares high homology with rice OsWRKY47 , and its expression is induced by low-temperature and ABA stress [ 52 ] . This observation further supports the synteny analysis inference: the collinear associations of the WRKY gene family among A. sesquipedale , P. aphrodite , and rice imply they may have undergone analogous segmental duplication events, thereby exhibiting functional conservation in key biological processes such as low-temperature stress responses. AsWRKY genes function in cold stresses The potential roles of AsWRKYs can be inferred by integrating their expression patterns across different tissues of A. sesquipedale with the known functions of WRKY genes in model plants (Fig. 1 ). In root development regulation, AtWRKY6 , AtWRKY11 , and AtWRKY23 modulate root development through protein-protein interactions in A. thaliana [ 53 , 54 , 55 ] . Notably, these genes cluster with AsWRKY21 in the phylogenetic tree and share root-preferential expression patterns (Fig. S4). These observations suggest that root-preferentially expressed AsWRKYs may act as key regulators of root development, participating in root responses to diverse stresses and potentially promoting nutrient uptake by regulating transporter gene expression [ 56 ] . In terms of leaf function, WRKY TFs in plant leaves regulate photosynthesis-related genes; for example, tomato ( S. lycopersicum ) SlWRKY17 modulates photosynthetic pigment accumulation by regulating genes encoding key enzymes in chlorophyll biosynthesis [ 57 ] . Additionally, leaf-specific WRKY genes have been implicated in complex defense signaling networks [ 58 , 59 ] . Moreover, referencing the mechanism by which WRKY genes influence leaf senescence through the regulation of senescence-associated genes (SAGs) [ 60 , 61 , 62 ] , these leaf-expressed WRKY genes may enhance biotic stress resistance and regulate leaf senescence by coordinating multiple hormone signaling pathways. Furthermore, AsWRKY44 and AsWRKY49 are highly expressed in both leaves and roots, a feature suggesting their potential role as critical regulators in the basal life processes of A. sesquipedale . Against the backdrop of escalating global climate change, plants face increasing threats from extreme environmental stresses such as drought, flooding, and low temperatures. As pivotal regulators in stress responses, the WRKY gene family not only mediates cold stress responses but also plays a key role in enhancing plant cold tolerance and environmental adaptability [ 63 ] . Analysis of the regulatory mechanisms of WRKY TFs under abiotic stress revealed the following patterns: transcriptomic data indicated that 45% of AsWRKYs in roots and leaves exhibited high expression levels at 0 h (control), followed by sustained downregulation to varying degrees after cold treatment (Fig. 7 ). Studies have demonstrated that when plants undergo abiotic stress, WRKY TFs can induce growth arrest through downregulated expression, thereby reducing energy expenditure to sustain fundamental physiological processes [ 64 , 65 ] . Importantly, a subset of AsWRKYs in roots and leaves displayed distinct temporal expression dynamics: 4 genes in leaves (Fig. 7 A) and 7 genes in roots (Fig. 7 B) exhibited rapid response characteristics, being rapidly induced upon cold treatment and maintaining expression until 12 h. This suggests their potential involvement in the early cold stress response by regulating downstream target gene expression [ 66 ] . Conversely, 15 genes in leaves and 4 genes in roots showed a distinct trend, with their expression levels accumulating continuously as cold treatment prolonged, peaking at 12 h, thus exhibiting characteristics of long-term response genes. This is analogous to the regulatory mechanism of VaWRKY65 in Vitis amurensis , which enhances plant cold tolerance by binding to the W-box in the promoter of VaBAM3 , sustainably upregulating its expression to promote soluble sugar accumulation [ 67 ] . To investigate the potential roles of AsWRKYs in cold stress regulation, the present study integrated phylogenetic analysis of AsWRKYs with cold stress-associated WRKY genes from four model plant species ( A. thaliana , S. lycopersicum , O. sativa , and G. max ) (Fig. 1 ). Combined with differential expression profiles of AsWRKYs under low-temperature stress, this approach aimed to dissect their underlying regulatory mechanisms. The cold stress-responsive functions of WRKY genes in these model systems have been extensively characterized, providing a robust framework for inferring the functional roles of AsWRKYs . The WRKY family exhibits evolutionary conservation in cold stress adaptation, as exemplified by soybean GmWRKY21 , which enhances cold stress tolerance when heterologously expressed in transgenic Arabidopsis [ 47 ] . Notably, AtWRKY33 is rapidly transcriptionally induced upon cold stress initiation, thereby initiating early stress-responsive signaling cascades in A. thaliana [ 68 ] . In tomato, SlWRKY33 enhances cold hardiness by directly targeting and activating genes encoding kinases, transcription factors, and molecular chaperones, such as CDPK11, MYBS3, and BAG6 [ 69 ] . Phylogenetic clustering analysis reveals that AsWRKY17 and AsWRKY30 closely cluster with AtWRKY33 and SlWRKY33 (Fig. 7 ), and exhibit sustained upregulation under cold stress, consistent with the prolonged regulatory dynamics mediated by SlWRKY33 . This suggests they may participate in cold stress responses through analogous target gene regulatory networks. In contrast, in A. thaliana , AtWRKY41 functions as a negative regulator by directly binding to W-box motifs in the promoters of CBF genes, repressing their expression and thereby attenuating plant freezing tolerance [ 70 ] . AsWRKY56 and AsWRKY58 are phylogenetically closely related to AtWRKY41 and exhibit transient upregulation under cold stress (Fig. 7 A). This immediate response profile aligns with their putative negative regulatory roles, suggesting they may modulate freezing tolerance by suppressing the expression of frost resistance-associated genes. Furthermore, other WRKY genes in tomato and rice regulate cold tolerance through distinct pathways: tomato SlWRKY45 and SlWRKY46 enhance reactive oxygen species (ROS) scavenging capacity by regulating antioxidant enzyme pathways, thereby improving cold tolerance in transgenic lines [ 71 , 72 ] ; SlWRKY50 indirectly enhances cold hardiness through hormone signaling networks by regulating jasmonic acid (JA) biosynthesis [ 73 ] . In rice, OsWRKY71 positively regulates cold tolerance via hierarchical control of downstream target gene networks [ 74 ] ; OsWRKY76 enhances cold tolerance through protein-protein interactions with OsbHLH148 , which transactivates OsDREB1B expression [ 75 ] . AsWRKY23 clusters phylogenetically with these rice and tomato WRKYs—particularly with OsWRKY71 —and maintains sustained expression under cold stress (Fig. 7 A). This indicates it may synergistically enhance plant cold tolerance through analogous mechanisms, such as modulating target gene networks or mediating protein interaction-based regulation. This study established functional associations between A. sesquipedale AsWRKY17 , AsWRKY23 , AsWRKY30 , AsWRKY56 , and AsWRKY58 and cold-responsive WRKY genes from model plants. It revealed that the WRKY family in this species may participate in cold stress responses via a multi-pathway regulatory network encompassing "positive regulation-negative regulation-coordinated regulation". These findings provide a foundation for subsequent genetic validation of cold tolerance mechanisms in A. sesquipedale . Additionally, this research clarified the evolutionary characteristics underlying functional divergence in cold responsiveness between AsWRKYs and WRKY genes from diverse plant lineages. The key genes identified herein can be directly applied to ex situ conservation, expansion of cultivation ranges, and breeding of stress-resistant A. sesquipedale varieties, thereby addressing the core issue of low-temperature constraints on its growth and development. Conclusions In this study, 70 AsWRKY genes were identified in A. sesquipedale , and a comprehensive analysis was conducted on these genes. First, through multiple sequence alignment combined with analyses of evolutionary relationships, conserved motifs, gene structures, cis-acting elements, gene duplication events, and collinearity, the characteristics of this gene family were systematically elucidated. Expression pattern analyses of AsWRKYs in two distinct tissues and under cold stress indicated that these genes may play important roles in the growth, development, and cold stress response of A. sesquipedale . Furthermore, based on integrated phylogenetic tree and expression profile analyses, five key genes potentially involved in regulating A. sesquipedale under cold stress were identified. In conclusion, this study establishes a foundation for functional characterization of the WRKY gene family in A. sesquipedale and provides an important reference for subsequent in-depth investigations into their molecular mechanisms underlying growth, development, and cold stress response. Declarations Ethics approval and consent to participate Not applicable. Plant material sampling adhered to institutional guidelines, and as no human/animal subjects or ethically regulated procedures were involved, formal ethics committee review was not applicable. Consent for publication Not applicable. Availability of data and materials Raw transcriptomic data have been deposited in the NCBI database (PRJNA1307821). All datasets analyzed during the current study are included in this published article and its supplementary information files. Competing interests The authors declare no competing interests. Funding This work was supported by grant from the Shanghai Landscaping and City Appearance Administrative Bureau (G242418). Author’ contributions CG and CH executed the experimental procedures, conducted data analysis, prepared the figures and tables, and drafted the initial manuscript. MFW and SSP contributed to experimental implementation and data interpretation. ZYN provided the experimental materials. MHZ and QNR were responsible for the compilation of selected figures and tables. YQS and RFP participated in drafting specific sections of the manuscript. TP and WCH conceptualized and designed the experiments, and provided critical input during the revision and review process. All authors reviewed and approved the final version of the manuscript. 