Systematic Analysis and Identification of Drought-Responsive Genes of the CAMTA Gene Family in Wheat (Triticum aestivum L.)

The calmodulin-binding transcription activator (CAMTA) is a Ca2+/CaM-mediated transcription factor (TF) that modulates plant stress responses and development. Although the investigations of CAMTAs in various organisms revealed a broad range of functions from sensory mechanisms to physiological activities in crops, little is known about the CAMTA family in wheat (Triticum aestivum L.). Here, we systematically analyzed phylogeny, gene expansion, conserved motifs, gene structure, cis-elements, chromosomal localization, and expression patterns of CAMTA genes in wheat. We described and confirmed, via molecular evolution and functional verification analyses, two new members of the family, TaCAMTA5-B.1 and TaCAMTA5-B.2. In addition, we determined that the expression of most TaCAMTA genes responded to several abiotic stresses (drought, salt, heat, and cold) and ABA during the seedling stage, but it was mainly induced by drought stress. Our study provides considerable information about the changes in gene expression in wheat under stress, notably that drought stress-related gene expression in TaCAMTA1b-B.1 transgenic lines was significantly upregulated under drought stress. In addition to providing a comprehensive view of CAMTA genes in wheat, our results indicate that TaCAMTA1b-B.1 has a potential role in the drought stress response induced by a water deficit at the seedling stage.


Introduction
Ca 2+ regulates many aspects of an organism's life cycle and functions as a signal messenger in response to environmental stimuli [1,2]. In plants, three major types of Ca 2+ -sensor proteins, including calmodulin (CaM)/CaM-like proteins, calcium-dependent protein kinases (CDPKs), and calcineurin B-like proteins (CBLs), are involved in the mechanism of Ca 2+ -dependent transcription regulation [3]. Additionally, Ca 2+ can bind to and control certain classes of transcription factors (TFs) [4]. For instance, calmodulin-binding transcription activators (CAMTAs) are well-studied CaM-binding TFs first identified in tobacco and later determined to exist in all multicellular organisms [5,6]. CAMTAs regulate more than 1000 genes in plants and are linked to environmental cues, with differential responses to various signals and stresses [5,7], as assumed to occur in other unknown gene families that are not yet investigated in many varieties of wheat [8,9]. Notably, CAMTA proteins participate in the rapid response to stress by efficiently transducing calcium signals [10]. Genetics data show that plant CAMTAs function in plant resistance against abiotic stresses by regulating many downstream genes [11][12][13][14][15][16][17][18]. name and ID, ORF length, number of amino acids and introns, predicted molecular weight (MW), isoelectric point (pI), and grand average of hydropathicity (GRAVY), are presented in Table S1. The length of predicted TaCAMTA proteins ranged from 138 (TaCAMTA5-B.2) to 1066 (TaCAMTA1b-A) amino acids, the molecular weight ranged from 16.21 (TaCAMTA5-B.2) to 119.32 (TaCAMTA1b-A.1) kDa, and pI ranged from 5.32 (TaCAMTA4a-D) to 9.57 (TaCAMTA5-B.2) ( Table S1).
The conserved domains of the CAMTAs, including a CG-1 DNA binding domain, a TIG domain, ankyrin repeats, IQ motifs, and CaMBD from the N terminus to the C terminus, were predicted in TaCAMTA proteins. TaCAMTA1b Table S2). All TaCAMTA proteins possessed at least four domains, except TaCAMTA5-B.2, which contained only a CG-1 DNA binding domain. TaCAMTAs contained a highly conserved domain structure and indicated that their function may be similar with multicellular organisms. Similar tertiary structures can reflect similar biological functions. We, therefore, predicted the tertiary structures for members of the CAMTA protein family via the SWISS-MODEL and Phyre2 online servers. The results indicated that CAMTA proteins mainly comprise α-helixes and random coils ( Figure 1C). Moreover, proteins in the same group were structurally similar, although some displayed β-sheets and certain variations in the random coils. Comparing the 3D structure and amino acid sequence of TuCAMTAs with TtCAMTAs, AetCAMTAs and TaCAMTAs showed that their core component appeared to be highly consistent, with all CAMTAs possessing at least four conserved domain structures, except for TaCAMTA5-B.2, which only had a CG-1 domain ( Figure S1). The evolutionary relationships among Triticum urartu Thüm. ex Gandilyan, Aegilops tauschii Coss, Triticum turgidum L., and common wheat with CAMTAs by examining 3D structure analysis can help to explain the origin and evolution of gene families and how they possess biological significance during species evolution [9,53,54].

