Abstract

   Calmodulin-like (CML) transcription factors function as calcium (Ca^2⁺)
   signal sensors and play a pivotal role in plant cold resistance.
   Although this gene family has been identified in various plant species,
   the characteristics of the CML gene family in Curcuma alismatifolia and
   its function under cold stress remain largely unknown. This study
   identified 202 CACML genes in the Curcuma alismatifolia genome, which
   were phylogenetically classified into four clades. Members within each
   clade shared conserved gene structures and motifs. Gene duplication
   analysis revealed that segmental duplication events (44 events) served
   as the primary driving force for the expansion of the CACML family.
   Most homologous gene pairs were subjected to strong purifying selection
   during evolution, while CACML86 underwent positive selection in both C.
   alismatifolia and A. viiosum. Promoter regions were enriched with
   cis-acting elements associated with growth and development,
   biotic/abiotic stress responses, and hormone responsiveness, suggesting
   diverse functional roles for CACML genes. Transcriptome analysis
   indicated that CACML140 regulates the cold stress response in Curcuma
   alismatifolia via the MAPK cascade signaling pathway. These findings
   enhance our understanding of CML-mediated cold stress responses in
   Curcuma alismatifolia and establish a framework for the systematic
   dissection of CML gene function and the regulatory network governing
   cold hardiness.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12870-025-06898-9.

   Keywords: Curcuma alismatifolia, CML genes, Cold tolerance,
   Transcriptomics, MAPK signaling pathway

Introduction

   Plants transmit external stimulus signals through messenger networks to
   internal systems and initiate adaptive responses [[34]1]. Calcium
   (Ca^2⁺) acts as an intracellular second messenger, orchestrating plant
   developmental programs, environmental acclimation, and adaptive
   responses to diverse abiotic and biotic stresses [[35]2]. When plants
   encounter diverse environmental challenges-including high temperature,
   drought, salinity, and pathogen infection fluctuations in cytosolic
   free calcium ([Ca^2⁺]cyt) concentrations trigger calcium signaling
   cascades [[36]3]. These Ca^2⁺ signals are typically sensed by Ca^2⁺
   sensors or calcium-binding proteins (CBPs), which undergo
   conformational changes upon Ca^2⁺ binding, subsequently regulating
   downstream target genes and propagating Ca^2⁺ signaling [[37]4].
   Distinct calcium-sensing proteins exhibit specialized capacities to
   decode differential Ca^2⁺ signatures generated by cytosolic free Ca^2⁺
   [[38]5]. Three major classes of EF-hand domain-containing proteins have
   been identified as Ca^2⁺ signal transducers in plants:
   calmodulin/calmodulin-like proteins (CaMs/CMLs), calcineurin B-like
   proteins (CBLs), and calcium-dependent protein kinases (CDPKs) [[39]6].
   Most calcium sensors utilize the conserved helix-loop-helix structural
   motif (EF-hand) to mediate Ca^2⁺ binding. Calmodulin (CaM), one of the
   most evolutionarily conserved and ubiquitously expressed proteins in
   eukaryotes, contains four canonical EF-hand domains [[40]7]. In
   contrast, calmodulin-like proteins (CMLs) are predominantly restricted
   to plants and select protists, displaying significant variation in
   EF-hand domain numbers (1–6) [[41]8]. CMLs are characterized by a Dx3D
   motif, whereas CaMs harbor four calcium-binding DxD motifs. The two
   α-helices interconnected by the Dx3D motif activate downstream
   regulatory networks upon calcium ion binding [[42]9].