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OsWRKY71 , a rice transcription factor, is involved in rice defense response. J Plant Physiol. 2007;164(8):969–79. Zhang M, Zhao R, Huang K, Huang S, Wang H, Wei Z, Li Z, Bian M, Jiang W, Wu T, Du X. The OsWRKY63 - OsWRKY76 - OsDREB1B module regulates chilling tolerance in rice. Plant J. 2022;112(2):383–98. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.docx Additional file 1: Fig. S1 Chromosomal localization and distribution of the WRKY Gene Family in A. sesquipedale . Fig. S2 Comprehensive Analysis of Conserved Motifs and Gene Structure in AsWRKY proteins. In the gene structure diagram, yellow boxes and lines represent exons and introns, respectively. Fig. S3 Prediction of cis-responsive elements in the 1500 bp upstream regulatory regions of AsWRKYs . Different cis-responsive elements are represented by different colored boxes. Fig. S4 Expression profile analysis of AsWRKYs in two tissues. Transcriptome data were used to measure the expression levels of AsWRKYs in roots, leaves. The colored scale for the different expression levels is shown. Additionalfile2.xlsx Additional file 2: Table. S1 Physicochemical properties and subcellular localization predictions of AsWRKY gene family members. 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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The blue characters and asterisks represent \u003cem\u003eAsWRKYs\u003c/em\u003e, red characters and circles represent \u003cem\u003eAtWRKYs\u003c/em\u003e, orange characters and check marks represent \u003cem\u003eSlWRKYs\u003c/em\u003e, green characters and triangles represent \u003cem\u003eOsWRKYs\u003c/em\u003e, and cyan characters and squares represent \u003cem\u003eGmWRKY21\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7505680/v1/1d001a49e7a9d08bc8bdfdee.png"},{"id":91460370,"identity":"aeef2e76-8b38-4bef-a8a7-89fe06299e13","added_by":"auto","created_at":"2025-09-16 17:04:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1478586,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment of WRKY domains of seven subfamilies \u003cem\u003eA.\u003c/em\u003e \u003cem\u003esesquipedale\u003c/em\u003e. The conserved WRKYGQK heptapeptide motif is highlighted in red boxes. The cysteine (C) residues in the zinc-finger motifs are marked with solid circles (●), and histidine (H) residues are marked with triangles (▲).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7505680/v1/a5bfbe7daf30b689697d6ee2.png"},{"id":91461188,"identity":"d77246e7-173b-427a-b7b5-50ddcbc374bf","added_by":"auto","created_at":"2025-09-16 17:12:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":464975,"visible":true,"origin":"","legend":"\u003cp\u003eConserved Motif Sequences of WRKY proteins in \u003cem\u003eA.\u003c/em\u003e \u003cem\u003esesquipedale\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7505680/v1/00c8f15951b16db83f007104.png"},{"id":91459825,"identity":"95d97427-dc21-4480-b885-f164f1888292","added_by":"auto","created_at":"2025-09-16 16:56:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":710169,"visible":true,"origin":"","legend":"\u003cp\u003eConserved motifs of the AsWRKY proteins arranged according to their phylogenetic relationships. Ten motifs were identified and were shown in different colors.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7505680/v1/64acfd57f3eda726f4ead6af.png"},{"id":91461189,"identity":"2b69eaea-9708-45da-b960-d4928fa4eda0","added_by":"auto","created_at":"2025-09-16 17:12:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":637850,"visible":true,"origin":"","legend":"\u003cp\u003eGenome-wide collinearity analysis of AsWRKY family members. Red lines denote intra-family collinear gene pairs, while the gray background highlights all syntenic blocks across the \u003cem\u003eA.\u003c/em\u003e \u003cem\u003esesquipedale\u003c/em\u003egenome.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7505680/v1/41129b310daed037c76788e8.png"},{"id":91460371,"identity":"965995b1-a940-4e0e-a507-c47afaf70f54","added_by":"auto","created_at":"2025-09-16 17:04:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":477008,"visible":true,"origin":"","legend":"\u003cp\u003eInter-species synteny analysis of AsWRKY proteins among \u003cem\u003eA.\u003c/em\u003e \u003cem\u003ethaliana\u003c/em\u003e (\u003cstrong\u003eA\u003c/strong\u003e), \u003cem\u003eP. aphrodite \u003c/em\u003e(\u003cstrong\u003eB\u003c/strong\u003e), and \u003cem\u003eO.\u003c/em\u003e \u003cem\u003esativa\u003c/em\u003e (\u003cstrong\u003eC\u003c/strong\u003e). Gray lines depict all collinear blocks between \u003cem\u003eA.\u003c/em\u003e \u003cem\u003esesquipedale\u003c/em\u003e and the respective genomes, while red lines specifically highlight orthologous \u003cem\u003eAsWRKY\u003c/em\u003e gene pairs.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7505680/v1/4d009052d3745785a3e9f3e6.png"},{"id":91459821,"identity":"06b7fe96-27ba-4757-af77-e32cf5b62d18","added_by":"auto","created_at":"2025-09-16 16:56:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":345901,"visible":true,"origin":"","legend":"\u003cp\u003eTissue-specific expression of AsWRKY transcription factors. Expression dynamics under cold Stress across time in leaf (\u003cstrong\u003eA\u003c/strong\u003e) and root (\u003cstrong\u003eB\u003c/strong\u003e). In scale bar, the expression of DEGs is indicated in red (high abundance) and green (low abundance).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7505680/v1/179b4919ba96b9c990d25031.png"},{"id":92570020,"identity":"08665fbf-2688-428c-af18-ff513e952e8a","added_by":"auto","created_at":"2025-10-01 07:32:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5365949,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7505680/v1/18a91420-9596-4c06-907b-97a25ef2b7c8.pdf"},{"id":91461191,"identity":"b2543c17-ee15-404e-ab4e-3032b60a48cd","added_by":"auto","created_at":"2025-09-16 17:12:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1332461,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 1: Fig. S1 Chromosomal localization and distribution of the WRKY Gene Family in \u003cem\u003eA. sesquipedale\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eFig. S2 Comprehensive Analysis of Conserved Motifs and Gene Structure in AsWRKY proteins. In the gene structure diagram, yellow boxes and lines represent exons and introns, respectively.\u003c/p\u003e\n\u003cp\u003eFig. S3 Prediction of cis-responsive elements in the 1500 bp upstream regulatory regions of \u003cem\u003eAsWRKYs\u003c/em\u003e. Different cis-responsive elements are represented by different colored boxes.\u003c/p\u003e\n\u003cp\u003eFig. S4 Expression profile analysis of \u003cem\u003eAsWRKYs\u003c/em\u003e in two tissues. Transcriptome data were used to measure the expression levels of \u003cem\u003eAsWRKYs\u003c/em\u003e in roots, leaves. The colored scale for the different expression levels is shown.\u003c/p\u003e","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7505680/v1/f2578dde07db78e9190125b2.docx"},{"id":91459824,"identity":"82c86223-32d5-4001-9e9f-b252b1c2537a","added_by":"auto","created_at":"2025-09-16 16:56:07","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16697,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 2: Table. S1 Physicochemical properties and subcellular localization predictions of AsWRKY gene family members.\u003c/p\u003e","description":"","filename":"Additionalfile2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7505680/v1/b224558ba76ce225577e0f20.