Chromosomal Location of TaCAMTAs
Seventeen wheat CAMTA genes were mapped on different wheat chromosomes using the available wheat genome annotation information. The chromosomal locations of these genes were determined using gene IDs from Ensembl Plants ( Figure S2). There were 5, 7, and 5 CAMTA genes located on A, B, and D sub-genomes, respectively. Most genes were distributed in chromosomes 2A/B/D; the remaining chromosomes each contained one of the identified genes. By contrast, no CAMTA gene was found in chromosomes 1A/B/D, 5A/D, 6A/B/D, and 7A/B/D. Therefore, we conclude CAMTA genes were unevenly distributed on all wheat chromosomes, indicating that gene duplication events might have occurred in wheat chromosomes 2A/B/D during evolution.

Phylogenetic Analysis of TaCAMTAs
To further explore the phylogenetic relationships among various species, all CAMTAs were aligned using ClustalW, and the maximum likelihood (ML) method was used to construct a phylogenetic tree. All CAMTA proteins from Arabidopsis thaliana, Oryza sativa, Zea mays, Glycine max, and wheat were grouped into three subfamilies (sub.) based on their homology, except for TaCAMTA5-B.2. The classification resulted in 26 proteins in sub. I, 11 in sub. II, and 16 in sub. III ( Figure 2). Sub. I was also the largest subfamily with CAMTA proteins from five different plant species. TaCAMTA1a-A/B/D were closely related to OsCAMTA1a.1 and OsCAMTA1a.2, whereas TaCAMTA1b-A, TaCAMTA1b-B.1, TaCAMTA1b-B.2, and TaCAMTA1b-D were closely linked with OsCAMTA1b.1 and OsCAMTA1b.2. Three 3:1 ortholog gene pairs with 100% bootstrap values were identified between wheat and rice: TaCAMTA4a-A/B/D, TaCAMTA4b-A/B/D, and TaCAMTA5-A/B.1/D were classified into OsCAMTA4a, OsCAMTA4b, and OsCAMTA5, respectively. Orthologous gene pairs of CAMTA were identified in monocots; no sister pairs of genes were identified in dicots. In

Chromosomal Location of TaCAMTAs
Seventeen wheat CAMTA genes were mapped on different wheat chromosomes using the available wheat genome annotation information. The chromosomal locations of TaCAMTA1b-B.2, and TaCAMTA1b-D were closely linked with OsCAMTA1b.1 and Os-CAMTA1b.2. Three 3:1 ortholog gene pairs with 100% bootstrap values were identified between wheat and rice: TaCAMTA4a-A/B/D, TaCAMTA4b-A/B/D, and TaCAMTA5-A/B.1/D were classified into OsCAMTA4a, OsCAMTA4b, and OsCAMTA5, respectively. Orthologous gene pairs of CAMTA were identified in monocots; no sister pairs of genes were identified in dicots. In summary, CAMTA genes from different species shared evolutionary divergence before the common ancestor of monocots and dicots.   It has been reported that more stress-related cis-elements are located in the promoter regions of wheat CAMTA genes than other plant species [42,55]. Here, we screened for cis-elements in the 2.0 kb promoter regions of the regulatory sequences of wheat CAMTA genes. Most cis-elements shown in Figure 3, especially stress-related ones such as ABRE, MYB, MYC, G-box, and TGACG-motif, were enriched in the promoters of TaCAMTAs. In total, 38 ABRE, 24 MYB, 18 MYC, and 27 G-box cis-elements were predicted in TaCAMTA promoters, indicating that wheat CAMTA genes may be more involved in the plant's response to drought stress. regions of wheat CAMTA genes than other plant species [42,55]. Here, we screened for ciselements in the 2.0 kb promoter regions of the regulatory sequences of wheat CAMTA genes. Most cis-elements shown in Figure 3, especially stress-related ones such as ABRE, MYB, MYC, G-box, and TGACG-motif, were enriched in the promoters of TaCAMTAs. In total, 38 ABRE, 24 MYB, 18 MYC, and 27 G-box cis-elements were predicted in TaCAMTA promoters, indicating that wheat CAMTA genes may be more involved in the plant's response to drought stress.