   Calmodulin-like proteins (CMLs) play pivotal roles in plant growth,
   development, and resistance to biotic/abiotic stresses [[43]10]. In
   Arabidopsis thaliana, 50 CML family members have been identified, with
   their functions demonstrated to rely on the abscisic acid
   (ABA)-dependent signaling pathway during abiotic stress responses
   [[44]11]. Regarding plant development, AtCML25 and AtCML36 regulate
   pollen tube elongation in A. thaliana, while cucumber CML25 promotes
   fruit expansion by modulating hormone signaling-related genes [[45]12].
   Overexpression of wheat TaCML20 enhances water-soluble carbohydrate
   accumulation, thereby increasing grain yield [[46]13]. CMLs also
   critically mediate abiotic stress tolerance. For instance, rice OsCML4
   improves drought resistance by reducing reactive oxygen species (ROS)
   levels [[47]14], whereas tomato ShCML44 overexpression confers dual
   tolerance to drought and cold stress [[48]15]. In Medicago truncatula,
   MtCML42 interacts with MtCBF1 and MtCBF4 to form a regulatory network
   that upregulates cold stress-responsive genes MtGolS1 and MtGolS2,
   thereby enhancing freezing tolerance [[49]16]. Vitis vinifera L. CML21
   exhibits divergent regulatory patterns under cold stress, underscoring
   the significance of genetic diversity in environmental adaptation
   [[50]17]. Despite systematic investigations of CML-mediated stress
   responses in model plants, the functional characterization and
   molecular mechanisms of CML family members in C. alismatifolia remain
   poorly understood.

   C. alismatifolia, a photophilic horticultural ornamental species in the
   Zingiberaceae family [[51]18], is primarily distributed across
   subtropical and tropical zones. This species demonstrates high
   ornamental value owing to its vividly colored, morphologically diverse
   inflorescences and complex aromatic profiles [[52]19]. However, its
   agricultural cultivation and reproductive development face substantial
   limitations in cold climatic conditions. Recent genomic advancements
   have enabled the complete assembly of the C. alismatifolia genome,
   thereby facilitating targeted investigations into Calmodulin-like (CML)
   gene families putatively associated with cold stress response pathways.
   In this study, we conducted a genome-wide screening to identify CML
   family members in C. alismatifolia, followed by systematic
   characterization of their structural properties, phylogenetic
   relationships, chromosomal assignments, and conserved domain
   architectures. To dissect the molecular basis of cold stress
   adaptation, we further profiled the transcriptional dynamics of select
   CML genes under low-temperature treatments. Through integrative
   analysis of these multi-dimensional datasets, our work aims to identify
   pivotal cold-responsive CML gene candidates, establishing a robust
   foundation for downstream functional characterization of CML-mediated
   cold tolerance mechanisms in this economically important ornamental
   species.

Materials and methods

Identification of members, gene physico-chemical properties

   Genomic and annotation files of C. alismatifolia were downloaded from
   the NCBI database, while Arabidopsis data were obtained from TAIR
   ([53]https://www.arabidopsis.org/). Candidate CACML proteins were
   identified by BlastP (V = 2.13.0) searching against the C.
   alismatifolia genome using A. thaliana CML (ATCML) sequences as queries
   (E-value < 1 × 10⁻^5). Protein sequences matching the EF-hand domain
   (PF00036) HMM profile were further retrieved (p-value < 1 × 10⁻^5)
   HMMsearch (V = 3.2.0). Overlapping sequences from both methods were
   validated for EF-hand motif presence using SMART, and only those
   containing canonical EF-hand motifs were retained as CACML proteins.
   Biochemical indices of CACML proteins, including isoelectric point,
   molecular weight, and grand average of hydropathicity, were calculated
   using Expasy tools ([54]https://www.expasy.org/).

Phylogenetic analysis of CACML gene family members

   Multiple sequence alignment of CACML and ATCML protein sequences was
   performed using Clustal software (V = 3.1). The aligned results were
   subsequently imported into MEGA (V = 11.0) for phylogenetic tree
   construction using the Maximum Likelihood (ML) method, with reliability
   assessed via 1000 bootstrap iterations. Additionally, a phylogenetic
   tree for CACML proteins was constructed using the same methodology and
   visualized using iTOL ([55]https://itol.embl.de/).

Analysis of gene structure, conserved motifs, structural domains and
cis-acting element of CACML gene family members

   Gene structure information of CACML genes was extracted from the C.
   alismatifolia genome annotation file, with positional data for CDS and
   UTR regions statistically analyzed. Gene structure visualization was
   performed using TBtools software
   ([56]https://github.com/CJ-Chen/TBtools-II) [[57]20]. CACML gene
   sequences were uploaded to the MEME
   ([58]http://memeesuite.org/tools/meme) and InterPro database
   ([59]https://www.ebi.ac.uk/interpro/), with the motif number set to 10
   for analysis. The 2000 bp upstream sequences of CACML genes were
   extracted as promoter regions, and cis-acting elements were identified
   using the PlantCARE database
   ([60]http://bioinformatics.psb.ugent.be/webtools/plantcare/html) and
   classified into functional categories.