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide identification of the WRKY gene family in Angraecum sesquipedale and exploration of its role in cold stress response","fulltext":[{"header":"Background","content":"\u003cp\u003eThe WRKY gene family represents a pivotal family of transcription factors (TFs) in plants, playing crucial roles in regulating plant development and orchestrating responses to diverse stresses. Structurally, WRKY proteins are characterized by a conserved domain architecture that includes one or two WRKY domains at the N-terminus and specific zinc-finger motifs at the C-terminus. Each WRKY domain encompasses the highly conserved amino acid sequence WRKYGQK, while the C-terminal region contains functionally essential zinc-finger structures that exhibit distinct structural variations. These variations are exemplified by the C2H2-type motifs (with the consensus sequence CX4-5CX22-23HXH) and C2HC-type motifs (with the consensus sequence CX7CX23HXC) \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Based on these structural variations, the WRKY family is classified into three major groups (Group I, Group II, and Group III), with Group II further subdivided into five subgroups \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWRKY TFs act as key regulators in plant growth and development, participating in a range of biological processes, including seed dormancy \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e and embryogenesis \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e to plant growth, senescence \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, and metabolism \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. For instance, in rice (\u003cem\u003eOryza sativa\u003c/em\u003e), \u003cem\u003eOsWRKY29\u003c/em\u003e acts as a repressor of seed dormancy by attenuating abscisic acid (ABA)-mediated responses \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eOsWRKY53\u003c/em\u003e modulates anther development through the transcriptional regulation of gibberellin (GA) metabolic genes, and its knockout mutants exhibit significantly enhanced cold tolerance under low-temperature stress \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. In addition, overexpression of \u003cem\u003eLcWRKY17\u003c/em\u003e promotes monoterpene biosynthesis \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. In \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eAtWRKY53\u003c/em\u003e serves as a convergence node for jasmonic acid (JA) signaling and age-dependent pathways; it directly upregulates the expression of senescence-associated genes, thereby functioning as a positive regulator of leaf senescence \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMembers of the WRKY family act as critical mediators in plant responses to both biotic and abiotic stresses, enabling plants to cope with bacterial infections \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, mechanical damage \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, and environmental stresses such as heat, drought, salinity, and cold \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. For instance, transgenic \u003cem\u003eArabidopsis\u003c/em\u003e overexpressing \u003cem\u003eVitis vinifera VvWRKY28\u003c/em\u003e exhibits significantly enhanced tolerance to both low-temperature and high-salinity stresses, highlighting its role in cross-stress adaptation \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Overexpression of wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) \u003cem\u003eTaWRKY19\u003c/em\u003e confers broad-spectrum tolerance to salt, drought, and freezing stresses in transgenic plants \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. In \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eAtWRKY25\u003c/em\u003e, \u003cem\u003eAtWRKY26\u003c/em\u003e, and \u003cem\u003eAtWRKY33\u003c/em\u003e cooperatively improve thermotolerance by regulating ethylene-activated signaling pathways \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Overexpression of maize (\u003cem\u003eZea mays\u003c/em\u003e) \u003cem\u003eZmWRKY106\u003c/em\u003e enhances drought and heat resistance, linking its function to multiple abiotic stress pathways \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Cucumber (\u003cem\u003eCucumis sativus\u003c/em\u003e) \u003cem\u003eCsWRKY46\u003c/em\u003e confers cold tolerance to transgenic plants and positively regulates the cold signaling pathway in an ABA-dependent manner \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. These WRKY TFs regulate downstream gene expression by specifically binding to W-box elements in the promoters of target genes, thereby activating or repressing transcription to enhance plant adaptation to adverse environments \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough the WRKY gene family has been extensively identified and functionally characterized in various plant species, research on orchids remains relatively scarce \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Existing studies have identified WRKY family members in \u003cem\u003eDendrobium officinale\u003c/em\u003e and analyzed their expression profiles under abiotic stress \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In \u003cem\u003eCymbidium sinense\u003c/em\u003e, \u003cem\u003eCsWRKY18\u003c/em\u003e regulates abiotic stress responses via an ABA-dependent pathway \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eAngraecum sesquipedale\u003c/em\u003e (Angraecinae, Orchidaceae), endemic to Madagascar, is renowned for its unique co-evolutionary relationship with pollinators, attributed to its extraordinarily long nectar spur. As a rare endemic orchid dependent on specific microhabitats, \u003cem\u003eA. sesquipedale\u003c/em\u003e is vulnerable to abiotic stresses such as low temperatures, making the WRKY family a pivotal target for investigating stress adaptation mechanisms. However, research on the WRKY gene family in \u003cem\u003eA. sesquipedale\u003c/em\u003e is currently lacking. Here, we performed genome-wide identification of WRKY genes in \u003cem\u003eA. sesquipedale\u003c/em\u003e (designated \u003cem\u003eAsWRKYs\u003c/em\u003e) and analyzed their conserved domains, phylogenetic relationships, gene structure, motif composition, cis-acting elements, gene duplication events, and collinearity. Additionally, transcriptome sequencing was used to investigate the role of \u003cem\u003eAsWRKYs\u003c/em\u003e in cold stress responses, aiming to provide valuable insights into the molecular mechanisms underlying cold tolerance.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of the AsWRKY Gene Family\u003c/h2\u003e\u003cp\u003eThe genome of \u003cem\u003eA. sesquipedale\u003c/em\u003e previously assembled by our research group was employed in this study \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eA. thaliana\u003c/em\u003e WRKY (\u003cem\u003eAtWRKYs\u003c/em\u003e) protein sequences were retrieved from The Arabidopsis Information Resource (TAIR) \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Sequences of \u003cem\u003eSlWRKY33\u003c/em\u003e (\u003cem\u003eSolyc09g014990\u003c/em\u003e), \u003cem\u003eSlWRKY45\u003c/em\u003e (\u003cem\u003eSolyc08g067360\u003c/em\u003e), \u003cem\u003eSlWRKY46\u003c/em\u003e (\u003cem\u003eSolyc08g067340\u003c/em\u003e), and \u003cem\u003eSlWRKY50\u003c/em\u003e (\u003cem\u003eSolyc06g068460\u003c/em\u003e) were obtained from the Phytozome database \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Sequences of \u003cem\u003eOsWRKY71\u003c/em\u003e (\u003cem\u003eOs02g0181300\u003c/em\u003e), \u003cem\u003eOsWRKY76\u003c/em\u003e (\u003cem\u003eOs09g0417600\u003c/em\u003e), and \u003cem\u003eGmWRKY21\u003c/em\u003e (NP_001237327.2) were acquired from the National Center for Biotechnology Information (NCBI) Protein database \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. The hidden Markov model (HMM) profile of the WRKY domain (PF03106) was retrieved from the Pfam database \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Candidate WRKY genes were identified by searching the local \u003cem\u003eA. sesquipedale\u003c/em\u003e protein database using HMMER 3.3.2 software with the parameter setting: E-value\u0026thinsp;\u0026lt;\u0026thinsp;1e-5. To ensure accuracy, the integrity of the WRKY domain (core sequence: WRKYGQK) and zinc-finger motifs within the sequences was further verified using SMART \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, NCBI CDD \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, and Pfam \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, leading to the final determination of the AsWRKY genes set. Based on the physical location information in the \u003cem\u003eA. sesquipedale\u003c/em\u003e genome annotation file, 70 WRKY genes were mapped to their respective chromosomes.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnalysis of Physicochemical Properties of the AsWRKY Gene Family\u003c/h3\u003e\n\u003cp\u003eThe Protein Parameter Calculator tool in TBtools software with default parameters was used to analyze the number of amino acids, molecular weight (MW), isoelectric point (pI), and instability index of \u003cem\u003eA. sesquipedale\u003c/em\u003e WRKY proteins (with an instability index\u0026thinsp;\u0026gt;\u0026thinsp;40 indicating unstable proteins). Subcellular localization was predicted using the WoLFPSORT tool \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003ewith default parameters, which infers subcellular compartment distribution based on amino acid composition and sorting signals (e.g., nuclear localization signals, chloroplast transit peptides).\u003c/p\u003e\n\u003ch3\u003ePhylogenetic Analysis and Sequence Alignment\u003c/h3\u003e\n\u003cp\u003eMultiple sequence alignment of \u003cem\u003eA. sesquipedale\u003c/em\u003e WRKY (\u003cem\u003eAsWRKY\u003c/em\u003e) protein sequences was performed using the MUSCLE algorithm in MEGA 11.0 software with the following parameters: gap opening penalty = -400, gap extension penalty\u0026thinsp;=\u0026thinsp;0, and default matrix. The alignment results, which highlight conserved amino acid residues, were visualized using GeneDoc software. A maximum likelihood (ML) phylogenetic tree was constructed in MEGA 11.0, with branch reliability evaluated via 1000 bootstrap replicates; other parameters were set to default. The phylogenetic tree was annotated (e.g., group labeling, branch coloring) and refined using the Interactive Tree of Life (iTOL) platform \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Based on topological congruence with the established phylogenetic framework of \u003cem\u003eAtWRKYs\u003c/em\u003e, \u003cem\u003eAsWRKYs\u003c/em\u003e were classified into major groups (I, II, III) and subgroups (IIa\u0026ndash;IIe).