Stage-and Tissue-Specific Expression Levels of TaCAMTA Genes
The determination of tissue-specific expression patterns of CAMTA genes could help us investigate their biological functions. We generated heatmaps of the expression levels of CAMTA genes in root, leaf, stem, and seed tissues using the WheatOmics platform, together with the iTAK and Expression Atlas databases. The results showed that the expression patterns varied between CAMTA genes. As shown in Figure S3, most orthologous genes in Arabidopsis, rice, and wheat exhibited similar expression patterns. The transcript levels of all the CAMTA genes were higher in the roots than in other organs, except for TaCAMTA5-B.2, OsCAMTA1a.2, and OsCAMTA1b.1. These results implicated that CAMTA family genes might have functional importance in root development.
We used qPCR to analyze the expression levels of TaCAMTA genes in the root and shoot of 2-week-old seedlings; in the root, stem, leaf, stamen, and spike at flowering during the reproductive stage; and in grains at 20 days after anthesis (during seed formation). Transcriptome profiling revealed diverse expression patterns, similarly to recent reports by Yang et al. [52]. Most CAMTA genes in wheat were predominantly expressed in shoots during the seedling stage, whereas higher expression levels were observed in leaves during the reproductive stage ( Figure 4). The overall expression, measured as gene expression in all organs at different developmental stages, produced inconsistent results, suggesting that TaCAMTA genes may play distinct roles during different stages of plant growth and development.
Transcriptome profiling revealed diverse expression patterns, similarly to recent reports by Yang et al. [52]. Most CAMTA genes in wheat were predominantly expressed in shoots during the seedling stage, whereas higher expression levels were observed in leaves during the reproductive stage ( Figure 4). The overall expression, measured as gene expression in all organs at different developmental stages, produced inconsistent results, suggesting that TaCAMTA genes may play distinct roles during different stages of plant growth and development.

Expression Profiles of Predicted CAMTA Sequences during Abiotic Stress
Extensive studies have shown that the expression of CAMTA genes responds to abiotic stress. To investigate the potential roles of TaCAMTAs in response to stresses and in the establishment of appropriate tolerance during the seedling stage, we analyzed the expression of 17 TaCAMTA genes in wheat seedlings subjected to salt, heat, drought, cold, and ABA stresses, as described in the Materials and Methods section. Our findings showed that all TaCAMTA genes responded to at least two stress-related treatments, with more than a two-fold change in expression. Under drought treatment, the expression of  Figure S4). Seedlings treated with NaCl showed no significant changes in the expression of most TaCAMTA genes, except for TaCAMTA1b-B.1/B.2, TaCAMTA4b-D, and TaCAMTA5-A/B.1, which increased to peak at 24 h post-treatment ( Figure S5). Additionally, most TaCAMTA gene expression patterns exhibited significant differences when subjected to ABA, heat, and cold (4 • C) stresses ( Figures S6-S8). These results indicated that TaCAMTAs responded to stresses, especially drought ( Figure 5). In seedlings, the rapid response of CAMTA genes to these external chemical and physical stimuli suggests that they participate in the cross-talk between multiple signal transduction pathways involved in stress tolerance. The relative expression levels in roots of two-week-old seedlings were normalized to the expression of the reference gene and set to 1 at 0 h. gene expression patterns exhibited significant differences when subjected to ABA, heat, and cold (4 °C) stresses ( Figures S6-S8). These results indicated that TaCAMTAs responded to stresses, especially drought ( Figure 5). In seedlings, the rapid response of CAMTA genes to these external chemical and physical stimuli suggests that they participate in the cross-talk between multiple signal transduction pathways involved in stress tolerance. The relative expression levels in roots of two-week-old seedlings were normalized to the expression of the reference gene and set to 1 at 0 h.