Chromosomal location and duplication events analysis of CACML gene

   Gene duplication events of CACML genes were analyzed using MCScanX
   software ([61]http://chibba.pgml.uga.edu/mcscan2/) [[62]21], with
   visualization performed in Circos. Collinear relationships of CML genes
   between C. alismatifolia, A. viiosum, and A. tsaoko were analyzed by
   MCScanX, and the results were visualized using TBtools software.
   Collinear CML gene pairs were statistically identified, and their Ka/Ks
   values were calculated using KaKs_Calculator (V = 3.0).

Tissue expression analysis of CACML gene family members

   C. alismatifolia were cultivated in the greenhouse of Guizhou
   University of Engineering Science (27°17′34.436″ N, 105°18′46.069″ E).
   For cold stress treatment, C. alismatifolia were transferred into two
   separate growth chambers maintained at 4 °C and 25 °C respectively.
   After 12 h of treatment, leaf samples from three biological replicates
   per treatment were collected for total RNA extraction. Sequencing
   libraries were constructed using 1 μg of RNA with the NEBNext Ultra RNA
   Library Prep Kit (NEB, Ipswich) for Illumina. Sequencing reads were
   aligned to the C. alismatifolia genome, and transcript assembly and
   FPKM value calculation were performed using the StringTie program
   (v. = 2.1.1) with default parameters. Differentially expressed mRNAs
   were identified using the DESeq2 package (v. = 1.33.5) in R software
   with default settings, where genes with |fold change|> 2 and
   P-value < 0.05 were defined as significantly altered.

   KEGG pathway enrichment analysis was conducted for differentially
   expressed genes (DEGs) meeting |fold change|> 2 and P < 0.05. Corrplot
   R package was performed to calculate gene correlations (R^2 > 0.9).
   Volcano plots, bubble enrichment maps, correlation heatmaps, and
   cluster heatmaps were generated using ggplot2 R package (V = 2.1).

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Analysis

   First-strand cDNA was synthesized from total RNA of samples using the
   HiScript®II cDNA Synthesis Kit. Primers were designed using NCBI primer
   blast([63]https://www.ncbi.nlm.nih.gov/tools/primer-blast/) with an
   amplified fragment length of 150–250 bp (Supplementary Table 2). The
   iTaq Universal SYBR®Green Supermix (Tiangen Biotech, Beijing) was used
   in a 20 µL reaction volume, with three biological replicates set for
   each sample. ACT2 expression level was used as an internal reference.
   Gene expression levels were detected on a Biosystems 7500 Real-Time PCR
   system (Thermo Fisher Scientific, Waltham), and expression differences
   were calculated using the 2^−ΔΔCT method.

Statistical analysis

   Statistical analyses and figure preparation were performed using
   GraphPad Prism 8.0 and Adobe Illustrator 2020.

Results

   A. thaliana AtCML protein sequences were selected to conduct BLASTP
   searches against the Curcuma alismatifolia genome. Candidate CML
   protein sequences were further filtered based on the EF-hand domain
   (PF00036), a structural signature characteristic of CML proteins. In
   this study, 202 CML genes were identified from the C. alismatifolia
   genome and systematically named CACML1-CACML202 according to their
   chromosomal positions (Supplementary Table 1). Sequence analysis
   revealed that all CACML proteins harbor a conserved EF-hand domain. The
   CML gene family displayed remarkable structural diversity, with protein
   lengths spanning 73–1447 amino acids (CACML134-CACML143). Molecular
   weight calculations showed values of 8.24–164.18 kDa
   (CACML134-CACML143), while isoelectric points (PI) ranged 3.64
   (CACML125)−9.77 (CACML176). 29 CACML proteins were classified as basic
   proteins (PI > 7).

   Chromosomal mapping of the 202 CACML genes revealed that 197 members
   were chromosomally localized (Fig. [64]1), with 5 genes remaining
   unassigned due to incomplete genome annotation. The 197 CACML genes
   exhibited non-uniform distribution across 16 chromosomes, with
   chromosome 1 harboring the highest number (29) and chromosome 15 the
   lowest (3). only a single tandem duplicate pair was identified within
   the CACML gene family.