\u003c/p\u003e\n\u003ch3\u003eGene Structure and Conserved Motif Analysis\u003c/h3\u003e\n\u003cp\u003eThe exon-intron structures of \u003cem\u003eAsWRKYs\u003c/em\u003e were visualized using the \"Gene Structure View\" tool in TBtools software, based on genome annotation files containing exon and intron position information. Conserved motifs within AsWRKY protein sequences were predicted using the MEME suite \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e with the following parameter settings: maximum number of motifs\u0026thinsp;=\u0026thinsp;10, motif width range\u0026thinsp;=\u0026thinsp;20 amino acids, motif E-value threshold\u0026thinsp;\u0026lt;\u0026thinsp;1e-5, and other parameters set to default.\u003c/p\u003e\n\u003ch3\u003eCis-Acting Element Analysis\u003c/h3\u003e\n\u003cp\u003ePromoter regions (1500 bp upstream of the transcription start site, with the transcription start site determined based on genome annotation files) were extracted using a custom Perl script. Cis-regulatory elements were identified using the PlantCARE database \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e, which provides comprehensive support for the annotation of plant cis-acting regulatory elements. Elements associated with stress responses and hormone regulation (e.g., ABRE, MYB binding sites) were filtered, and the identified cis-elements were visualized (colored by element type) using the \"Visualize Cis-elements\" tool in TBtools software.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eGene Duplication and Synteny Analysis\u003c/h2\u003e\u003cp\u003eGene duplication events were analyzed using the MCScanX algorithm integrated in TBtools with the following parameter settings: sequence similarity\u0026thinsp;\u0026ge;\u0026thinsp;70% and alignment length coverage\u0026thinsp;\u0026ge;\u0026thinsp;80%. Paralogous gene pairs and segmental duplication blocks were identified based on protein sequence similarity and genomic synteny, with a threshold for excluding tandem duplications set as gene spacing\u0026thinsp;\u0026lt;\u0026thinsp;5 genes. Genomic data of \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003ePhalaenopsis aphrodite\u003c/em\u003e, and \u003cem\u003eO. sativa\u003c/em\u003e were retrieved from the NCBI Genome database \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Interspecific synteny maps, with connecting lines representing homologous gene pairs, were generated using Advanced Circos software with default parameters \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePlant Materials\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eAngraecum sesquipedale\u003c/em\u003e seedlings were obtained from the orchid nursery of Shanghai Chenshan Botanical Garden, China. For cold stress treatment, nine seedlings with uniform growth status (plant height: 10\u0026thinsp;\u0026plusmn;\u0026thinsp;1 cm; 3\u0026ndash;4 leaves) were transferred to a 4\u0026deg;C growth chamber, maintained under the same light, photoperiod, and humidity conditions as the control environment. Leaf and root samples were collected at 0 h (control), 6 h, and 12 h post-treatment, with three seedlings allocated per time point and each seedling serving as an independent biological replicate. Immediately after collection, tissue samples were flash-frozen in liquid nitrogen for 5 min and stored at -80\u0026deg;C in an ultra-low temperature refrigerator to preserve RNA integrity. RNA extraction and transcriptome sequencing were conducted by MetWare Biotechnology Co., Ltd. (Wuhan, China) using the Illumina sequencing platform.\u003c/p\u003e\n\u003ch3\u003eTranscriptome Analysis\u003c/h3\u003e\n\u003cp\u003eRaw sequencing reads were filtered using FASTP software \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e with the following parameter settings: removal of reads containing Illumina universal adapter sequences, reads with N content exceeding 10%, and low-quality reads (reads in which bases with a Qphred value\u0026thinsp;\u0026le;\u0026thinsp;20 accounted for more than 50% of the total read length) to obtain clean data. Clean reads were aligned to the reference genome of \u003cem\u003eA. sesquipedale\u003c/em\u003e from our laboratory's previous study \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e using HISAT software \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. The expression level of each gene was quantified as its TPM (Transcripts Per Million) value, with differentially expressed genes (DEGs) identified using the criteria: |log₂Fold Change| \u0026ge; 1 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Inter-sample clustering and visualization were performed using the \"pheatmap\" package in R software.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eIdentification of the WRKY Gene in\u003c/b\u003e \u003cb\u003eA. sesquipedale\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 70 WRKY genes (\u003cem\u003eAsWRKY1\u003c/em\u003e\u0026ndash;\u003cem\u003eAsWRKY70\u003c/em\u003e) were identified in the \u003cem\u003eA. sesquipedale\u003c/em\u003e genome and systematically named based on their chromosomal locations (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). To lay a foundation for functional analysis, a comprehensive physicochemical characterization of these genes was conducted, with key parameters determined, including amino acid (aa) length, molecular weight (MW), isoelectric point (pI), instability index, aliphatic index, hydropathy (Grand Average of Hydropathy, GRAVY), and predicted subcellular localization (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe amino acid lengths of \u003cem\u003eAsWRKYs\u003c/em\u003e ranged from 73 residues (\u003cem\u003eAsWRKY50\u003c/em\u003e) to 665 residues (\u003cem\u003eAsWRKY65\u003c/em\u003e), with corresponding molecular weights of 8.48 kDa and 72.97 kDa, respectively. The pI values spanned from 4.76 (\u003cem\u003eAsWRKY54\u003c/em\u003e) to 10.17 (\u003cem\u003eAsWRKY25\u003c/em\u003e), with a mean of 7.51, indicating a slight bias toward alkalinity. Instability index analysis classified most proteins as unstable, except for \u003cem\u003eAsWRKY10\u003c/em\u003e, \u003cem\u003eAsWRKY13\u003c/em\u003e, \u003cem\u003eAsWRKY47\u003c/em\u003e, \u003cem\u003eAsWRKY50\u003c/em\u003e, \u003cem\u003eAsWRKY63\u003c/em\u003e, \u003cem\u003eAsWRKY65\u003c/em\u003e, \u003cem\u003eAsWRKY67\u003c/em\u003e, and \u003cem\u003eAsWRKY70\u003c/em\u003e. The aliphatic index varied between 46.93 (\u003cem\u003eAsWRKY5\u003c/em\u003e) and 106.95 (\u003cem\u003eAsWRKY45\u003c/em\u003e). All \u003cem\u003eAsWRKYs\u003c/em\u003e exhibited negative GRAVY values, consistent with hydrophilic properties. Subcellular localization predictions indicated nuclear localization for the majority of AsWRKY proteins.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic and Multiple Sequence Alignment Analysis of the AsWRKY Gene Family\u003c/h2\u003e\u003cp\u003eTo explore the evolutionary relationships among members of the AsWRKY gene family and identify potential cold-responsive genes, a phylogenetic tree was constructed using MEGA 11.0 software, incorporating WRKY gene sequences from \u003cem\u003eA. sesquipedale\u003c/em\u003e, \u003cem\u003eA. thaliana\u003c/em\u003e (72 genes), \u003cem\u003eS. lycopersicum\u003c/em\u003e (4 genes), \u003cem\u003eO. sativa\u003c/em\u003e (2 genes), and \u003cem\u003eG. max\u003c/em\u003e (1 gene) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, the selected WRKY genes from tomato, rice, and soybean have been previously reported to be associated with plant cold stress responses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnalysis results showed that the 70 \u003cem\u003eAsWRKYs\u003c/em\u003e could be classified into three major groups based on the established grouping criteria for \u003cem\u003eAtWRKYs\u003c/em\u003e: Group I (14 genes), Group II (45 genes), and Group III (11 genes). Group II was further subdivided into five subgroups: IIa (4 genes), IIb (3 genes), IIc (25 genes), IId (3 genes), and IIe (10 genes). Phylogenetic tree analysis not only clarified the evolutionary relationships among family members but also provided potential evidence for functional conservation. Notably, WRKY proteins with analogous functions typically exhibit a clustered distribution pattern. Specifically, the cold-responsive WRKY genes from rice and tomato clustered on the same branch as \u003cem\u003eAsWRKY23\u003c/em\u003e, \u003cem\u003eAsWRKY45\u003c/em\u003e, \u003cem\u003eAsWRKY46\u003c/em\u003e, and \u003cem\u003eAsWRKY48\u003c/em\u003e, all of which belong to Group IIa. Additionally, \u003cem\u003eAsWRKY1\u003c/em\u003e and \u003cem\u003eAsWRKY55\u003c/em\u003e showed a close phylogenetic relationship with \u003cem\u003eG. max GmWRKY21\u003c/em\u003e, while \u003cem\u003eAsWRKY56\u003c/em\u003e and \u003cem\u003eAsWRKY58\u003c/em\u003e clustered with \u003cem\u003eA. thaliana AtWRKY41\u003c/em\u003e on a separate branch.