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. 2026 Mar 1;16(3):e73178. doi: 10.1002/ece3.73178

Evolutionary and Functional Characterization of an Auxin Methyltransferase (CsIAMT) in Cucumber Reveals Its Role in Stress Adaptation and Development

Xinjie Zhang 1, Yang Zhou 1, Mengxin Chen 1, Jingwen Li 1, Lisi Jiang 1, Ken Li 2, Lin Hao 1,✉, Wei Fu 1,✉
PMCID: PMC12950355  PMID: 41777356

ABSTRACT

The expansion and functional diversification of gene families are key drivers of phenotypic innovation in plants. The SABATH family of methyltransferases, involved in growth regulation and stress responses, provides an ideal system to study such evolutionary processes. In this study, we identified and functionally characterized a novel member of this family in cucumber, designated CsIAMT. Phylogenetic and synteny analyses confirmed that CsIAMT shares a common ancestry with conserved IAA methyltransferase (IAMT) orthologs across angiosperms. Evolutionary analysis revealed that CsIAMT has undergone strong purifying selection (dN/dS < 1), indicating high functional conservation despite deep evolutionary divergence. Functional validation in transgenic tobacco plants revealed a decrease in IAA content accompanied by a significant increase in MeIAA. Expression profiling under various stress conditions showed that CsIAMT is notably up‐regulated under biotic stresses—such as powdery mildew and nematode infection—while exhibiting variable responses to abiotic treatments. These findings collectively establish CsIAMT as a conserved IAA methyltransferase in cucumber and suggest its potential role in integrating auxin metabolism with biotic stress responses, highlighting the evolutionary conservation of core enzymatic functions within the SABATH family amid lineage‐specific regulatory diversification.

Keywords: abiotic stress, biotic stress, conservative evolution, cucumber, IAMT, SABATH

The plant SABATH methyltransferase family drives phenotypic diversity evolution through gene duplication and new functionalization. Among them, IAMT, as the most ancient member of this family, is widely present in various species. The study identified a potential auxin methyltransferase homologous to IAMT (CsIAMT) in cucumber, which can catalyze IAA methylation to regulate auxin balance. Moreover, RNA‐seq analysis indicated that this gene responded significantly more strongly to biotic stress than abiotic stress.

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1. Introduction

Climate change acts as a powerful selective force, driving ecological and evolutionary responses in plant populations (Valdés‐Florido et al. 2024). Rising global temperatures and the increasing frequency of extreme climatic events pose significant threats to agricultural systems and natural ecosystems (Nepal et al. 2024; Robinson 2021). These abiotic pressures are often compounded by shifts in biotic interactions, such as altered pest and disease dynamics, which collectively contribute to substantial crop yield losses and arable land reduction (Costa and Farrant 2019; Langridge et al. 2021). In this context, understanding the molecular mechanisms underlying plant adaptation to combined stresses is crucial for informed crop breeding strategies aimed at enhancing climate resilience (Langridge et al. 2021).

The plant hormone auxin, primarily indole‐3‐acetic acid (IAA), is a central regulator of growth and developmental processes (Zhang et al. 2022). Its spatiotemporal activity is finely tuned not only through biosynthesis, transport, and degradation but also via chemical modifications such as methylation (Zhang et al. 2009, 2022; Yang et al. 2008). The conversion of IAA to its methyl ester (MeIAA) represents a key regulatory node, influencing auxin homeostasis, stability, and signaling (Goto et al. 2022; Abbas et al. 2018; Qin et al. 2005; Li et al. 2008). This reaction is catalyzed by auxin methyltransferase (IAMT), an enzyme whose evolutionary history and functional diversification warrant detailed investigation to fully elucidate auxin regulatory networks (Koeduka et al. 2024).

IAMT is one of the ancient members of the plant SABATH gene family (Zhao et al. 2008), which is conservative in the process of evolution. The SABATH gene family is an enzyme family characterized by the methylation of various substrates (plant hormones, secondary metabolites, etc.) (Wang et al. 2025). Comparative studies suggest that functional diversification within this family often results from changes in specific active‐site residues, leading to altered substrate specificity (Huang et al. 2012). A classic example is the substitution of a tryptophan (Trp226 in CbSAMT) with a glycine in IAMT, which remodels the active site to accommodate IAA, a change conserved in IAMT orthologs from Arabidopsis to poplar (Qin et al. 2005; Zubieta et al. 2003). Currently, the IAMT gene has been identified in various plants, such as Arabidopsis thaliana (Qin et al. 2005), poplar (Zhao et al. 2007), lotus (Goto et al. 2022), spruce (Chaiprasongsuk et al. 2018), and rice (Zhao et al. 2008), etc. Although IAMT is evolutionarily conserved across multiple plant lineages, its functional contribution to ecological adaptation in cucumber ( Cucumis sativus L.)—specifically in mediating trade‐offs under combined biotic and abiotic stresses—remains largely unexplored.