Fig. 1.

   [65]Fig. 1
   [66]Open in a new tab

   Chromosomal localization of CACML genes in C. alismatifolia. Note:
   Chromosome sizes are indicated by the leftmost vertical scale (Mb);
   chromosome numbers are positioned on the left side of each bar; gene
   pairs highlighted in red represent tandem duplicate pairs

   To explore the evolutionary relationships and functional divergence of
   the CACML gene family, a phylogenetic tree was constructed using 202
   CACML sequences and 50 ATCML (Fig. [67]2). Phylogenetic analysis
   grouped the CACML genes into four distinct clades (Clade 1–4), with
   Clade 3 containing the largest number of members (83) and Clade 1 the
   smallest (26). Notably, Clade 4 lacked A. thaliana, suggesting
   potential species-specific functional innovations within this Clade.

Fig. 2.

   [68]Fig. 2
   [69]Open in a new tab

   Phylogenetic tree of the CML gene family in C. alismatifolia and A.
   thaliana. Note: The tree was constructed using the Maximum likelihood
   (ML) method with 1000 bootstrap replicates

   To investigate the evolutionary relationships and functional divergence
   of the CACML gene family, a phylogenetic tree was constructed using 202
   CACML sequences and 50 AtCML sequences from A. thaliana (Fig. [70]2).
   Phylogenetic analysis classified CACML genes into four distinct clades
   (Clade 1–4), each containing CACML members. Clade 3 exhibited the
   largest membership (83 genes), while Clade 1 contained the fewest (26
   genes). Clade 4 lacked A. thaliana homologs, suggesting potential
   species-specific functional divergence within this clade. High sequence
   similarity observed among CML proteins from diverse plant species
   implies conserved functional roles and shared evolutionary trajectories
   across clade.

   Previous studies have demonstrated correlations between gene structural
   features, expression patterns, and functional divergence [[71]22].
   Using MEME, we identified 10 conserved motifs among the 202 CACML genes
   (Fig. [72]3A). Motifs 2 and 4 corresponded to EF-hand domains, which
   were universally conserved across all CACML proteins. All 10 motifs
   were retained in Clade 4 members, whereas most CACML proteins in Clades
   1–3 comprised Motifs 2, 4, 8, and 10. The shared motif composition
   among phylogenetically closely related protein family members suggests
   potential functional differentiation between CACML proteins in Clades
   1–3 and Clade 4. These findings indicate that conserved motif
   organization may underpin functional coherence within phylogenetic
   clade.

Fig. 3.

   [73]Fig. 3
   [74]Open in a new tab

   Conserved motifs and gene structure analysis of 202 CACML genes. Note:
   a Schematic diagram of conserved motifs in CACML protein sequences,
   where different colored boxes (1–10) represent distinct conserved
   motifs. b Exon–intron-untranslated region (UTR) structure of CACML
   genes, with exons shown as pink boxes, introns as black lines, and UTRs
   as green boxes

   To further elucidate structural variations in CACML genes, we analyzed
   their conserved motifs, exon–intron architectures, and untranslated
   regions (UTRs). Results revealed that most CACML genes possess two UTRs
   and multiple exons. Specifically, members of Clade 1 and Clade 4
   exhibited relatively fewer exons (0–16) (Fig. [75]3 B). In contrast,
   Clade 2 and Clade 3 displayed more complex gene structures, with
   CACML141 in Clade 3 containing the highest number of exons (34),
   indicative of evolutionary diversification. The simpler gene
   architectures of Clade 1 and Clade 4 members suggest their potential
   for rapid transcriptional activation due to reduced intron splicing
   steps, thereby shortening stress response latency. Structural
   divergence of CACML genes across clades may be associated with
   lineage-specific functional specialization.