\u003c/p\u003e\u003cp\u003eTo further investigate the evolutionary relationships among the structural domains of AsWRKY proteins across the seven subgroups, multiple sequence alignment of the highly conserved 60-residue WRKY domains was performed to assess structural conservation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The WRKYGQK heptapeptide was highly conserved across most AsWRKY proteins, although notable substitutions were observed in specific members. For instance, \u003cem\u003eAsWRKY1\u003c/em\u003e, \u003cem\u003eAsWRKY10\u003c/em\u003e, and \u003cem\u003eAsWRKY37\u003c/em\u003e contained a WRKYGKK substitution, while \u003cem\u003eAsWRKY40\u003c/em\u003e exhibited an Arg6-to-Asn (R6N) substitution, resulting in a modified WNKYGQK sequence within its DNA-binding domain. Additionally, three Group IIc members (\u003cem\u003eAsWRKY17\u003c/em\u003e, \u003cem\u003eAsWRKY55\u003c/em\u003e, and \u003cem\u003eAsWRKY63\u003c/em\u003e) possessed truncated WRKY domains, whereas three Group I genes (\u003cem\u003eAsWRKY50\u003c/em\u003e, \u003cem\u003eAsWRKY65\u003c/em\u003e, and \u003cem\u003eAsWRKY70\u003c/em\u003e) displayed a single WRKY domain architecture. Furthermore, 8 AsWRKYs (e.g., \u003cem\u003eAsWRKY18\u003c/em\u003e, \u003cem\u003eAsWRKY59\u003c/em\u003e, \u003cem\u003eAsWRKY60\u003c/em\u003e) contained non-canonical zinc-finger motifs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eConserved Motif and Gene Structure Analysis of the AsWRKY Gene Family\u003c/h2\u003e\u003cp\u003eTo further explore the conservation and divergence of protein motif composition, the online tool MEME was used to analyze the conserved motifs of \u003cem\u003eAsWRKYs\u003c/em\u003e. Ten distinct motifs were identified, designated as Motif 1 to Motif 10, each consisting of 20 amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Across all \u003cem\u003eAsWRKYs\u003c/em\u003e, the number of motifs per protein ranges from 2 to 8, with Motif 1 and Motif 3 collectively constituting the conserved domains that harbor the WRKYGQK sequence and zinc-finger structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Motif 1, Motif 2, and Motif 3 are highly conserved, present in 79% of all AsWRKY proteins. As noted earlier, these motifs form the structural foundation of the WRKY domain, underscoring their importance for core family functionality. Motif 5, Motif 6, and Motif 9 are uniquely present in Group I AsWRKY proteins. Notably, \u003cem\u003eAsWRKY50\u003c/em\u003e, \u003cem\u003eAsWRKY55\u003c/em\u003e, \u003cem\u003eAsWRKY63\u003c/em\u003e, \u003cem\u003eAsWRKY65\u003c/em\u003e, \u003cem\u003eAsWRKY66\u003c/em\u003e, and \u003cem\u003eAsWRKY70\u003c/em\u003e all lack Motif 1, which typically contains the \"WRKYGQK\" signature sequence. This absence may indicate functional divergence or alternative modes of action for these members, necessitating further investigation into their DNA-binding capacity or regulatory roles.\u003c/p\u003e\u003cp\u003eTo further explore the structural diversity and evolutionary conservation of AsWRKY proteins, an analysis of their exon-intron organizations was conducted (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The 70 \u003cem\u003eAsWRKYs\u003c/em\u003e exhibited significant structural heterogeneity, with the number of exons ranging from 1 to 10 and introns from 0 to 9. Notably, genes clustered within the same phylogenetic group tended to share consistent numbers of coding exons, reflecting evolutionary conservation. Among these, \u003cem\u003eAsWRKY65\u003c/em\u003e displayed the most complex structure, containing 10 exons and 9 introns, while \u003cem\u003eAsWRKY50\u003c/em\u003e and \u003cem\u003eAsWRKY70\u003c/em\u003e were intronless.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCis-Acting Element Analysis\u003c/h2\u003e\u003cp\u003eAnalysis of the 1500 bp promoter regions upstream of AsWRKY genes identified a diverse array of cis-acting elements, which were functionally categorized into hormone-responsive, stress-responsive, light-responsive, and growth/development-related elements (Fig. S3). Hormone-associated elements included ABRE (abscisic acid response element), AuxRR-core (auxin signaling element), CGTCA-motif and TGACG-motif (methyl jasmonate/MeJA responsiveness elements), GARE-motif and TATC-box (gibberellin regulatory elements), TCA-element (salicylic acid response element), and TGA-element (auxin signaling element). Stress-related elements encompassed MBS (drought response element), LTR (low-temperature response element), GC-motif (anoxia tolerance element), WUN-motif (wound signaling element), and ARE (antioxidant defense element). Light-responsive elements comprised G-Box, ACE, Sp1, and GT1-motif. Notably, 20 \u003cem\u003eAsWRKYs\u003c/em\u003e contained LTR cis-elements.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eGene Duplication Events and Synteny Analysis\u003c/h2\u003e\u003cp\u003eA total of 68 \u003cem\u003eAsWRKYs\u003c/em\u003e are unevenly distributed across 17 chromosomes of \u003cem\u003eA. sesquipedale\u003c/em\u003e, while the remaining two genes (\u003cem\u003eAsWRKY69\u003c/em\u003e, \u003cem\u003eAsWRKY70\u003c/em\u003e) are located on unanchored scaffolds (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Chromosome 1 contained the highest number of \u003cem\u003eAsWRKYs\u003c/em\u003e (9 members), making it a hotspot for family localization. In contrast, Chromosomes 8 and 15 each harbored only one AsWRKY gene, representing the lowest gene density (excluding unlocalized scaffolds). Notably, no AsWRKY genes were detected on chromosomes 10 and 19. Gene duplication is a major driver of gene family expansion; analysis of AsWRKY genes identified 21 homologous gene pairs among the 70 members. \u003cem\u003eAsWRKY26\u003c/em\u003e and \u003cem\u003eAsWRKY31\u003c/em\u003e, located on chromosome 5, form a pair of tandemly duplicated genes, while the remaining 20 homologous gene pairs are derived from segmental duplication.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo elucidate evolutionary mechanisms, syntenic relationships were analyzed between \u003cem\u003eA. sesquipedale\u003c/em\u003e and three reference species: \u003cem\u003eA. thaliana\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), \u003cem\u003eP. aphrodite\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), and \u003cem\u003eO. sativa\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Comparative synteny mapping identified 14 collinear gene pairs with \u003cem\u003eA. thaliana\u003c/em\u003e, 40 with \u003cem\u003eP. aphrodite\u003c/em\u003e, and 48 with \u003cem\u003eO. sativa\u003c/em\u003e. Compared to the dicotyledonous plant \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eA. sesquipedale\u003c/em\u003e exhibits higher genomic homology with \u003cem\u003eP. aphrodite\u003c/em\u003e (a fellow Orchidaceae member) and \u003cem\u003eO. sativa\u003c/em\u003e (another monocotyledonous species).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eExpression patterns of AsWRKY gene family in response to cold stress\u003c/h2\u003e\u003cp\u003eLow temperature is a critical environmental stress factor constraining plant growth, development, and geographical distribution. To investigate the potential roles of \u003cem\u003eAsWRKYs\u003c/em\u003e in the cold stress response of \u003cem\u003eA. sesquipedale\u003c/em\u003e, we analyzed the expression patterns of 70 \u003cem\u003eAsWRKYs\u003c/em\u003e in leaves and roots based on seedling transcriptome data (Fig. S3) and examined their expression profiles under 4\u0026deg;C cold stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The results showed that 42.8% (30/70) of \u003cem\u003eAsWRKYs\u003c/em\u003e were weakly expressed or not expressed in both tissues. Meanwhile, seven genes (\u003cem\u003eAsWRKY2\u003c/em\u003e, \u003cem\u003eAsWRKY6\u003c/em\u003e, \u003cem\u003eAsWRKY21\u003c/em\u003e, \u003cem\u003eAsWRKY26\u003c/em\u003e, \u003cem\u003eAsWRKY30\u003c/em\u003e, \u003cem\u003eAsWRKY33\u003c/em\u003e, and \u003cem\u003eAsWRKY40\u003c/em\u003e) exhibited root-preferential expression, whereas \u003cem\u003eAsWRKY1\u003c/em\u003e, \u003cem\u003eAsWRKY7\u003c/em\u003e, \u003cem\u003eAsWRKY10\u003c/em\u003e, and \u003cem\u003eAsWRKY66\u003c/em\u003e displayed leaf-preferential expression. Additionally, \u003cem\u003eAsWRKY44\u003c/em\u003e and \u003cem\u003eAsWRKY49\u003c/em\u003e showed high expression levels in both tissues (log₂(TPM\u0026thinsp;+\u0026thinsp;1)\u0026thinsp;\u0026gt;\u0026thinsp;1.5).\u003c/p\u003e\u003cp\u003eUnder low-temperature stress, most \u003cem\u003eAsWRKYs\u003c/em\u003e responded in both roots and leaves. Among these, 45% (32/70) of \u003cem\u003eAsWRKYs\u003c/em\u003e were downregulated to varying degrees in both tissues as the duration of low-temperature treatment increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Notably, in leaves, the expression levels of 19 \u003cem\u003eAsWRKYs\u003c/em\u003e (\u003cem\u003eAsWRKY4\u003c/em\u003e, \u003cem\u003eAsWRKY9\u003c/em\u003e, \u003cem\u003eAsWRKY16\u003c/em\u003e, \u003cem\u003eAsWRKY17\u003c/em\u003e, \u003cem\u003eAsWRKY21\u003c/em\u003e, \u003cem\u003eAsWRKY25\u003c/em\u003e, \u003cem\u003eAsWRKY23\u003c/em\u003e, \u003cem\u003eAsWRKY30\u003c/em\u003e, \u003cem\u003eAsWRKY32\u003c/em\u003e, \u003cem\u003eAsWRKY36\u003c/em\u003e, \u003cem\u003eAsWRKY37\u003c/em\u003e, \u003cem\u003eAsWRKY41\u003c/em\u003e, \u003cem\u003eAsWRKY44\u003c/em\u003e, \u003cem\u003eAsWRKY49\u003c/em\u003e, \u003cem\u003eAsWRKY56\u003c/em\u003e, \u003cem\u003eAsWRKY58\u003c/em\u003e, \u003cem\u003eAsWRKY59\u003c/em\u003e, \u003cem\u003eAsWRKY61\u003c/em\u003e, and \u003cem\u003eAsWRKY67\u003c/em\u003e) increased rapidly over time or showed a sustained increase to varying degrees, with peak expression observed at 6 h or 12 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Similarly, in roots, 11 \u003cem\u003eAsWRKYs\u003c/em\u003e (\u003cem\u003eAsWRKY7\u003c/em\u003e, \u003cem\u003eAsWRKY13\u003c/em\u003e, \u003cem\u003eAsWRKY21\u003c/em\u003e, \u003cem\u003eAsWRKY30\u003c/em\u003e, \u003cem\u003eAsWRKY31\u003c/em\u003e, \u003cem\u003eAsWRKY32\u003c/em\u003e, \u003cem\u003eAsWRKY43\u003c/em\u003e, \u003cem\u003eAsWRKY44\u003c/em\u003e, \u003cem\u003eAsWRKY49\u003c/em\u003e, \u003cem\u003eAsWRKY52\u003c/em\u003e, and \u003cem\u003eAsWRKY61\u003c/em\u003e) exhibited similar dynamic expression patterns under cold stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eUnder natural conditions, plant growth, development, and reproduction are frequently challenged by both biotic and abiotic stresses. To cope with these adverse factors, plants have evolved elaborate regulatory mechanisms, with the WRKY gene family serving as a particularly representative example \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Its members are extensively involved in diverse physiological processes, including the regulation of plant growth and development, secondary metabolite synthesis, and stress signal transduction \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Although the functions of the WRKY family in model angiosperms have been relatively well characterized, systematic research on their molecular mechanisms in orchids remains scarce, especially in \u003cem\u003eA. sesquipedale\u003c/em\u003e, a species with unique evolutionary significance.