Figure 5.
TaCAMTA transcript levels in response to drought, cold, NaCl, ABA, and heat treatments. Venn diagram for upregulated (left) and downregulated (right) TaCAMTA genes after exposure to different abiotic stresses or ABA treatment. Each section of the diagrams shows genes that are specific for a single stress (drought, salt, heat, cold, or ABA) or that are shared across multiple conditions.

TaCAMTA1b-B.1 Regulates Prominent Drought-Responsive Genes
A preliminary investigation was undertaken to investigate the functions of the TaC-AMTA gene family in wheat that included the determination of expression patterns in seedlings subjected to stress conditions. Collectively, the data obtained suggested that TaCAMTA genes respond to various stresses, but especially to drought conditions. Transcripts of all TaCAMTA genes were modified under moderate dehydration during the seedling stage; the expression of TaCAMTA1b-B.1 markedly increased following drought stress, emphasizing that TaCAMTA1b-B.1 is a strong candidate to function in stress re- Figure 5. TaCAMTA transcript levels in response to drought, cold, NaCl, ABA, and heat treatments. Venn diagram for upregulated (left) and downregulated (right) TaCAMTA genes after exposure to different abiotic stresses or ABA treatment. Each section of the diagrams shows genes that are specific for a single stress (drought, salt, heat, cold, or ABA) or that are shared across multiple conditions.

TaCAMTA1b-B.1 Regulates Prominent Drought-Responsive Genes
A preliminary investigation was undertaken to investigate the functions of the TaCAMTA gene family in wheat that included the determination of expression patterns in seedlings subjected to stress conditions. Collectively, the data obtained suggested that TaCAMTA genes respond to various stresses, but especially to drought conditions. Transcripts of all TaCAMTA genes were modified under moderate dehydration during the seedling stage; the expression of TaCAMTA1b-B.1 markedly increased following drought stress, emphasizing that TaCAMTA1b-B.1 is a strong candidate to function in stress responses. We constructed overexpression and RNAi vectors and produced transgenic specimens in T2 generation to further understand the function of TaCAMTA1b-B.1. All transgenic lines were identified at the transcriptional level using qPCR ( Figure 6B).
We examined the growth response of transgenic plants to drought stress. No significant difference in the phenotype was observed among the RNAi, overexpression, and wild-type (WT) plants before drought treatment. However, after 2 weeks of drought treatment, the overexpression plants had more green leaves than WT plants, whereas the RNAi plants did not grow as well ( Figure 6A). The survival rate was higher in overexpression plants than in WT plants when exposed to drought conditions but lower in RNAi plants after re-watering ( Figure 6C). These data demonstrate that the TaCAMTA1b-B.1 gene positively regulates drought tolerance in transgenic wheat.
Plants with a high capacity for water retention can better survive drought or dehydration stress. The water loss from isolated leaves reflects, to a certain extent, the water status of the entire plant. During 0-24 h of a dehydration treatment, overexpression plants showed a low water loss rate (WLR), whereas RNAi plants had high WLR compared with WT leaves ( Figure 6D). These results indicated that TaCAMTA1b-B.1 promotes the plant's ability to retain water under dehydration conditions. We also determined that the MDA contents were lower in overexpression plants but higher in RNAi plants than in WT plants, with or without exposure to drought ( Figure 6E). These analyses confirmed that tolerance to dehydration increased in TaCAMTA1b-B.1-OE lines but decreased in TaCAMTA1b-B.1-RNAi lines.
The expression levels of drought stress-related genes, such as MYB33, NAC69-1, and NAC69-3, are used to assess the capacity of wheat plants to respond to drought conditions. Here, we measured the relative expression levels of these three genes in WT and transgenic plants grown under normal and drought conditions. The qPCR results showed that the expression levels of stress-related genes were upregulated in both WT and transgenic lines after drought treatment, but that they were significantly higher in overexpressed transgenic plants than in WT plants ( Figure 6F). Therefore, we conclude that TaCAMTA1b-B.1 may regulate stress-related genes to promote drought resistance in plants. Int sponses. We constructed overexpression and RNAi vectors and produced transgenic specimens in T2 generation to further understand the function of TaCAMTA1b-B.1. All transgenic lines were identified at the transcriptional level using qPCR ( Figure 6B). We examined the growth response of transgenic plants to drought stress. No significant difference in the phenotype was observed among the RNAi, overexpression, and wild-type (WT) plants before drought treatment. However, after 2 weeks of drought treatment, the overexpression plants had more green leaves than WT plants, whereas the RNAi plants did not grow as well ( Figure 6A). The survival rate was higher in overexpression plants than in WT plants when exposed to drought conditions but lower in RNAi plants after re-watering ( Figure 6C). These data demonstrate that the TaCAMTA1b-B.1 gene positively regulates drought tolerance in transgenic wheat.
Plants with a high capacity for water retention can better survive drought or dehydration stress. The water loss from isolated leaves reflects, to a certain extent, the water Data are presented as the mean ± SE of three different biological replicates and three technical replicates. The asterisks indicate a significant difference from control values at p < 0.05 (*) and p < 0.01(**), measured using the t-test.