   Promoter analysis of the 2000 bp upstream regulatory regions of 202
   CACML genes identified abiotic/biotic stress-responsive elements,
   phytohormone-responsive elements, and growth/development-related
   cis-elements (Fig. [76]4, A,B). Stress-responsive and
   phytohormone-related elements were the most abundant, with MYC elements
   involved in cold response showing the highest frequency (1094)
   (Fig. [77]4, A). Notably, CACML187 contained the largest number of MYC
   cis-acting elements (21) (Fig. [78]4, A). Among growth/developmental
   elements, Box4 (644 occurrences) was most prevalent, associated with
   light signal perception and transduction. Within hormone-responsive
   elements, jasmonic acid-related CGTCA-motif (829 instances) dominated
   (Fig. [79]4, A). These findings collectively indicate that the CACML
   gene family plays regulatory roles in turmeric's responses to diverse
   abiotic/biotic stresses and growth/developmental processes
   (Fig. [80]5).

Fig. 4.

   [81]Fig. 4
   [82]Open in a new tab

   Cis-elements in the CACML promoters. Note: The vertical axis represents
   CACML genes, while the horizontal axis shows different types of
   cis-acting elements. The text annotations denote the number of
   corresponding cis-acting elements, with red color indicating higher
   abundance

Fig. 5.

   [83]Fig. 5
   [84]Open in a new tab

   Intraspecies homology analysis of CACML genes. Note: Black lines
   represent duplicated CACML gene pairs, while gray lines indicate whole
   genome duplicated gene pairs

   To investigate the evolutionary driving forces underlying the expansion
   of the CACML family, we performed genome-wide collinearity analysis on
   202 CACML. A single tandem duplication event (CACML132-CACML149) was
   identified. Furthermore, 44 segmental duplication events involving 83
   CACML genes were detected. The predominance of segmental duplication
   events over tandem duplications suggests that segmental duplication
   served as the primary evolutionary mechanism driving CACML gene family
   expansion in C. alismatifolia.

   To further characterize the evolutionary trajectory of CML genes in C.
   alismatifolia, interspecific collinearity analysis was performed among
   three Zingiberaceae species (C. alismatifolia, Amomum viiosum, and
   Amomum tsaoko) and A. thaliana. A total of 161 orthologous pairs were
   identified between C. alismatifolia and A. tsaoko, with 148 orthologs
   detected between C. alismatifolia and A. viiosum (Fig. [85]6). These
   orthologous relationships primarily exhibited one-to-many or
   many-to-many collinear patterns, e.g., CACML20 shared two orthologs in
   both A. tsaoko and C. alismatifolia, indicating potential functional
   redundancy. Notably, no collinearity was observed between A. tsaoko/C.
   alismatifolia genes and the previously identified tandem duplication
   pair, suggesting that this tandem duplication event underwent
   independent evolution in the C. alismatifolia genome.

Fig. 6.

   [86]Fig. 6
   [87]Open in a new tab

   Collinear CML genes between C. alismatifolia and A. viiosum, and
   between C. alismatifolia and A. tsaoko, C. alismatifolia and A.
   thaliana are shown. Red lines represent collinear gene pairs

   To further characterize the evolutionary trajectory of CML genes in C.
   alismatifolia, interspecific collinearity analysis was performed among
   three Zingiberaceae species (C. alismatifolia, A. viiosum, and A.
   tsaoko) and A. thaliana. A total of 161 orthologous pairs were
   identified between C. alismatifolia and A. tsaoko, with 148 orthologs
   detected between C. alismatifolia and A. viiosum (Fig. [88]6). These
   orthologous relationships primarily exhibited one-to-many or
   many-to-many collinear patterns, e.g., CACML20 shared two orthologs in
   both A. tsaoko and C. alismatifolia, indicating potential functional
   redundancy. Notably, no collinearity was observed between A. tsaoko/C.
   alismatifolia genes and the previously identified tandem duplication
   pair, suggesting that this tandem duplication event underwent
   independent evolution in the C. alismatifolia genome. Only three pairs
   of orthologous CML genes exist between C. alismatifolia and Arabidopsis
   thaliana (CACML118-ATCML49, CACML127-ATCML11, CACML155-ATCML48). These
   three CACML genes also exhibit synteny with A. viiosum and A. tsaoko.
   This interspecies synteny indicates that these three CACML genes have
   been conserved during evolution.