\u003c/p\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eStructural conservation and divergence of AsWRKY genes\u003c/h2\u003e\u003cp\u003eGenomic screening identified 70 putative AsWRKY proteins, with physicochemical characterization revealing substantial heterogeneity in encoded protein attributes, including molecular mass (8.48\u0026ndash;72.97 kDa) and isoelectric point (pI 4.76\u0026ndash;10.17) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Such biophysical divergence suggests adaptive specialization for context-dependent stress responses. Subcellular localization predictions indicated nuclear enrichment for the majority of AsWRKY proteins, congruent with their canonical roles as transcriptional regulators and consistent with orthologous studies in other vascular plants \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eThe evolutionary relationship of AsWRKY genes\u003c/h2\u003e\u003cp\u003eTo explore the evolutionary relationships of the AsWRKY gene family, a phylogenetic tree was constructed alongside the \u003cem\u003eA. thaliana\u003c/em\u003e WRKY gene family, classifying the 70 \u003cem\u003eAsWRKYs\u003c/em\u003e into three major groups (I, II, III), with Group II further subdivided into five subgroups (IIa\u0026ndash;IIe) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This classification is consistent with observations of the WRKY gene family in other plant species \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Members within the same group exhibit high similarity in conserved domains and gene structures, reflecting the relative evolutionary stability of WRKY family genes in \u003cem\u003eA. sesquipedale\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eNotably, several \u003cem\u003eAsWRKYs\u003c/em\u003e cluster closely with cold stress-associated WRKY genes from four other plant species, suggesting they may participate in cold stress responses through analogous mechanisms. Additionally, several Group I \u003cem\u003eAsWRKYs\u003c/em\u003e (e.g., \u003cem\u003eAsWRKY50\u003c/em\u003e, \u003cem\u003eAsWRKY65\u003c/em\u003e, \u003cem\u003eAsWRKY70\u003c/em\u003e) possess only one WRKY domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), a phenomenon also reported in cassava \u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e and maize \u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe study further revealed that the highly conserved WRKYGQK motif in specific \u003cem\u003eAsWRKYs\u003c/em\u003e (e.g., \u003cem\u003eAsWRKY1\u003c/em\u003e, \u003cem\u003eAsWRKY10\u003c/em\u003e, \u003cem\u003eAsWRKY37\u003c/em\u003e) has mutated to WRKYGKK, mirroring WRKY domain mutations in soybean \u003cem\u003eGmWRKY6\u003c/em\u003e and \u003cem\u003eGmWRKY21\u003c/em\u003e. Such mutations impair normal binding to the W-box element (TTGACC/T), significantly reducing or even eliminating the ability to interact with target gene promoters \u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Interestingly, mutations in the WRKY domain and zinc finger structures are primarily concentrated in subgroup IIc, consistent with previous reports of greater sequence diversity in plant subgroup IIc \u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. This pattern suggests a potential association with functional divergence during plant adaptation to diverse ecological niches.\u003c/p\u003e\u003cp\u003ePromoter cis-acting elements provide insights into the potential regulatory mechanisms and biological functions of genes. Analysis of \u003cem\u003eAsWRKYs\u003c/em\u003e identified numerous hormone-responsive motifs (e.g., ABRE, TGA elements) and stress-responsive motifs (e.g., MBS, LTR) (Fig. S3). These findings suggest that \u003cem\u003eAsWRKYs\u003c/em\u003e may be involved in mediating plant hormone crosstalk and environmental stress adaptation processes. Regarding gene family expansion mechanisms, both tandem duplication and segmental duplication contribute to the expansion of the WRKY gene family. In \u003cem\u003eA. sesquipedale\u003c/em\u003e, segmental duplication events (20 pairs) were far more frequent than tandem duplication events (1 pair) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e), consistent with macroevolutionary patterns reported for WRKY families in other plant species \u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. This indicates that segmental duplication is the primary driver of WRKY gene family expansion during the evolution of \u003cem\u003eA. sesquipedale\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eInterspecific synteny reflects evolutionary relationships between species. The phylogenetic relationship between \u003cem\u003eA. sesquipedale\u003c/em\u003e and \u003cem\u003eP. aphrodite\u003c/em\u003e, as well as with \u003cem\u003eO. sativa\u003c/em\u003e, is significantly closer than that with \u003cem\u003eA. thaliana\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003e), resulting in greater genomic structural conservation and more abundant collinear gene pairs among the former three. Notably, previous studies have shown that the \u003cem\u003eCymbidium goeringii CgWRKY57\u003c/em\u003e gene shares high homology with rice \u003cem\u003eOsWRKY47\u003c/em\u003e, and its expression is induced by low-temperature and ABA stress \u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. This observation further supports the synteny analysis inference: the collinear associations of the WRKY gene family among \u003cem\u003eA. sesquipedale\u003c/em\u003e, \u003cem\u003eP. aphrodite\u003c/em\u003e, and rice imply they may have undergone analogous segmental duplication events, thereby exhibiting functional conservation in key biological processes such as low-temperature stress responses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eAsWRKY genes function in cold stresses\u003c/h2\u003e\u003cp\u003eThe potential roles of \u003cem\u003eAsWRKYs\u003c/em\u003e can be inferred by integrating their expression patterns across different tissues of \u003cem\u003eA. sesquipedale\u003c/em\u003e with the known functions of WRKY genes in model plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In root development regulation, \u003cem\u003eAtWRKY6\u003c/em\u003e, \u003cem\u003eAtWRKY11\u003c/em\u003e, and \u003cem\u003eAtWRKY23\u003c/em\u003e modulate root development through protein-protein interactions in \u003cem\u003eA. thaliana\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. Notably, these genes cluster with \u003cem\u003eAsWRKY21\u003c/em\u003e in the phylogenetic tree and share root-preferential expression patterns (Fig. S4). These observations suggest that root-preferentially expressed \u003cem\u003eAsWRKYs\u003c/em\u003e may act as key regulators of root development, participating in root responses to diverse stresses and potentially promoting nutrient uptake by regulating transporter gene expression \u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn terms of leaf function, WRKY TFs in plant leaves regulate photosynthesis-related genes; for example, tomato (\u003cem\u003eS. lycopersicum\u003c/em\u003e) \u003cem\u003eSlWRKY17\u003c/em\u003e modulates photosynthetic pigment accumulation by regulating genes encoding key enzymes in chlorophyll biosynthesis \u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. Additionally, leaf-specific WRKY genes have been implicated in complex defense signaling networks \u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e. Moreover, referencing the mechanism by which WRKY genes influence leaf senescence through the regulation of senescence-associated genes (SAGs) \u003csup\u003e[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/sup\u003e, these leaf-expressed WRKY genes may enhance biotic stress resistance and regulate leaf senescence by coordinating multiple hormone signaling pathways. Furthermore, \u003cem\u003eAsWRKY44\u003c/em\u003e and \u003cem\u003eAsWRKY49\u003c/em\u003e are highly expressed in both leaves and roots, a feature suggesting their potential role as critical regulators in the basal life processes of \u003cem\u003eA. sesquipedale\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eAgainst the backdrop of escalating global climate change, plants face increasing threats from extreme environmental stresses such as drought, flooding, and low temperatures. As pivotal regulators in stress responses, the WRKY gene family not only mediates cold stress responses but also plays a key role in enhancing plant cold tolerance and environmental adaptability \u003csup\u003e[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/sup\u003e. Analysis of the regulatory mechanisms of WRKY TFs under abiotic stress revealed the following patterns: transcriptomic data indicated that 45% of \u003cem\u003eAsWRKYs\u003c/em\u003e in roots and leaves exhibited high expression