In this study, we identified and characterized the cucumber IAA methyltransferase, designated CsIAMT. Phylogenetic and synteny analyses confirmed its orthology with known IAMT genes and indicated a conserved genomic context. Evolutionary analysis revealed strong purifying selection (dN/dS < 1), suggesting high functional constraint. Enzymatic function was validated through heterologous expression in tobacco, which confirmed its ability to methylate IAA to MeIAA. Expression profiling demonstrated that CsIAMT is more responsive to biotic stresses, such as powdery mildew and nematode infection, than to abiotic treatments. These results establish CsIAMT as a conserved functional IAMT in cucumber and suggest a specific role for this enzyme in auxin‐related stress responses, providing a foundation for further investigation into its contribution to plant adaptation.

1.1. Identification and Phylogenetic Analysis of CsIAMT Gene

To identify potential IAMT genes within the cucumber SABATH family, we constructed a phylogenetic tree based on the SABATH protein sequences of Arabidopsis, rice, tomato, and cucumber using the maximum likelihood method. The results showed that AtIAMT and OsIAMT clustered together within a subclade of Group A. The two tomato genes in this subclade were also consistent with previously reported identifications of tomato SABATH family members (Wei et al. 2021). Notably, the cucumber gene CsaV3_7G020980 was the only cucumber member present in this subclade (Figure 1a). In terms of sequence similarity, we performed a Blastp search using the AtIAMT protein sequence against the Cucurbitaceae genome database and obtained five homologous genes, among which CsaV3_7G020980 showed the highest similarity to AtIAMT, exceeding 70% (Table S1). Further phylogenetic analysis of these five candidate genes along with other SABATH family members from various species revealed that all IAMT proteins from different species clustered within a single independent branch, and only cucumber CsaV3_7G020980 co‐clustered with IAMT proteins from other species in this branch (Figure 1b), which was consistent with the results from Figure 1a. Conserved motif analysis indicated that the motif compositions of SABATH family members across different species were highly similar, particularly the conserved motif pattern of CsaV3_7G020980, which was almost identical to that of IAMT proteins from other species (Figure 1c). Phylogenetic relationship analysis further demonstrated that CsaV3_7G020980 was most closely related to lotus, followed by poplar. This finding aligns with previous studies indicating that IAMT from different species can form a monophyletic group (Zhao et al. 2007). Subsequently, we performed a comparative analysis of genomic synteny between cucumber and other species. The results revealed that cucumber shares conserved syntenic regions exclusively with Arabidopsis. Within these regions, the cucumber gene CsaV3_7G020980 was found to be syntenic with AtIAMT from Arabidopsis. Integrating evidence from phylogenetic clustering, conserved motif analysis, sequence similarity, and synteny relationships, we identified CsaV3_7G020980 as an evolutionarily conserved IAA methyltransferase in cucumber ( Cucumis sativus L.) and designated it as CsIAMT. Evolutionary analysis revealed that CsIAMT and its orthologs have been under intense purifying selection throughout their diversification (Table S2). Despite high levels of synonymous divergence (dS > 1.0 in most comparisons), indicative of ancient gene duplication events, the dN/dS ratios (ω) were consistently and significantly less than 1. Specifically, the ω value between cucumber CsIAMT and AtIAMT1 was 0.14, while comparisons with rice and poplar orthologs yielded values of 0.24 and 0.16, respectively. These exceptionally low ω values demonstrate strong functional constraint, preserving the critical amino acid sequence necessary for IAMT enzymatic activity over deep evolutionary time.

FIGURE 1.

FIGURE 1

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(a) Phylogenetic tree of the SABATH family constructed with cucumber, Arabidopsis, and other species. The orange squares, purple stars, green triangles, and blue circles represent the SABATH family genes from Arabidopsis thaliana , rice, tomato, and cucumber, respectively. The values in the top right corner indicate the bootstrap support values. (b) Phylogenetic analysis of candidate genes of cucumber and the identified and named members of the SABATH family. (c) Conserved motifs of IAMT for each species. (d) Alignment of cucumber and Arabidopsis thaliana . The sequence information of all genes is provided in Tables S3 and S4.