   Ka/Ks ratio analysis revealed that the majority of Zingiberaceae CML
   genes have been subjected to purifying selection (Ka/Ks < 1)
   (Supplementary Table 3). A notable exception was CACML86, which showed
   contrasting evolutionary pressures, its ortholog in A. viiosum
   experienced positive selection (Ka/Ks > 1), while the A. tsaoko paralog
   remained under purifying selection (Ka/Ks < 1).

   Ka/Ks ratio analysis indicates that the CML genes in C. alismatifolia
   and A. thaliana, as well as most CML genes in Zingiberaceae, underwent
   purifying selection (Ka/Ks < 1) (Supplementary Table 3). A notable
   exception was CACML86, which showed contrasting evolutionary pressures,
   its ortholog in A. viiosum experienced positive selection (Ka/Ks > 1),
   while the A. tsaoko paralog remained under purifying selection
   (Ka/Ks < 1).

   To further investigate the potential molecular mechanisms of CACML gene
   involvement in cold stress response in C. alismatifolia, six samples
   were selected for transcriptome analysis. Six cDNA libraries were
   constructed, with post-filtering data showing Q20 and Q30 values both
   exceeding 95%, and over 90% of clean reads successfully mapped to the
   C. alismatifolia genome. Differentially expressed genes (DEGs) were
   identified and visualized through volcano plots. Compared with the
   control group, 6,909 DEGs (3,191 upregulated and 3,718 downregulated)
   were detected in cold-treated samples (Fig. [89]7A). KEGG pathway
   enrichment analysis of these 6,909 DEGs revealed the top 10
   significantly enriched pathways based on Padjust values. The results
   demonstrated significant enrichment of DEGs in the MAPK signaling
   pathway, plant hormone signal transduction, and nitrogen metabolism
   (Fig. [90]7B).

Fig. 7.

   [91]Fig. 7
   [92]Open in a new tab

   Cold-resistant transcriptome analysis of C. alismatifolia. Note: A
   Volcano plot of differentially expressed genes in the cold-resistant
   transcriptome. B KEGG enrichment bubble map of differentially expressed
   genes in the cold-resistant transcriptome. C Heatmap of CACML gene
   expression levels in the cold-resistant transcriptome. D Correlation
   diagram between MAPK signaling pathway-enriched genes and CACML140

   Expression profiling of CACML genes in this dataset (Fig. [93]7, C)
   identified CACML140 as a significantly upregulated gene enriched in the
   MAPK signaling pathway. Co-expression analysis within this pathway
   revealed positive correlations between CACML140 and MKK7/MKK9, whereas
   RCAR11 and MKK7 showed negative correlations (Fig. [94]7, D).
   Collectively, these transcriptomic data suggest that CACML140 modulates
   the MAPK signaling cascade during cold stress in C. alismatifolia.

   To validate RNA-seq findings, qRT-PCR analysis was performed to
   characterize the expression profiles of CACML140 and MAPK signaling
   components (MKK7, MKK9, and RCAR11) in leaves subjected to 4 °C cold
   treatment for 12 h (Fig. [95]8). Statistical analysis revealed
   significant upregulation of CACML140, CACML142, CACML93, MKK7, OXI1,
   WRKY20 and MKK9, whereas RCAR11, EBF1, CAT2.1 showed marked
   downregulation under cold stress. These results suggest that CACML140
   and the MAPK signaling cascade play critical roles in cold acclimation
   of C. alismatifolia.

Fig. 8.

   [96]Fig. 8
   [97]Open in a new tab

   Relative expression of genes related to the MAPK signaling pathway in
   response to cold treatment. Error bars represent standard error, **
   denote highly significant differences (p < 0.01)

Discussion

   Calmodulin-like proteins (CMLs), ubiquitous calcium-binding sensors in
   eukaryotes, regulate plant growth, development, and responses to biotic
   and abiotic stress signals [[98]23]. In this study, 202 CACML genes
   were identified in C. alismatifolia. Each CACML gene possesses at least
   one EF-hand motif, conforming to the conserved structural
   characteristics of typical calmodulin-like proteins. With the
   increasing availability of high-quality plant genomes, the CML gene
   family has been systematically identified at the whole-genome level,
   revealing considerable variation in gene number across species: Glycine
   max (41 GmCMLs) [[99]24], common bean (111 PvCMLs) [[100]25], and
   peanut (191 AhCMLs) [[101]26]. The number of CML genes may depend on
   selective pressures and functional demands encountered during plant
   evolution [[102]27]. Given that most CACML genes also contain introns
   in their structure, it is speculated that CACMLs may have undergone
   distinct evolutionary pressures to facilitate adaptation and function
   in diverse ecological niches inhabited by C. alismatifolia.