levels at 0 h (control), followed by sustained downregulation to varying degrees after cold treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Studies have demonstrated that when plants undergo abiotic stress, WRKY TFs can induce growth arrest through downregulated expression, thereby reducing energy expenditure to sustain fundamental physiological processes \u003csup\u003e[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eImportantly, a subset of \u003cem\u003eAsWRKYs\u003c/em\u003e in roots and leaves displayed distinct temporal expression dynamics: 4 genes in leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) and 7 genes in roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) exhibited rapid response characteristics, being rapidly induced upon cold treatment and maintaining expression until 12 h. This suggests their potential involvement in the early cold stress response by regulating downstream target gene expression \u003csup\u003e[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]\u003c/sup\u003e. Conversely, 15 genes in leaves and 4 genes in roots showed a distinct trend, with their expression levels accumulating continuously as cold treatment prolonged, peaking at 12 h, thus exhibiting characteristics of long-term response genes. This is analogous to the regulatory mechanism of \u003cem\u003eVaWRKY65\u003c/em\u003e in \u003cem\u003eVitis amurensis\u003c/em\u003e, which enhances plant cold tolerance by binding to the W-box in the promoter of \u003cem\u003eVaBAM3\u003c/em\u003e, sustainably upregulating its expression to promote soluble sugar accumulation \u003csup\u003e[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTo investigate the potential roles of \u003cem\u003eAsWRKYs\u003c/em\u003e in cold stress regulation, the present study integrated phylogenetic analysis of \u003cem\u003eAsWRKYs\u003c/em\u003e with cold stress-associated WRKY genes from four model plant species (\u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eS. lycopersicum\u003c/em\u003e, \u003cem\u003eO. sativa\u003c/em\u003e, and \u003cem\u003eG. max\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Combined with differential expression profiles of \u003cem\u003eAsWRKYs\u003c/em\u003e under low-temperature stress, this approach aimed to dissect their underlying regulatory mechanisms. The cold stress-responsive functions of WRKY genes in these model systems have been extensively characterized, providing a robust framework for inferring the functional roles of \u003cem\u003eAsWRKYs\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThe WRKY family exhibits evolutionary conservation in cold stress adaptation, as exemplified by soybean \u003cem\u003eGmWRKY21\u003c/em\u003e, which enhances cold stress tolerance when heterologously expressed in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Notably, \u003cem\u003eAtWRKY33\u003c/em\u003e is rapidly transcriptionally induced upon cold stress initiation, thereby initiating early stress-responsive signaling cascades in \u003cem\u003eA. thaliana\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]\u003c/sup\u003e. In tomato, \u003cem\u003eSlWRKY33\u003c/em\u003e enhances cold hardiness by directly targeting and activating genes encoding kinases, transcription factors, and molecular chaperones, such as CDPK11, MYBS3, and BAG6 \u003csup\u003e[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]\u003c/sup\u003e. Phylogenetic clustering analysis reveals that \u003cem\u003eAsWRKY17\u003c/em\u003e and \u003cem\u003eAsWRKY30\u003c/em\u003e closely cluster with \u003cem\u003eAtWRKY33\u003c/em\u003e and \u003cem\u003eSlWRKY33\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003e), and exhibit sustained upregulation under cold stress, consistent with the prolonged regulatory dynamics mediated by \u003cem\u003eSlWRKY33\u003c/em\u003e. This suggests they may participate in cold stress responses through analogous target gene regulatory networks.\u003c/p\u003e\u003cp\u003eIn contrast, in \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eAtWRKY41\u003c/em\u003e functions as a negative regulator by directly binding to W-box motifs in the promoters of CBF genes, repressing their expression and thereby attenuating plant freezing tolerance \u003csup\u003e[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eAsWRKY56\u003c/em\u003e and \u003cem\u003eAsWRKY58\u003c/em\u003e are phylogenetically closely related to \u003cem\u003eAtWRKY41\u003c/em\u003e and exhibit transient upregulation under cold stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). This immediate response profile aligns with their putative negative regulatory roles, suggesting they may modulate freezing tolerance by suppressing the expression of frost resistance-associated genes.\u003c/p\u003e\u003cp\u003eFurthermore, other WRKY genes in tomato and rice regulate cold tolerance through distinct pathways: tomato \u003cem\u003eSlWRKY45\u003c/em\u003e and \u003cem\u003eSlWRKY46\u003c/em\u003e enhance reactive oxygen species (ROS) scavenging capacity by regulating antioxidant enzyme pathways, thereby improving cold tolerance in transgenic lines \u003csup\u003e[\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]\u003c/sup\u003e; \u003cem\u003eSlWRKY50\u003c/em\u003e indirectly enhances cold hardiness through hormone signaling networks by regulating jasmonic acid (JA) biosynthesis \u003csup\u003e[\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]\u003c/sup\u003e. In rice, \u003cem\u003eOsWRKY71\u003c/em\u003e positively regulates cold tolerance via hierarchical control of downstream target gene networks \u003csup\u003e[\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]\u003c/sup\u003e; \u003cem\u003eOsWRKY76\u003c/em\u003e enhances cold tolerance through protein-protein interactions with \u003cem\u003eOsbHLH148\u003c/em\u003e, which transactivates \u003cem\u003eOsDREB1B\u003c/em\u003e expression \u003csup\u003e[\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eAsWRKY23\u003c/em\u003e clusters phylogenetically with these rice and tomato WRKYs\u0026mdash;particularly with \u003cem\u003eOsWRKY71\u003c/em\u003e\u0026mdash;and maintains sustained expression under cold stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). This indicates it may synergistically enhance plant cold tolerance through analogous mechanisms, such as modulating target gene networks or mediating protein interaction-based regulation.\u003c/p\u003e\u003cp\u003eThis study established functional associations between \u003cem\u003eA. sesquipedale AsWRKY17\u003c/em\u003e, \u003cem\u003eAsWRKY23\u003c/em\u003e, \u003cem\u003eAsWRKY30\u003c/em\u003e, \u003cem\u003eAsWRKY56\u003c/em\u003e, and \u003cem\u003eAsWRKY58\u003c/em\u003e and cold-responsive WRKY genes from model plants. It revealed that the WRKY family in this species may participate in cold stress responses via a multi-pathway regulatory network encompassing \"positive regulation-negative regulation-coordinated regulation\". These findings provide a foundation for subsequent genetic validation of cold tolerance mechanisms in \u003cem\u003eA. sesquipedale\u003c/em\u003e. Additionally, this research clarified the evolutionary characteristics underlying functional divergence in cold responsiveness between \u003cem\u003eAsWRKYs\u003c/em\u003e and WRKY genes from diverse plant lineages. The key genes identified herein can be directly applied to ex situ conservation, expansion of cultivation ranges, and breeding of stress-resistant \u003cem\u003eA. sesquipedale\u003c/em\u003e varieties, thereby addressing the core issue of low-temperature constraints on its growth and development.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, 70 AsWRKY genes were identified in \u003cem\u003eA. sesquipedale\u003c/em\u003e, and a comprehensive analysis was conducted on these genes. First, through multiple sequence alignment combined with analyses of evolutionary relationships, conserved motifs, gene structures, cis-acting elements, gene duplication events, and collinearity, the characteristics of this gene family were systematically elucidated. Expression pattern analyses of \u003cem\u003eAsWRKYs\u003c/em\u003e in two distinct tissues and under cold stress indicated that these genes may play important roles in the growth, development, and cold stress response of \u003cem\u003eA. sesquipedale\u003c/em\u003e. Furthermore, based on integrated phylogenetic tree and expression profile analyses, five key genes potentially involved in regulating \u003cem\u003eA. sesquipedale\u003c/em\u003e under cold stress were identified. In conclusion, this study establishes a foundation for functional characterization of the WRKY gene family in A. \u003cem\u003esesquipedale\u003c/em\u003e and provides an important reference for subsequent in-depth investigations into their molecular mechanisms underlying growth, development, and cold stress response.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. Plant material sampling adhered to institutional guidelines, and as no human/animal subjects or ethically regulated procedures were involved, formal ethics committee review was not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw transcriptomic data have been deposited in the NCBI database (PRJNA1307821).\u0026nbsp;All datasets analyzed during the current study are included in this published article and its supplementary information files.\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\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grant from the Shanghai Landscaping and City Appearance Administrative Bureau (G242418).