1.2. Sequence Alignment and Structural Characterization

CsaV3_7G020980 is located on chromosome 7 of cucumber and encodes a protein sequence consisting of 385 amino acids. Its length is approximately the same as that of IAMT in other species. Furthermore, like the IAMT of other species, CsaV3_7G020980 also contains three introns and four exons. To verify whether CsaV3_7G020980 is CsIAMT, we selected the protein sequences of AtIAMT, LjIAMT, PtIAMT, OsIAMT, and CbSAMT for sequence comparison. The SAM binding site in CbSAMT has been identified (Zubieta et al. 2003). As shown in the Figure 2a, the amino acids at the SAM binding site in CsaV3_7G020980 are similar to those at the SAM binding site in CbSAMT. In CbSAMT, Trp226 is one of the amino acids that form the salicylic acid binding site. In AtIAMT, LjIAMT, PtIAMT, and OsIAMT, the Trp226 residue is replaced by a Gly residue. This creates a large and spacious pocket for recognizing and binding to the indole ring of IAA (Zhao et al. 2007). In CsaV3_7G020980, the same substitution also occurred at this site. This not only provides more sufficient evidence to support the significance of this site in the binding with IAA, but also, based on its structure and the conservation with the known IAMT, the gene CsaV3_7G020980 is named as CsIAMT. Subsequently, we further predicted the three‐dimensional structure of the proteins based on the protein sequences of cucumbers and various species of IAMT. Meanwhile, the Ramachandran plot indicates that the conformation of this model is reasonable (Figure 2b). As shown in the Figure 2b, even though the species are different, the three‐dimensional structure of IAMT is mostly similar, and its mutation site scaffolds are divided into two categories. CsIAMT, AtIAMT, and PtIAMT belong to one category, while OsIAMT, LjIAMTa, and LjIAMTb belong to another category.

FIGURE 2.

FIGURE 2

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(a) The structural‐based sequence alignment of IAMT and other identified species IAMT and CbSAMT. The green box represents the indole ring. (b) 3D structure of each species IAMT proteins along with Ramachandran plots. The 3D structure and ramachandran plot of the protein predicted based on the protein sequences of cucumber and various species of IAMT.

1.3. Functional Validation and Expression Patterns of CsIAMT in Cucumber

To further verify whether this gene encodes an IAMT protein in cucumber, we treated plants with a set of potential substrates, including IAA, benzoic acid (BA), salicylic acid (SA), gibberellin (GA), and the auxin‐like compound indole‐3‐butyric acid (IBA). The results showed that CsaV3_7G020980 exhibited significantly different response patterns to various hormonal stimuli. Notably, exogenous IAA treatment induced a dramatic upregulation of CsaV3_7G020980 expression by several hundred‐fold at 6 h, far exceeding the magnitude of change caused by any other hormone treatment (Figure 3a). In contrast, changes in CsaV3_7G020980 expression in plants treated with BA, SA, and GA remained within a few‐fold range. By 12 h, except for an approximately 7‐fold upregulation under SA treatment, changes in most other treatments were not significant. Although IBA could also regulate the expression of CsaV3_7G020980 to some extent, its induction level was relatively low (Figure 3a). These results indicate that IAA possesses remarkable specificity and efficiency in activating CsaV3_7G020980, meaning this gene is specifically activated primarily when IAA serves as the substrate, while BA, SA, and GA lack similar inducing capacity. Therefore, we designated CsaV3_7G020980 as CsIAMT.

FIGURE 3.

FIGURE 3

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(a) Expression analysis of the CsIAMT gene. The gene relative expression was calculated using the 2−ΔΔCt method with CsActin as an internal control, and the value represents the mean ±SE of three biological replicates. Asterisks indicated values that are significantly different from 0h (“ns” indicates insignificant, * p < 0.05, ** p < 0.01, and *** p < 0.001**** p < 0.0001, one‐way ANOVA). (b) GC–MS analysis of the enzymatic product formed by CsIAMT proteins using different hormones as substrates. (c) The expression levels of CsIAMT were analyzed under different developmental and hormonal conditions. First, its expression in the third leaf was compared at 14 and 28 days after sowing (both at the three‐leaf stage). Second, the dynamic expression in the newest leaf versus an older leaf was measured at 0 and 6 h after hormone treatment, with data collected from three randomly selected seedlings per group. Additionally, the relative expression level of CsIAMT in young fruits was determined, normalized to its expression in root tissues. The relative expression levels were determined based on three independent biological replicates, with values presented as the mean ± standard deviation (SD). Statistical significance was assessed using one‐way ANOVA: **** p < 0.0001.