Molecular Mechanisms of CAMTA Duplication
Gene duplication is one of the most important steps in the evolution of complex genes from simple ones [56]. A domain is a well-defined region within a protein that either performs a specific function within a protein [57] or induces modifications at the structural level via changes in nucleotide and amino acid sequence to influence the evolution of the protein [58]. Internal repeats often correspond to functional or structural domains within the proteins, and an exact correspondence exists between gene exons and the structural domains of the protein product [56]. The segments are structurally very complex (larger blocks composed of small units or modules of duplications), and the abundance of segmental duplications is one of the primary forces driving the evolution of genes with new functions. The association of these segments with chromosomal instability and the rapid evolution of new functions occur in tandem with duplication events and are documented as segmental duplications [59]. Bread wheat exhibits allopolyploidy (AABBDD) via hybridization followed by the merging of genomic content of diverging species, with gene expansion through polyploidization coming from whole-genome duplication and small-scale duplication [60]. Although the genome-wide identification of the CAMTA family has been completed in wheat [52], we identified 17 CAMTA genes in wheat and characterized two new members of the TaCAMTA family, TaCAMTA5 Figure 1). Notably, it is clear that TaCAMTA5 had orthologs in the A genome lineage (Triticum urartu), the D genome lineage (Aegilops tauschii), and tetraploid wheat (Triticum turgidum) ( Figure S1). TaCAMTA5-B.2 only contains the CG-1 domain (for any protein to be characterized as CAMTA, the presence of the CG-1 domain is obligatory [7]); however, TaCAMTA5-B.1 has multiple characteristic domains except for the CG-1 domain (the CaMBD domain is implicated in the association of Ca 2+ -loaded CaM to CAMTAs [7]). A previous study has reported the evolution and diversity of the CAMTA family from six Chlorophyta genomes and 35 plant genomes and showed that not every gene contained both a CG-1 domain and a CaMBD domain [61,62]. This evidence indicated the probable function of CAMTA proteins TaCAMTA5-B.1 and TaCAMTA5-B.2 ( Figure 1B).
We also explored whether TaCAMTA5-B.1 and TaCAMTA5-B.2 belong to the CAMTA gene family. We cloned the coding regions of the TaCAMTA5 genes, fused them with the pGBKT7 and pGADT7 vectors separately, and transformed each of these constructs individually into the yeast strain Y2HGold. The transformants carrying each construct grew well on selective SD medium lacking tryptophan (SD/−Trp or SD/−Trp−Leu). Transformants carrying TaCAMTA5-B.1 and TaCAMTA5-B.2 did not grow on SD/−Trp−His medium, indicating that these genes had no transactivation activity ( Figure 7A). In addition, TaCAMTA5-B.1 interacted with TaCaM, as confirmed by the yeast two-hybrid assay, whereas TaCAMTA5-B.2 interacted with neither TaCaM nor TaCAMTA5-B.1 ( Figure 7B). These results suggest that TaCAMTA5-B.1 and TaCAMTA5-B.2 may be involved in the Ca 2+ -CaM-CAMTA binding model and provided valuable insight into CAMTA with respect to its molecular functions.