   Chromosomal localization and subcellular prediction analyses revealed
   that 197 CACML proteins are unevenly distributed across C.
   alismatifolia chromosomes, with chromosome 1 harboring the highest
   number. This distribution pattern aligns with previously reported CML
   gene distributions in other plant species [[103]28]. Phylogenetic
   analysis using A. thaliana CML genes classified CACML genes into four
   distinct clades (Clade 1–4). Notably, Clade 4 lacked A. thaliana
   orthologs, indicating that dicotyledonous CML genes underwent
   functional diversification during evolution to adapt to environmental
   selection pressures and acquire novel functions. All four clades
   retained motif 2 and 4(Dx3D domain), with Clade 4 uniquely containing
   all 10 conserved motifs. This expanded motif repertoire likely
   contributes to specialized protein functions within Clade 4.

   Tandem duplication and segmental duplication are major factors driving
   the expansion of gene families [[104]29]. Genes arising from these
   duplication events frequently undergo subsequent sequence and
   functional divergence, enabling plants to adapt to diverse
   environmental challenges [[105]30]. In C. alismatifolia, 44 segmental
   duplication events and one tandem duplication pair (CACML132-CACML149)
   were identified. As observed in other monocots, segmental duplication
   serves as the primary driver for CML gene family expansion in
   monocotyledonous plants.

   Inter-species synteny analysis between C. alismatifolia and A. tsaoko,
   A. viiosum, and the eudicot model A. thaliana revealed distinct
   evolutionary patterns. Complex homologous relationships were observed
   with A. tsaoko and A. viiosum, yielding 161 and 148 syntenic gene
   pairs, respectively. In contrast, only three syntenic gene pairs were
   identified with A. thaliana, likely reflecting the distant evolutionary
   divergence between monocots and eudicots. tandem duplications in C.
   alismatifolia exhibited no syntenic relationship with those in A.
   tsaoko or A. viiosum, indicating independent evolution of these
   tandemly duplicated genes with potential species-specific functional
   innovations. Synteny of ATCML11 was further detected in other eudicots
   (Brassica oleracea [[106]31] and Phoebe bournei [[107]32]),
   demonstrating its ancestral conservation across monocot and eudicot
   lineages. The widespread occurrence of one-to-many or many-to-many
   homologies (e.g., CACML20 sharing two homologs in both A. viiosum and
   A. tsaoko) suggests these genes arose either from duplication events in
   a common ancestor or through independent convergent evolution. The
   conservation of such homologous genes may underpin essential biological
   functions conserved across Zingiberaceae species, including growth,
   development, and stress responses.

   Positive selection (Ka/Ks > 1) drives adaptive evolution in response to
   specific environmental pressures or ecological interactions.
   Conversely, purifying selection (Ka/Ks < 1) indicates conserved
   functional constraints [[108]33]. The majority of CACML genes in C.
   alismatifolia underwent purifying selection (Ka/Ks < 1), demonstrating
   strong functional constraints. This evolutionary pattern purging
   deleterious mutations and preserving gene function integrity suggests
   their involvement in fundamental biological processes essential for
   plant survival and reproduction [[109]34]. CML genes in plants such as
   Passiflora edulis [[110]35] and Ralstonia solanacearum [[111]26]
   consistently exhibit purifying selection. In contrast, CACML86 diverged
   from other CACML genes, experiencing positive selection in both C.
   alismatifolia and A. viiosum. Shared ecological niches or evolutionary
   histories may underlie this convergent adaptive trajectory in CACML86
   between the two species.