To further elucidate the relationship between CsIAMT expression changes and hormone metabolism, we quantified endogenous IAA and its methyl ester (MeIAA) levels in plants 12 h after hormone treatment using liquid chromatography. In the IAA treatment group, not only was the exogenously applied IAA rapidly converted and absorbed, but the contents of endogenous IAA and MeIAA also reached the highest levels among all treatments (Figure 3b). The significant accumulation of IAA and MeIAA induced by CsIAMT suggests that the introduction of exogenous IAA not only directly provides substrate but may also activate CsIAMT expression, thereby driving the methylation conversion of endogenous IAA to MeIAA. In contrast, under other hormone treatments, although CsIAMT expression levels fluctuated, no significant changes in IAA/MeIAA metabolite concentrations comparable to those observed under IAA treatment were detected (Figure 3b).

In summary, this study confirms that IAA is the most effective and specific transcriptional inducer of the CsIAMT gene, and its upregulated expression is closely associated with a significant increase in endogenous IAA and MeIAA levels. This specific regulatory pattern indicates that the protein encoded by CsIAMT primarily functions as an IAA methyltransferase in cucumber, directly involved in maintaining the metabolic homeostasis of auxin, particularly its active form IAA and its methylated form MeIAA. The relatively weaker regulatory effects of other hormones on CsIAMT expression may reflect complex cross‐interactions within plant hormone signaling networks rather than these hormones acting directly as donors in the methylation reaction.

Furthermore, this study analyzed the expression patterns of CsIAMT during leaf dynamic development and in young organs. qRT‐PCR detection of leaves at different time points during the three‐leaf stage, as well as young and mature leaves from plants at the same stage, showed that the expression abundance of CsIAMT was significantly higher when the third leaf had just emerged (Day 14) compared to when it was fully mature (Day 28), with a difference of several dozen‐fold (Figure 3c). Simultaneously, sampling analysis of plants at the single‐leaf and three‐leaf stages at the same time points (0 and 6 h after watering) indicated that, regardless of treatment, CsIAMT expression in young leaves was significantly higher than in mature leaves collected from the same plant at the same time (Figure 3c). These results suggest that CsIAMT may be preferentially involved in regulating auxin metabolism during early leaf expansion or differentiation. Previous research from our group also showed that CsIAMT is expressed in roots, leaves, stamens, pistils, tendrils, and young fruits, with significantly higher expression levels particularly in young fruits (Zhang et al. 2025). We further validated CsIAMT expression in young fruits and found its transcript level was several dozen‐fold higher than in root tissues, indicating that this gene may play an important regulatory role in organ development. In summary, we speculate that the expression of the CsIAMT in cucumber shows significant developmental stage specificity; it is highly expressed in actively growing tissues or organs, and its expression level gradually decreases as the tissues mature.

1.4. Functional Validation of CsIAMT as an IAA Methyltransferase in Transgenic Tobacco

To validate the predicted IAA methyltransferase activity of CsIAMT, we performed heterologous expression in tobacco. Western blot analysis confirmed the successful accumulation of the CsIAMT protein in transgenic lines (Figure 4a). Subsequent metabolite profiling revealed a significant metabolic shift in these overexpression plants: the IAA content decreased from 0.45 mg/mL in the wild type to 0.19 mg/mL (a reduction of approximately 57%), while the MeIAA content increased markedly from 3.1 to 10.9 mg/mL (a 3.5‐fold elevation) (Figure 4b). This complementary decrease in the substrate (IAA) and accumulation of the product (MeIAA) provides direct in vivo evidence that CsIAMT catalyzes the methylation of IAA to MeIAA, confirming its function as an IAA methyltransferase.

FIGURE 4.

FIGURE 4

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Functional validation of CsIAMT in transgenic tobacco plants. (a) Western blot analysis of CsIAMT protein expression in tobacco leaves using an anti‐FLAG antibody. (b) Endogenous levels of indole‐3‐acetic acid (IAA) and its methyl ester (MeIAA) in Control and CsIAMT‐OE plants. Data represent mean ± SD of three biological replicates. Asterisks indicate significant differences compared to the WT control (Student's t‐test, **** p < 0.0001).