Basic Characteristics of CAMTAs in Wheat
Phylogenetic analysis provided insight into the evolutionary relationships among the CAMTA family members across species, as well as putative functional assignments. Most CAMTA genes possess a conserved structure and a similar function [52]. Based on our phylogenetic analyses (bootstrap value = 100%), six sister pairs of genes between wheat and rice were identified as ortholog genes, suggesting that the functions of these TaCAMTAs might be similar to those of CAMTAs in rice. No sister pair of genes was identified between monocots and dicots ( Figure 2). A comparison of the CAMTA gene family among Triticeae species (e.g., Aegilops tauschii, Triticum urartu, Triticum turgidum, and hexaploid bread wheat) showed that TaCAMTA1b had no ortholog in the A genome lineage and that the 3D structure prediction, combined motif analysis, and sequence alignment exhibited similar patterns. Most CAMTA tertiary structures were homologous (especially in the A, B, and D sub-genomes) but structurally different in their progenitors ( Figure S1). These results indicate that intraspecies originate from whole-genomic duplication events and chromosomal arrangements from multiple evolutionary lineages through the combination of differentiated genomes. The evolution of CAMTA family members diverges across plant species [40]. Recently, CAMTAs from plant species were reported to play essential roles in abiotic stress responses [21]. Gene expression profiling in silico and qPCR analyses showed significant species specificity, tissue specificity, and developmental stage specificity at the transcription level (Figure 4 and Figure S3). The 17 TaCAMTAs were expressed differentially in various tissues subjected to hormone treatment or abiotic stresses (Figures S4-S8). Under drought stress conditions, TaCAMTAs were significantly expressed at all time points during the seedling stage. Moreover, several cis-acting elements known to be related to drought stress were discovered in the promoter region. These observations led us to hypothesize the involvement of TaCAMTAs in drought stress regulation. with the pGBKT7 and pGADT7 vectors separately, and transformed each of these constructs individually into the yeast strain Y2HGold. The transformants carrying each construct grew well on selective SD medium lacking tryptophan (SD/−Trp or SD/−Trp−Leu). Transformants carrying TaCAMTA5-B.1 and TaCAMTA5-B.2 did not grow on SD/−Trp−His medium, indicating that these genes had no transactivation activity ( Figure  7A). In addition, TaCAMTA5-B.1 interacted with TaCaM, as confirmed by the yeast twohybrid assay, whereas TaCAMTA5-B.2 interacted with neither TaCaM nor TaCAMTA5-B.1 ( Figure 7B). These results suggest that TaCAMTA5-B.1 and TaCAMTA5-B.2 may be involved in the Ca 2+ -CaM-CAMTA binding model and provided valuable insight into CAMTA with respect to its molecular functions.