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCG and CH executed the experimental procedures, conducted data analysis, prepared the figures and tables, and drafted the initial manuscript. MFW and SSP contributed to experimental implementation and data interpretation.\u0026nbsp;ZYN\u0026nbsp;provided the experimental materials. MHZ and QNR were responsible for the compilation of selected figures and tables. YQS and RFP participated in drafting specific sections of the manuscript. TP and WCH conceptualized and designed the experiments, and provided critical input during the revision and review process. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely appreciate Li Shao from the Horticulture Department of Shanghai Chenshan Botanical Garden for their substantial contributions in providing essential experimental materials and critical technical support during the course of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRushton PJ, Somssich IE, Ringler P, Shen QJ. 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BMC Plant Biol. 2021;21(1):458.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNiu F, Cui X, Zhao P, Sun M, Yang B, Deyholos MK, Li Y, Zhao X, Jiang YQ. \u003cem\u003eWRKY42\u003c/em\u003e transcription factor positively regulates leaf senescence through modulating SA and ROS synthesis in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Plant J. 2020;104(1):171\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu Y, Qi Y, Xu J, Dai X, Chen J, Dong CH, Xiang F. \u003cem\u003eArabidopsis WRKY71\u003c/em\u003e regulates ethylene-mediated leaf senescence by directly activating \u003cem\u003eEIN2\u003c/em\u003e, \u003cem\u003eORE1\u003c/em\u003e and \u003cem\u003eACS2\u003c/em\u003e genes. Plant J. 2021;107(6):1819\u0026ndash;1836.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen W, Zheng Y, Wang J, Wang Z, Yang Z, Chi X, Dai L, Lu G, Yang Y, Sun B. Ethylene-responsive \u003cem\u003eSbWRKY50\u003c/em\u003e suppresses leaf senescence by inhibition of chlorophyll degradation in sorghum. New Phytol. 2023;238(3):1129\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFei J, Wang YS, Cheng H, Su YB, Zhong YJ, Zheng L. The \u003cem\u003eKandelia obovata\u003c/em\u003e transcription factor \u003cem\u003eKoWRKY40\u003c/em\u003e enhances cold tolerance in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e. BMC Plant Biol. 2022;22(1):274.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStock J, Br\u0026auml;utigam A, Melzer M, Bienert GP, Bunk B, Nagel M, Overmann J, Keller ERJ, Mock HP. The transcription factor \u003cem\u003eWRKY22\u003c/em\u003e is required during cryo-stress acclimation in \u003cem\u003eArabidopsis\u003c/em\u003e shoot tips. J Exp Bot. 2020;71(16):4993\u0026ndash;5009.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDu P, Wang Q, Yuan DY, Chen SS, Su YN, Li L, Chen S, He XJ. WRKY transcription factors and OBERON histone-binding proteins form complexes to balance plant growth and stress tolerance. EMBO J. 2023;42(19):e113639.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen S, Xu L, Wang Y, Mao B, Zhang X, Song Q, Cui F, Ma Y, Dong J, Wang K, Bi H, Liu L. \u003cem\u003eRsWRKY40\u003c/em\u003e coordinates the cold stress response by integrating \u003cem\u003eRsSPS1\u003c/em\u003e-mediated sucrose accumulation and the CBF-dependent pathway in radish (\u003cem\u003eRaphanus sativus L\u003c/em\u003e). Mol Hortic. 2025;5(1):14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeng L, Zhou H, Tan L, Li Q, Hou Y, Li W, Kafle S, Liang J, Aryal R, Liang Z, Xin H. \u003cem\u003eVaWRKY65\u003c/em\u003e contributes to cold tolerance through dual regulation of soluble sugar accumulation and reactive oxygen species scavenging in \u003cem\u003eVitis amurensis\u003c/em\u003e. Hortic Res. 2025;12(4):uhae367.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFu QT, Yu DQ. Expression profiles of \u003cem\u003eAtWRKY25\u003c/em\u003e, \u003cem\u003eAtWRKY26\u003c/em\u003e and \u003cem\u003eAtWRKY33\u003c/em\u003e under abiotic stresses. Yi Chuan. 2010;32(8):848\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuo M, Yang F, Liu C, Zou J, Qi Z, Fotopoulos V, Lu G, Yu J, Zhou J. A single-nucleotide polymorphism in \u003cem\u003eWRKY33\u003c/em\u003e promoter is associated with the cold sensitivity in cultivated tomato. New Phytol. 2022;236(3):989\u0026ndash;1005.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang X, Li Z, Shi Y, Liu Z, Zhang X, Gong Z, Yang S. Strigolactones promote plant freezing tolerance by releasing the \u003cem\u003eWRKY41\u003c/em\u003e mediated inhibition of CBF/DREB1 expression. EMBO J. 2023;42(19):e112999.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFan B, Ren M, Wang YF, Dang FF, Chen GL, Cheng GT, Yang JY, Sun HR. Functions of \u003cem\u003eSlWRKY45\u003c/em\u003e in re sponse to low-temperature and drought stress in tomato. Chin Bull Bot 2025,60(02):186\u0026ndash;203.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun HR, Dang FF, Ren M, Zhang JN, Fan B, Chen GL, Cheng GT, Wang YF. Function analysis of \u003cem\u003eSlWRKY46\u003c/em\u003e in regulating tomato response to low temperature stress. Acta Horticulturae Sinica. 2024;51(12):2758\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang L, Chen H, Chen G, Luo G, Shen X, Ouyang B, Bie Z. Transcription factor \u003cem\u003eSlWRKY50\u003c/em\u003e enhances cold tolerance in tomato by activating the jasmonic acid signaling. Plant Physiol. 2024;194(2):1075\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu X, Bai X, Wang X, Chu C. \u003cem\u003eOsWRKY71\u003c/em\u003e, a rice transcription factor, is involved in rice defense response. J Plant Physiol. 2007;164(8):969\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang M, Zhao R, Huang K, Huang S, Wang H, Wei Z, Li Z, Bian M, Jiang W, Wu T, Du X. The \u003cem\u003eOsWRKY63\u003c/em\u003e-\u003cem\u003eOsWRKY76\u003c/em\u003e-\u003cem\u003eOsDREB1B\u003c/em\u003e module regulates chilling tolerance in rice. Plant J. 2022;112(2):383\u0026ndash;98.\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":"Angraecum sesquipedale, WRKY gene family, transcription factor, cold tolerance","lastPublishedDoi":"10.21203/rs.3.rs-7505680/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7505680/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003e\u003cem\u003eAngraecum sesquipedale\u003c/em\u003e, commonly known as Darwin\u0026rsquo;s orchid, is a distinctive ornamental species uniquely susceptible to biotic and abiotic stresses, primarily owing to its confinement to specialized habitats. WRKY transcription factors play pivotal roles in plant stress responses, growth and development regulation, and secondary metabolism. However, research on this gene family in \u003cem\u003eA. sesquipedale\u003c/em\u003e remains unreported.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIn this study, 70 WRKY genes (\u003cem\u003eAsWRKYs\u003c/em\u003e) were identified in \u003cem\u003eA. sesquipedale\u003c/em\u003e through genome-wide analysis. Phylogenetic analysis, integrating WRKY genes from four model plants (\u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eSolanum lycopersicum\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, and \u003cem\u003eGlycine max\u003c/em\u003e), classified these 70 \u003cem\u003eAsWRKYs\u003c/em\u003e into three major groups, with Group Ⅱ further subdivided into five subgroups. Genes within the same group exhibited high conservation in gene structure and motif composition. Tissue expression profiling revealed that several \u003cem\u003eAsWRKYs\u003c/em\u003e (\u003cem\u003eAsWRKY21\u003c/em\u003e and \u003cem\u003eAsWRKY49\u003c/em\u003e) exhibit root-preferential expression, suggesting their potential involvement in regulating root growth and development in \u003cem\u003eA. sesquipedale\u003c/em\u003e. The expression patterns under cold stress showed that 7 genes in roots and 4 genes in leaves exhibited early rapid responses, while 15 genes in leaves and 4 genes in roots exhibited long-term sustained response features. Integrating expression patterns with phylogenetic relationships, key candidate genes potentially implicated in cold stress regulation, including \u003cem\u003eAsWRKY17\u003c/em\u003e, \u003cem\u003eAsWRKY23\u003c/em\u003e, \u003cem\u003eAsWRKY30\u003c/em\u003e, \u003cem\u003eAsWRKY56\u003c/em\u003e, and \u003cem\u003eAsWRKY58\u003c/em\u003e, were identified.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study identified 70 WRKY genes in \u003cem\u003eA. sesquipedale\u003c/em\u003e, elucidated their classification, evolutionary characteristics, and expression patterns, and unveiled the potential mechanisms of \u003cem\u003eAsWRKYs\u003c/em\u003e in cold stress responses. The findings establish a foundation for understanding the evolutionary adaptability of \u003cem\u003eAsWRKYs\u003c/em\u003e and the cold regulatory network in \u003cem\u003eA. sesquipedale\u003c/em\u003e, and lay a foundation for molecular breeding and genetic improvement of stress resistance in orchids.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification of the WRKY gene family in Angraecum sesquipedale and exploration of its role in cold stress response","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-16 16:56:02","doi":"10.21203/rs.3.rs-7505680/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":"d148c535-269a-4096-908a-f5b05562d3cb","owner":[],"postedDate":"September 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-01T07:24:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-16 16:56:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7505680","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7505680","identity":"rs-7505680","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}