1.5. Expression Profiling of CsIAMT in Response to Diverse Stresses

To elucidate the expression patterns of CsIAMT under various environmental challenges, we analyzed its transcript levels across eight distinct RNA‐seq datasets from cucumber, encompassing both abiotic and biotic stresses. The expression values were normalized across all samples using a global median scaling method to enable direct cross‐experiment comparison. CsIAMT exhibited dynamic yet distinct regulatory patterns under different stress conditions (Figure 5). Under abiotic stresses, the gene responded in a stress‐specific manner. High‐temperature treatment triggered a strong and time‐dependent induction, with expression peaking at 6 h (Figure 5a). In contrast, low temperature led to a progressive downregulation, reaching its lowest level by 12 h (Figure 5b). Waterlogging stress resulted in a significant suppression of CsIAMT expression after 7 days (Figure 5c). Meanwhile, salt stress elicited only a marginal change in its transcript abundance (Figure 5d). The response to biotic stresses revealed more complex temporal dynamics. Upon root‐knot nematode infection, CsIAMT expression was initially suppressed at early time points (1 day) but was strongly induced thereafter, showing high expression levels at 2 and 3 days post‐infection (Figure 5e). A similar pattern was observed during downy mildew infection, where expression was low at early stages but increased substantially by the 6th day (Figure 5f). Powdery mildew infection consistently upregulated CsIAMT expression (Figure 5g). Conversely, infection with angular leaf spot caused a marked downregulation of CsIAMT, with similarly low expression levels observed at both 1 and 3 days post‐infection (Figure 5h). In summary, CsIAMT is differentially regulated by a wide spectrum of stresses. It is notably induced by high temperature and specific pathogen infections, but suppressed by cold, waterlogging, and angular leaf spot. The induction magnitude, particularly under biotic stresses such as powdery mildew and nematode infection at later stages, often exceeded that observed under abiotic challenges. This comprehensive transcriptome‐based analysis underscores a potent and significant role for CsIAMT in cucumber's response to biotic stressors, highlighting its potential importance in plant defense signaling.

FIGURE 5.

FIGURE 5

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Expression level of CsIAMT under various stress treatments. (a) Expression pattern diagram of CsIAMT in response to high‐temperature stress. (b) Expression pattern diagram of CsIAMT in response to low‐temperature stress. (c) Expression pattern diagram of CsIAMT in response to waterlogging stress. (d) Expression pattern diagram of CsIAMT in response to salt stress. (e) Expression pattern diagram of CsIAMT in response to rhizosphere nematode stress. (f) Expression pattern diagram of CsIAMT in response to powdery mildew stress. (g) Expression pattern diagram of CsIAMT in response to powdery mildew stress. (h) Expression pattern diagram of CsIAMT in response to angular leaf spot stress. The abscissa denotes the time points after stress treatment. The expression levels were normalized across all samples using a global median scaling method to enable cross‐experiment comparison. Values represent the global‐median scaled FPKM. The asterisks indicate significant differences between the two groups (ns represents no significant difference, * indicates a p < 0.05, ** indicates a p < 0.01, *** indicates a p < 0.001, **** indicates a p < 0.0001. A one‐way analysis of variance was used).

2. Materials and Methods

2.1. Materials and Pretreatment

The experiment was conducted in the botany laboratory of Shenyang Normal University. The materials used in the experiment were cucumbers ( Cucumis sativus L.), and the seeds of Zhongnong 26 came from the Institute of Vegetables and Flowers of the Chinese Academy of Agricultural Sciences. After germinating in the incubator, the seeds were cultivated in a seedling pot (10 × 10 cm). The soil ratio was: nutrient soil: vermiculite: perlite = 7: 4: 2. Ensure to apply 1 times the amount of MS nutrient solution once a week. During the rest of the time, use water for cultivation.

Cucumber seedlings at the two‐true‐leaf stage were used for hormone treatments. Stock solutions of IAA and IBA were prepared in a minimal volume of 1 mol/L NaOH, then diluted with distilled water to final working concentrations (IAA: 1 mg/L; IBA: 1 mg/L). BA, GA, and SA were directly dissolved in distilled water to final concentrations of 0.5 mmol/L, 5 μmol/L, and 0.5 mmol/L, respectively. The pH of all working solutions was adjusted to 5.8. For each treatment, a volume of 100 mL of hormone solution was applied to the soil of each pot via root drench. Control plants received an equal volume of solvent‐adjusted distilled water (pH 5.8). Each treatment group consisted of 15 individual plants. The second true leaf was collected from each plant at 0, 6, and 12 h post‐treatment, immediately frozen in liquid nitrogen, and stored at −80°C for RNA extraction. For tissue sampling, a separate batch of 10–15 plants was grown to the fruiting stage. Young fruit and root tissues were collected 3 days after artificial pollination of the female flowers. All samples were immediately frozen and stored as above for subsequent qRT‐PCR analysis.