Basic Characteristics of CAMTAs in Wheat
Phylogenetic analysis provided insight into the evolutionary relationships among the CAMTA family members across species, as well as putative functional assignments. Most CAMTA genes possess a conserved structure and a similar function [52]. Based on our

TaCAMTA1b-B.1 Regulates the Expression of Stress-Associated Genes in Response to Drought
TFs regulate virtually every aspect of the plant's response to different stresses. We previously demonstrated that TaCAMTA1b-B.1 is orthologous to AtCAMTA1, which is involved in stress response in Arabidopsis. The camta1 mutant showed drought sensitivity, and AtCAMTA1 regulates a broad spectrum of stress-inducible genes such as RD26, ERD7, RAB18, LTPs, COR78, CBF1, and HSPs [7,24]. In this study, we detected drought tolerance in TaCAMTA1b-B.1 overexpression lines but drought sensitivity in TaCAMTA1b-B.1 RNAi lines ( Figure 6A). TaMYB33, TaNAC69-1, and TaNAC69-3 overexpression in wheat positively regulated the drought stress response, and the water loss rate in transgenic plants was low compared with WT plants under drought treatment [63,64]. Similar results were obtained in our study; the expression levels of the drought-related genes MYB33, NAC69-1, and NAC69-3 were compared between transgenic and WT wheat plants under drought or normal conditions. We speculated that TaCAMTA1b-B.1 might improve the drought tolerance of wheat by regulating TFs related to drought stress through Ca 2+ -mediated signal transduction. Our results are consistent with those of previous studies, which reported that other CAMTA genes positively regulated drought stress tolerance [19,[32][33][34]37,[39][40][41]52]. There is much evidence to suggest that TaCAMTA1b-B.1 may function to improve the plant's ability to retain water under drought conditions. The results demonstrated that the TaCAMTA1b-B.1 gene could help reveal the molecular mechanism by which wheat induces a response to drought stress at seedling stage. However, there is no evidence that suggests that TaCAMTAs are induced by the drought stress of adult plants. Our study has provided useful information and insights into the functional divergence of CAMTA genes, which is worthy of further investigation. CAMTA genes are an appropriate candidate to be exploited as a plant genetic engineering tool in future studies.

Chromosomal Location and Sequence Analysis of CAMTA Genes
Each chromosomal position and gene length was extracted from the Ensembl wheat genome database (http://ensembl.gramene.org/Triticum_aestivum/Info/Index (accessed on 23 May 2020)) to confirm and indicate the locations of the CAMTA genes in the chromosome. Next, all data about the chromosomal position and length of CAMTA genes were uploaded to Map Gene2 Chromosome v2 (http://mg2c.iask.in/mg2c_v2.0/ (accessed on 24 May 2020)) to obtain specific chromosomal locations. The 2 kb upstream promoter sequences of all TaCAMTA transcripts were obtained from the Ensembl Plants database and submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 25 May 2020)) for sequence analysis and to predict and locate their cis-acting regulatory elements.

Multiple Sequence Alignment and Phylogenetic Analysis of CAMTA
The amino acid sequences from four plant species were aligned using ClustalW. The phylogenetic tree was constructed using the MEGAX software and maximum likelihood (ML) method with 1000 bootstrap replications. Furthermore, the phylogenetic tree was visu-ally enhanced using EvolView (https://www.evolgenius.info//evolview/#login (accessed on 26 May 2020))

Gene Structure and Conserved Motif Analysis of Wheat CAMTAs
The CDS and DNA sequences of TaCAMTAs were obtained from Ensembl Plants, and the gene structures were analyzed online via the Gene Structure Display Server v2.0 (GSDS) (http://gsds.cbi.pku.edu.cn/ (accessed on 27 May 2020)). In addition, the protein domain structures were obtained using the Domain Illustrator software (http://dog.biocuckoo.org/ (accessed on 28 May 2020)).

