The published CMLs gene sequences in Arabidopsis and rice were obtained from the TAIR database (https://www.arabidopsis.org) (accessed on 04 December 2024) and the TIGR database (http://rice.plantbiology.msu.edu) to identify members of the CMLs gene family in the tobacco genome. Then, 32 OsCMLs and 50 AtCMLs proteins were used as query sequences to perform BLASTP search (E-value < e − 5), and the redundant and repetitive sequences were removed manually. Meanwhile, the HMMER (PF13499) was utilized as a keyword in the databases above to conduct searches. The NCBI Conserved Domain Database (http://www.ncbi.nlm.nih.gov/cdd) (accessed on 04 December 2024), InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan5/) (accessed on 04 December 2024), and SMART (http://smart.embl-heidelberg.de/) (accessed on 04 December 2024) were used to predict the structural domains of EF-hands, eliminating protein sequences that lack EF-hands or contain other functional domains. Additionally, AtCaM2 was used to guarantee the NtCMLs by acting as usual CaMs and ensuring that the amino acid identity was less than 80%. The nucleotide and predicted amino acid sequences of the discovered genes, which were named NtCML1 through NtCML75, were used for additional investigation.

ExPASyProtParam (http://web.expasy.org/protparm/) (accessed on 04 December 2024) was used to estimate the physicochemical properties of NtCMLs, such as the number of amino acids, theoretical point (pI), grand average of hydropathicity (GRAVY), and aliphatic index. SMART was used to forecast the number of EF-hands (https://smart.embl.de/smart/set_mode.cgi?NORMAL=1) (accessed on 04 December 2024). Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) (accessed on 04 December 2024) was used to predict subcellular localization.

MEME (http://meme-suite.org/index.html) (accessed on 04 December 2024) was used to study the conserved domains, and ten motifs were chosen. The exon and intron structures of NtCMLs were ascertained using the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/) (accessed on 04 December 2024).

Using 1000 bootstrap replicates and the neighbor-joining method in MEGA7, the phylogenetic tree was constructed for evolutionary analysis. The NtCML proteins were classified based on the evolutionary relationships of 50 AtCML proteins. Evolutionary tree landscaping can be achieved using the website ChiPlot (https://www.chiplot.online/) (accessed on 04 December 2024).

PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) (accessed on 04 December 2024) was used to examine the 2000 bp upstream sequences of NtCMLs genes to identify the cis-acting elements in the promoter region of NtCMLs genes. The Simple BioSequence Viewer from TBtools is used to visualize.

The online website mg2c (http://mg2c.iask.in/mg2c_v2.1/) (accessed on 04 December 2024) was utilized to visualize the chromosomal position. TBtools software was used to examine the homologous relationship of NtCML genes. The Multiple Collinearity Scan toolbox (MCScanX) examined gene duplication occurrences.

Tobacco K326 tissue data from 19 tissues was downloaded from the EMBL-EBI website. The corresponding gene expression profile data were obtained by comparing the sample numbers of NtCMLs gene family members. The data was plotted using the Heat Map tool in the software TBtools.

The tobacco cultivar ‘Yunyan87’ was sown in soil and allowed to grow at room temperature (26°C) until the plants had six or seven leaves. The plants were then divided into two groups. The P seedlings were grown in an artificial climate chamber and were treated with cold acclimation therapy for three days at a temperature of 12°C (night/day). The other group of seedlings that were not cold-acclimated is known as N. The N and P seedling groups were treated normally for 7 days and recorded as NCT and PCT. NCT and PCT seedlings were put through cold treatment (8°C) for three days and recorded as NCL and PCL. Each treatment involved the collection of three biological samples, which were immediately frozen in liquid nitrogen and stored at −80°C until they could be examined further.

The Spin Column Plant Total RNA Purification Kit (Sangon Biotech, Shanghai, China) was used after the frozen samples had been thoroughly ground to powder in liquid nitrogen. 1% agarose gel electrophoresis was used to assess the quality of the RNA. The All-In-One 5X RT Master Mix (ABM, Shanghai, China) is then used for the cDNA synthesis. The qPrimerDB qPCR Primer Database online resource was utilized to design the primers used in the RT-qPCR. Table 1 lists the particular primers used for qRT-PCR.Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was carried out in real-time utilizing a CFX-1000 Real-Time System (BioRad) and SYBR Premix Ex Taq II (TaKaRa, China). Three duplicates of the real-time PCR were conducted. We used the N, NCT, and NCL treatments as the baseline to calculate P, PCT, and PCL expression. The N, NCT, and NCL treatments were utilized as a baseline to calculate P, PCT, and PCL expression. The e 2^−ΔΔCt technique was used to analyze relative expression [31]. The data shows the average of three biological replicates.