2.2. Construction of Phylogenetic Tree

To elucidate the evolutionary position of the candidate gene CsaV3_7G020980, a two‐step phylogenetic analysis was conducted. First, a maximum likelihood phylogenetic tree was constructed using the identified SABATH family protein sequences from four representative species— Arabidopsis thaliana , rice, tomato, and cucumber. This initial analysis placed CsaV3_7G020980 within a specific clade of the SABATH family. Subsequently, to investigate its potential functional assignment as an IAA methyltransferase, a more focused phylogenetic reconstruction was performed. This analysis included the candidate gene alongside previously characterized SABATH members with known or putative IAMT function, such as AtIAMT, LjIAMT, PtIAMT, OsIAMT, as well as other methyltransferases, such as CbSAMT. Multiple sequence alignment was carried out using the Muscle algorithm in MEGA 11, and the phylogenetic relationship was inferred using the maximum likelihood method (Tamura et al. 2021). The phylogenetic tree was subsequently visualized and refined using the iTOL online platform (https://itol.embl.de/).

2.3. Identification of Potential IAMT Genes in Cucumber

In order to search for the possible IAMT gene in the cucumber SABATH gene family, the protein sequence of AtIAMT was used to conduct a BLASTp search on the cucumber database (http://cucurbitgenomics.org/), with the e‐value set to less than 1e‐10. Five candidate genes were obtained.

2.4. Identification and Analysis of Genomic Synteny

To identify homologous gene pairs and explore collinearity within the SABATH gene family of cucumber, MCScanX (Multiple Collinearity Scan toolkit) was used with default parameters. Furthermore, synteny relationships of orthologous SABATH genes were analyzed between cucumber and three reference species—Arabidopsis, rice, and tomato—to infer potential gene functions and evolutionary conservation. The syntenic connections among cucumber, Arabidopsis, rice, and tomato were also investigated under the same default settings of MCScanX. Finally, TBtools was employed to generate homologous association charts visualizing the syntenic relationships of SABATH genes across these four species.

2.5. Genetic Selection and Evolution Analysis

To assess the selective constraints acting on the IAMT genes, pairwise estimates of the nonsynonymous (dN) and synonymous (dS) substitution rates were calculated. The coding sequences (CDS) of CsIAMT and its orthologs from Arabidopsis thaliana (AtIAMT1), Oryza sativa (OsIAMT1), and Populus trichocarpa (PtIAMT) were aligned at the codon level using the Clustal Omega algorithm. The dN/dS ratio (ω) was then computed for each pair using the Nei‐Gojobori method with Jukes‐Cantor correction, implemented in the Biopython library.

2.6. Sequence Alignment

The protein sequences of IAMT and CsaV3_7G020980 identified for each species were aligned using MEGA11, and then viewed through ESPript (https://espript.ibcp.fr/ESPript/ESPript/index.php). Subsequently, based on the protein sequences of each species IAMT, the three‐dimensional structures and Ramachandran plots of the proteins were generated using SWISS‐MODEL (swissmodel.expasy.org) (Waterhouse et al. 2018). For detailed specific information, please refer to Table S5.

2.7. RNA Isolation, cDNA Synthesis and Quantitative Real‐Time PCR Analysis

RNA was extracted using the Total RNA Extraction Kit (Promega, USA), and complementary DNA (cDNA) was synthesized employing the PrimeScript RT Reagent Kit (TaKaRa, Japan), following the manufacturers' standardized procedures. The expression levels of target genes were then quantified using the LightCycler 96 Real‐Time PCR System (Roche, Switzerland). Gene expression analysis was based on the amplification profiles obtained under defined thermal cycling conditions. The 2−ΔΔCT method was applied to calculate the relative mRNA expression, normalizing the data against internal control amplification. With the Control group designated as the reference point (value = 1), the expression patterns of other experimental groups were comparatively analyzed against CK. Detailed information regarding the gene‐specific primer sequences is provided in Tables S6 and S7.

2.8. Identification of Hormones and MeIAA

Select cucumber plants with consistent growth, weigh 1 g of the leaves, then cut them and add them to 80% methanol. Grind them on ice and dilute to a total volume of 8 mL. Then, it was placed in a 4°C refrigerator for 12 h, followed by centrifugation for 20 min, and the supernatant was collected. Extract three times with 80% methanol, combine the supernatants and dry them in a 4°C constant temperature box under light protection. After drying, add 100% methanol to dissolve the sample, make up the volume to 5 mL, centrifuge and filter the supernatant. Different concentrations of hormone solutions were prepared and injected separately into the GC column along with the above samples. After obtaining the data, standard curves were made based on different hormone solutions to calculate the concentrations of hormones and MeIAA. The information of the standard curve can be found in Table S8.

2.9. Vector Construction and Transient Expression in Tobacco

The coding sequence of CsIAMT was cloned into the plant overexpression vector pRI101 under the control of the cauliflower mosaic virus (CaMV) 35S promoter. After verification by sequencing, the recombinant plasmid was introduced into Agrobacterium tumefaciens strain GV3101 via electroporation. For transient expression, the constructed Agrobacterium strain was injected into leaves of Nicotiana benthamiana using an Agrobacterium‐mediated transient transformation method.