   Cis-acting element analysis of 2000 bp upstream promoter regions
   revealed diverse regulatory motifs in CACML genes. Abundant abiotic and
   biotic stresses and phytohormone responsive cis-acting elements suggest
   multifunctional roles in environmental adaptation and hormonal
   signaling. The prevalence of MYC cis-acting elements (1094),
   particularly in CACML187 (21), highlights its potential as a key
   regulator in cold stress response pathways [[112]36]. These elements
   likely activate downstream cold tolerance genes through MYC-mediated
   transcriptional regulation. Jasmonic acid responsive CGTCA-motifs (829)
   dominate hormone-related elements, indicating strong regulation by
   jasmonic acid signaling [[113]37]. This suggests CACML genes coordinate
   plant defense responses against herbivores, pathogens, and
   developmental processes involving jasmonic acid [[114]38].

   Drought, cold, and other abiotic stresses significantly impact the
   growth and development of C. alismatifolia [[115]39]. Understanding the
   gene regulatory network underlying its response to cold stress is
   crucial for its development [[116]40]. Cold stress induces dynamic
   changes in intracellular calcium ion (Ca^2⁺) concentration in plants.
   Calmodulin-like proteins (CMLs) act as calcium sensors [[117]41]. They
   bind Ca^2⁺ ions to form Ca^2⁺-CML complexes, which interact with
   downstream target proteins [[118]42]. These interactions modulate the
   expression of cold-responsive genes, thereby initiating the plant's
   cold stress response. AtCML9, 18, 24, and 37 are all involved in
   responses to abiotic stress in A. thaliana [[119]43]. Soybean GsCML27
   is induced by salt stress, and its heterologous expression enhances
   salt tolerance [[120]44]. Tomato ShCML44 is induced by multiple abiotic
   stresses, and overexpression of ShCML44 improves tolerance to both cold
   and salt stress [[121]45]. Collectively, these findings indicate that
   CML genes function as key regulators enhancing plant resistance to
   abiotic stress. In this study, transcriptomic analysis of C.
   alismatifolia under cold stress identified 6,909 DEGs. KEGG pathway
   enrichment analysis revealed significant enrichment in the MAPK
   signaling pathway and nitrogen metabolism. The plant MAPK signaling
   pathway is associated with signal transduction under cold stress. For
   instance, the Arabidopsis AtCRLK1-AtMEKK1/2-AtMPK4/6 cascade enhances
   cold tolerance by antagonizing the AtMPK3/6 pathway [[122]46], while
   Medicago truncatula MtCTLK1 or Medicago falcata MfCTLK1 influences cold
   resistance via the CBF transcriptional cascade, antioxidant defense,
   and proline accumulation [[123]47]. CACML140 exhibited the most
   significant differential expression and was enriched within the MAPK
   cascade signaling pathway. The above results indicate that CACML140
   regulates the cold tolerance of C. alismatifolia through the MAPK
   cascade signaling pathway, a conclusion consistent with validation by
   qRT-PCR.

Conclusion

   This study identified 202 CML genes in C. alismatifolia, all containing
   EF-hand domains. Based on phylogenetic relationships, they were
   classified into four distinct clades. Clade 4 contained the complete
   motif composition, and genes within the same subgroup exhibited similar
   exon–intron structures. Segmental duplication events were identified as
   the primary driver for the expansion of the CACML gene family. Most
   genes underwent purifying selection (Ka/Ks < 1). CACML86 experienced
   positive selection in both C. alismatifolia and A. viiosum, suggesting
   a potential shared evolutionary history between these species. Promoter
   analysis revealed that CACML promoters are enriched in cis-elements
   responsive to abiotic/biotic stress, plant hormones, and
   growth/development. RNA-Seq and qRT-PCR results indicated that C.
   alismatifolia may respond to cold stress via CACML140, potentially
   through the MAPK cascade signaling pathway. This research provides a
   theoretical foundation for deciphering the cold stress regulatory
   network in C. alismatifolia, particularly elucidating the regulatory
   role of the CACML gene family. The findings not only enrich the
   understanding of the evolutionary history and functional studies of
   plant CML gene families but also offer valuable candidate gene
   resources for the future development of cold-tolerant germplasm.

Supplementary Information

   [124]Supplementary Material 1.^ (23KB, xlsx)
   [125]Supplementary Material 2.^ (9.8KB, xlsx)
   [126]Supplementary Material 3.^ (28.7KB, xlsx)

Acknowledgements