A network of 75 NtCMLs protein sequences was analyzed using the STRING online server (https://cn.string-db.org/) (accessed on 04 December 2024).

SPSS26.0 data processing software is utilized to perform variance analysis and significance experiments on experimental data. Origin2021 software is employed to perform mapping analysis on experimental data.

A total of 75 NtCMLs genes were retrieved from the genome of Tobacco and were named in the order of their chromosomal locations (NtCML1 to NtCML40 and NtCML41 to NtCML75) (Table 2). The results were confirmed by analyzing the deduced peptides using Pfam, InterProScan, and SMART databases. Physicochemical property analysis showed that the number of amino acids in the NtCMLs proteins ranged from 79 AA (NtCML39) to 283 AA (NtCML6) and the pI ranged from 3.97 (NtCML41) to 9.63 (NtCML10). The Aliphatic index ranged from 58.10 (NtCML29) to 104.84 (NtCML57). The NtCMLs shared 23%~78% identity with AtCaM2. Most NtCMLs proteins contained two to four EF-hand domains. The GRAVY values of most NtCMLs proteins were negative, indicating that NtCMLs proteins in cucumber are hydrophilic. Subcellular localization demonstrates that most NtCMLs are found in the cell membrane and cytoplasm.

Motifs are important in identifying Transcription Factor Binding Sites, which helps understand the mechanisms that regulate gene expression [32]. The MEME tool was employed to locate conserved motifs for a more thorough examination of the NtCML proteins (Fig. 1A). Motif 1 and motif 2 were present in all 75 NtCMLs family members. Motifs 3, 4, 5, 6, and 8 are most commonly observed in the N-terminus and motifs 1, 2, and 7 are present in the C-terminus. Some paralogous proteins contained different motifs, such as NtCML14 and NtCML54, NtCML12 and NtCML31, NtCML6 and NtCML9, NtCML41 and NtCML50, while NtCML18 and NtCML59, NtCML38 and NtCML49, NtCML48 and NtCML74, NtCML11 and NtCML37, NtCML40 and NtCML55 had the same motif. Motif 9 is exclusively in NtCML3 and NtCML27.

Most NtCMLs belong to the PTZ00184 superfamily, which is also known as the EF-hand protein superfamily (Fig. 1B), Introns can increase transcript levels, and exons as enhancers are crucial in protein synthesis [33,34]. We analyzed the exon-intron structure of NtCMLs genes to describe their conservation and differences (Fig. 1C). According to the findings, NtCMLs contain between one and eight exons, and the most of their members lack introns.

To further comprehend the links between these compounds, phylogenetic analysis was performed using the recovered tobacco protein sequences, we constructed the phylogenetic tree with 125 NtCMLs protein sequences, including 75 sequences from tobacco (NtCMLs) and 50 from Arabidopsis (AtCMLs). These NtCMLs protein were classified into Five subgroups (Group I–Group V) (Fig. 2). The smallest subgroup was Group I which consisted of 4 CMLs (3 AtCMLs and 1 NtCMLs). The largest subgroup was Group V which consisted of 49 CMLs (19 AtCMLs and 30 NtCMLs). Group II included 18 CMLs members (3 AtCMLs and 15 NtCMLs). The Group III included 17 CML members (7 AtCMLs and 10 NtCMLs). The Group IV included 37 CMLs members (18 AtCMLs and 19 NtCMLs). Furthermore, the majority of NtCML was homologous to Arabidopsis. The findings suggest that CMLs are conserved across all plant species.

Gene expression is significantly influenced by parallel trans-regulatory factors and cis-elements. To better understand transcriptional and functional control of the NtCMLs genes, the extraction of 2000-bp upstream sequences of the NtCMLs coding areas was performed by analyzing cis-acting elements (Fig. 3). The primary cis-acting factors linked to plant growth and development, biotic and abiotic stressors, light, and hormones (Fig. 4). Among the elements that respond to plant hormones are abscisic acid (ABRE), methyl jasmonate (CGTCA-motif, TGACG-motif), ethylene (ERE), and salicylic acid (TCA). Abiotic and biotic stressors such as low temperature (LTR), drought (MBS), anaerobic (ARE), and W-box are associated with the element type. Other components of the stress response have also been identified, such as the light element G-box and the trauma response element WUN-motif. The cis-element analysis highlighted the regulatory complexity of the NtCMLs gene family, suggesting that the most of NtCMLs genes are essential for plant development and growth, as well as hormone and stress responses.