To validate the expression of the CsIAMT protein, total soluble protein was extracted from the agroinfiltrated tobacco leaves and separated by SDS PAGE. Subsequently, Western blot analysis was performed using an Anti‐FLAG antibody to confirm the successful expression of the target protein.

2.10. Transcriptome Analysis of the CsIAMT Gene

In order to study the specific expression of the CsIAMT gene under different stresses in cucumbers, RNA‐seq data from different stress conditions were obtained using the accession numbers low temperature (PRJNA438923), high temperature (PRJNA634519) (Chen et al. 2020), salt and silicon (PRJNA477930) (Zhu et al. 2019), waterlogging (PRJNA678740) (Kęska et al. 2021) and root‐knot nematode (PRJNA419665) (Wang et al. 2018), downy mildew (PRJNA285071) (Burkhardt and Day 2016), powdery mildew (PRJNA321023) (Xu et al. 2017), and angular leaf spot (PRJNA704621) (Słomnicka et al. 2021) from the cucumber genome database (http://cucurbitgenomics.org/). All relevant metadata, including tissue type, developmental stage, and specific stress treatment details, are now compiled in Table S9. The published transcriptome data of cucumber was combined with the cucumber V3 genome data for re‐analysis using RNA‐seq. To integrate and compare transcriptomic data from independent experiments, we performed cross‐sample normalization of FPKM values to correct for technical batch effects. Specifically, a global distribution‐based scaling method was employed (Bolstad et al. 2003; Ritchie et al. 2015). First, the median FPKM value (Median i) of reliably expressed genes (FPKM > 1) was calculated for each sample. Next, the global median (Global Median) across all sample‐specific Median_i values was determined. A scaling factor (Scale i) for each sample was then defined as Global_Median/Median i. Finally, the comparable expression level of the target gene CsIAMT in any given sample was obtained by multiplying its original FPKM value by the corresponding Scale i. This approach aimed to remove systematic biases introduced by differences in sequencing depth and library construction across experiments, thereby enabling meaningful comparison of expression levels under different stress treatments.

3. Discussion

Cucumber is a globally significant vegetable crop, valued both nutritionally and economically (Grumet et al. 2021; Mirzwa‐Mróz et al. 2024). In the context of climate change and increasing human dependence on major crops, cucumber faces escalating pressures from abiotic stressors such as drought and heat, as well as from emerging biotic threats like pathogen outbreaks (Grumet et al. 2021; Aparna et al. 2023; Parada‐Rojas and Quesada‐Ocampo 2022). Within this ecological and evolutionary framework, deciphering and utilizing key genetic regulatory modules to enhance crop adaptability and resilience in fluctuating environments is crucial for the sustainability of agricultural systems (Peng et al. 2021; Dong et al. 2022; He et al. 2022).

The SABATH gene family represents an evolutionarily conserved system for chemical diversification in plants, with IAMT being one of its most ancestral members (Guo et al. 2020; Wen et al. 2025). Phylogenetic analysis indicates that IAMT orthologs across species share a common origin and form a well‐supported, monophyletic clade (Figure 1). Strong signals of purifying selection (dN/dS < 1) and conserved microsynteny, particularly between cucumber and Arabidopsis (Table S1), underscore the functional preservation of this gene lineage over evolutionary time. To validate its predicted function, CsaV3_7G020980 was heterologously expressed in tobacco. We observed that in the transgenic overexpression lines, IAA content decreased while MeIAA increased markedly (Figure 4), supporting its identification as CsIAMT and confirming its IAA methyltransferase activity. Furthermore, qRT‐PCR analysis revealed that CsIAMT expression was particularly high in young fruits and newly developed leaves—tissues that are key sites for auxin synthesis and distribution. This spatiotemporal expression pattern suggests that CsIAMT may fine‐tune active auxin levels by methylating IAA to MeIAA, thereby contributing to regulated organ development (Zhao 2010; Casanova‐Sáez et al. 2021; Li et al. 2025). The attenuation of CsIAMT expression during leaf maturation further aligns with its putative function in coordinating developmental transitions, consistent with observations in AtIAMT1 mutants (Qin et al. 2005).