Vector Construction and Plant Transformation
The coding sequence of TaCAMTA1b-B.1 was amplified from leaf cDNA using the specific primers TaCAMTA1b-B.1-F and TaCAMTA1b-B.1-R to generate overexpression lines. Following sequence verification, the full-length CDS was introduced to the expression vector pUBI-GFP using an adaptor containing KpnI (NEB, Beijing, China) and BamHI (NEB, Beijing, China) digestion sites. RNAi lines were generated by cloning a 184 bp fragment amplified from TaCAMTA1b-B.1 cDNA into the pCUB vector through BamHI restriction sites in the sense orientation and SacI (NEB, Beijing, China) restriction sites in the antisense orientation. The resulting plasmids were transformed into Agrobacterium strain EHA105 and subsequently introduced into the wheat cultivar Jingdong18 (J18). Agrobacteriummediated transformation to obtain transgenic plants following the protocol described by Wang et al. [65,66]. The primers used for vector construction are listed in Table S3.

Plant Materials and Stress Treatments
Winter wheat cultivar J18 was obtained from Institute of Hybrid Wheat, Beijing Academy of Agriculture and Forestry Sciences (http://123.127.160.133/index (accessed on 25 April 2019)). Wheat plants were grown in hydroponic boxes containing sterile water in a suitable incubator (Panasonic, Ehime, Japan) for 2 weeks under the following conditions: 25 • C/12 h light and 15 • C/12 h darkness. The root and shoot of 2-week-old seedlings in the root, stem, leaf, stamen, and spike at flowering during the reproductive stage; and in grains at 20 days after anthesis (during seed formation), were used to analyze tissue specificity. Whole seedlings were subjected to different abiotic stresses and hormone treatments. Seedlings were subjected to salt (incubated in 200 mM NaCl), drought (incubated in 25% (w/v) PEG-6000), ABA (sprayed with 100 mM), and cold (grown at 4 • C) treatments for 0 h, 1 h, 2 h, 5 h, 10 h, and 24 h [67] and heat (grown at 40 • C) treatment for 0, 1/4, 1/2, 1, 2, and 4 h [68]. The shoot tissue was collected at each time point. No treated plants were used as controls.
Seeds were sown in pots containing soil for 2 weeks to study the response of wheat plants to drought stress (Approximately 12 seeds of each genotype were spread on six pots). The 2-week-old seedlings were subjected to drought conditions by withholding water for 14 days. Control plants were watered normally. After 14 days of drought stress, the plants were watered and allowed to recover for 10 days before observing their survival response. The water loss rate and MDA contents of the leaves were determined according to the method described by [69,70], and the experiment was repeated independently three times. All samples were immediately frozen in liquid nitrogen and stored at −80 • C for subsequent analysis.

RNA Isolation and Quantitative Real-Time RT-PCR
Total RNA was extracted using Trizol. First-strand cDNA was synthesized using the Takara RNA extraction kit (Cat. No. RR047A, Dalian, China) according to the manufacturer's instructions. SYBR qPCR Master Mix (Code. Q311-02, Nanjing, China) was used to perform RT-qPCR in Multiplate™ 96-well PCR plates (Bio-Rad, California, USA). Reaction systems were prepared in 20-µL volumes as follows: 10 µL of 2× Taq Pro Universal SYBR qPCR Master Mix, 1 µL of forward primer, 1 µL of reverse primer, 2 µL of cDNA, and 6 µL of RNase-free water. Each sample was tested using three technical replicates to ensure the accuracy of results.

Conclusions
In conclusion, 17 CAMTA genes have been identified and characterized in wheat. Our current research has provided new information of TaCAMTAs and described two new members of the family (TaCAMTA5-B.1 and TaCAMTA5-B.2). The responsiveness of TaCAMTA genes to various abiotic stresses and hormones during the seedling stage demonstrate that TaCAMTAs are mainly induced by the drought stress. In particular, TaCAMTA1b-B.1 played an important role in the drought stress response induced by a water deficit at the seedling stage. Further studies are still needed to reveal the functional significance of TaCAMTA genes in wheat, an important global crop with large and complex genome.