The protein sequences of the discovered NtCMLs were used to determine their chromosomal locations. The findings revealed that tobacco has 18 chromosomes, which contain 40 NtCMLs genes (Fig. 5). Particularly, chromosome 17 has the most genes, followed by chromosome 12, which contains five NtCML genes, and chromosome 4 contains four NtCML genes. Ten Chromosomes contain only one NtCMLs genes (Chromosomes 1, 2, 5, 6, 8, 9, 13, 14, 15, 24). The results demonstrated that NtCMLs were randomly distributed across different chromosomes.

To determine the NtCMLs gene duplication of the NtCMLs genes, the segmental duplication events in the NtCMLs gene family were conducted. 12 NtCMLs genes were predicted to be segmentally duplicated on chromosomes 3, 8, 12, 15, 17, 19 and 22. The tobacco genome include seven segmental duplicated gene pairs and two tandemly duplicated gene pairs (Fig. 6). These duplicate genes are probably caused by intra- or inter-chromosomal segmental duplication of other genes.

A comparative analysis of the genomes of tobacco and Arabidopsis was conducted to gain a better understanding of the evolution of NtCMLs genes (Fig. 7). 22 pairs of orthologous genes were present between tobacco and Arabidopsis, there was one-to-many or many-to-one collinearity between the NtCMLs and the AtCMLs (Table 3). For instance, four genes (NtCML5, MtCML28, NtCML25, NtCML26) had two homologous genes in Arabidopsis, while one gene (NtCML27) had three orthologous genes. The existence of ancient gene pairs during the divergence of Arabidopsis and tobacco has been demonstrated, and their functions may be similar.

To determine the possible roles of NtCMLs, the public transcription data for several tobacco K326 tissues, including seed, shoot, flower, stem, and root was downloaded. The different expression patterns in most of the 19 tissues and developmental stages were analyzed (Fig. 8). Some NtCMLs genes exhibited a tissue-specific expression pattern. Eight NtCMLs genes (NtCML19, NtCML41, NtCML50, NtCML73, NtCML6, NtCML10, NtCML38, NtCML49) were highly expressed in the seed, NtCML21, NtCML24, NtCML66, NtCML8, and NtCML29 were highly expressed in flowers, three NtCMLs genes (NtCML70, NtCML36, NtCML47) were highly expressed in young shoot. Furthermore, some NtCMLs genes showed similar expression patterns, including NtCML75 and NtCML30, NtCML41 and NtCML50, NtCML62 and NtCML4, NtCML54 and NtCML14. The findings suggested that these genes may have similar functions in plant growth and development.

Ten NtCMLs genes were examined for their expression in response to cold acclimation (Fig. 9). The transcript levels of NtCML8, NtCML18, NtCML12, NtCML23, NtCML27, and NtCML28 were significantly upregulated following exposure to cold conditions. In contrast, transcripts for NtCML22 and NtCML53 showed a significant reduction. However, no significant changes were observed in the expression of NtCML30 and NtCML3 under cold stress when compared with the other genes. After 7 days of cold acclimation, we found that NtCML3, NtCML12, NtCML18, NtCML27, and NtCML28 were still significantly upregulated, while NtCML8 and NtCML30 were significantly down-regulated. However, NtCML22, NtCML23, and NtCML53 had no significant change. However, when exposed to low-temperature stress once more, NtCML3, NtCML12, NtCML22, NtCML18, NtCML23, NtCML27, NtCML28, NtCML30, and NtCML53 significantly upregulated. These findings imply that the NtCMLs gene has distinct functions in the cold acclimation recovery process.

In this study, 75 NtCMLs proteins were analyzed using STRING to predict the protein interaction network in tobacco (Fig. 10). 13 NtCMLs proteins were identified as participating in the interaction network, and four CMLs proteins exhibited correlations with more than four other CMLs. Notably, CML46 was associated with ten NtCMLs proteins. The protein-protein associations suggested that some NtCMLs proteins are likely co-expressed based on findings from Arabidopsis research. NtCML4, NtCML59, and NtCML2 demonstrated a close protein interaction and exhibited potential co-expression and co-occurrence patterns. The analysis of the protein interaction network indicated that NtCMLs regulate downstream gene expression through interactions with other proteins, thereby providing a valuable resource for further research.