Beyond development, auxin signaling is intimately linked to stress responses (Korver et al. 2018). Based on previously published data (Zhang et al. 2025) and our transcriptome analysis, CsIAMT can be induced by both biotic and abiotic stresses. However, its induction level was more pronounced under biotic stress, which might suggest a specific role in the cucumber disease resistance pathway. Pathogenic bacteria can promote infection by manipulating the auxin signaling pathway of the host plant (Lyons et al. 2015). The upregulation of CsIAMT could represent a host counterstrategy, potentially operating through mechanisms such as reducing bioactive auxin levels to limit pathogen exploitation or generating MeIAA as a defensive signal that may intersect with salicylic acid (SA)‐mediated resistance (Koo et al. 2008; Spoel and Dong 2024).

While this study provides molecular evidence for the evolutionary conservation (dN/dS < 1), stress‐responsive expression, and catalytic function of CsIAMT, its precise ecological and evolutionary significance warrants careful interpretation. The observed stronger induction of CsIAMT under biotic stress—against the backdrop of strong purifying selection—suggests that this conserved biochemical module may have been recruited for a specific role in defense responses. From an evolutionary perspective, the conservation of IAMT across plant lineages, coupled with its substrate specificity for IAA, implies that fine‐tuning auxin homeostasis through methylation may represent an adaptive mechanism under pathogen pressure (Fu et al. 2015; Zeng et al. 2024). Functionally, the rapid upregulation of CsIAMT following pathogen challenge could modulate free IAA levels, thereby limiting pathogen manipulation of auxin signaling, while the resulting MeIAA might act as a defensive metabolite or signal. In summary, this study defines CsIAMT as an evolutionarily conserved component of auxin metabolism that is integrated into stress‐responsive networks and likely contributes to adaptive regulation under biotic pressure.

4. Conclusion

In this paper, we identified the cucumber auxin methyltransferase CsIAMT through BLASTp analysis of the cucumber database, phylogenetic relationships, and sequence alignment. Exogenous hormone treatment further confirmed that the specific substrate of CsIAMT is indole‐3‐acetic acid (IAA). The analysis of tissue‐specific expression profiles revealed that CsIAMT exhibits a dual regulatory pattern in both space and time: in the spatial dimension, this gene is highly expressed in young tissues or organs; in the temporal dimension, the expression level of the new leaves is significantly higher than that of mature leaves, suggesting that it participates in the early development process of cucumber organs by mediating IAA‐methylation. What is particularly important is that based on transcriptome data analysis, CsIAMT shows a significantly stronger response characteristic in biotic stress compared to abiotic stress, laying a foundation for subsequent studies on enhancing the stress resistance of cucumber by improving CsIAMT (Figure 6).

FIGURE 6.

FIGURE 6

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The mechanism of changes in CsIAMT, IAA, and MeIAA in cucumber.

Author Contributions

Xinjie Zhang: data curation (equal), formal analysis (equal), investigation (equal), methodology (equal), writing – original draft (equal). Yang Zhou: data curation (equal), investigation (equal), methodology (equal). Mengxin Chen: investigation (equal), methodology (equal). Jingwen Li: conceptualization (equal), methodology (equal). Lisi Jiang: methodology (equal), project administration (equal). Ken Li: formal analysis (equal), supervision (equal), writing – review and editing (equal). Lin Hao: formal analysis (equal), supervision (equal), writing – review and editing (equal). Wei Fu: funding acquisition (equal), methodology (equal), project administration (equal), writing – original draft (equal), writing – review and editing (equal).

Funding

The study was supported by Liaoning Provincial universities Basic Research funds special fund (LJ202410166039) and Liaoning Provincial Science and Technology Plan Joint Project (2025‐MSLH‐618).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1: ece373178‐sup‐0001‐Tables.docx.

ECE3-16-e73178-s001.docx (209.7KB, docx)

Acknowledgments

We are grateful to Dr. Sai Wang for her expert guidance and critical technical assistance in designing and performing the tobacco transient expression assays, which were essential for the functional validation in this study.

Contributor Information

Lin Hao, Email: haolinwj2001@163.com.

Wei Fu, Email: fu260242@126.com.

Data Availability Statement

The data that support the findings of this study are available in Cucurbit Genomics Database at http://cucurbitgenomics.org/, reference number PRJNA438923, PRJNA634519, PRJNA477930, PRJNA678740, PRJNA419665, PRJNA285071, PRJNA321023, PRJNA704621.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1: ece373178‐sup‐0001‐Tables.docx.

ECE3-16-e73178-s001.docx (209.7KB, docx)

Data Availability Statement

The data that support the findings of this study are available in Cucurbit Genomics Database at http://cucurbitgenomics.org/, reference number PRJNA438923, PRJNA634519, PRJNA477930, PRJNA678740, PRJNA419665, PRJNA285071, PRJNA321023, PRJNA704621.

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