Background

Ca2+ signals, one of the most important secondary messengers in plants, are widely involved in many adaptive and developmental processes [1]. In plants, there are three main classes of Ca2+ sensors to decode and transmit the Ca2+ signals, including calmodulin (together with calmodulin-like proteins) (CaMs/CMLs), calcium-dependent protein kinases (CDPKs), and calcineurin B-like proteins (CBLs) [2]. Most of the calmodulin/calmodulin-like proteins execute their biological functions by binding to calmodulin-binding proteins (CaMBPs), including transcription factors, protein kinases, ion channels, and enzymes, with the exception of CaM7, which can act as a transcription factor to directly regulate the expression of the HY5 gene [3]. To our knowledge, plant CaMs can regulate at least 90 transcription factors, including calmodulin-binding transcription activators (CAMTAs) [4].

CAMTA proteins are characterized by several conserved domains, including a unique DNA-binding domain (CG-1), a transcription factor immunoglobulin-like DNA-binding domain (TIG), ankyrin repeats (ANK), IQ motifs (IQXXXRGXXXR), and a Ca2+-dependent calmodulin-binding domain (CaMBD) [5,6,7]. To date, the CAMTA gene family has been identified in a wide variety of plant species, such as Arabidopsis (Arabidopsis thaliana, six members) [7], tomato (Solanum lycopersicum, seven members) [8], rice (Oryza sativa, seven members) [9], grape (Vitis vinifera, 10 members) [10], maize (Zea mays, nine members) [11], soybean (Glycine max, 15 members) [12], rape (Brassica napus, 18 members) [13], alfalfa (Medicago sativa, seven members) [14], poplar (Populus trichocarpa, seven members) [4], and citrus (Citrus sinensis and Citrus clementina, nine members) [15].

CAMTAs have been shown to be extensively involved in plant growth and developmental regulation, as well as in biotic and abiotic stress tolerance. In Arabidopsis, CAMTA1 and CAMTA2 work in concert with CAMTA3 to directly bind to the promoter of C-repeat binding factor2 (CBF2) to induce expression, leading to increased plant freezing tolerance [16, 17]. While AtCAMTA1 also positively regulates drought responses by regulating a few stress-responsive genes, including responsive to dehydration26 (RD26), early response to dehydration7 (ERD7), responsive to ABA18 (RAB18), lipid transfer proteins (LTPs), cold-regulated78 (COR78), CBF1, and heat shock proteins (HSPs) [18], AtCAMTA3 can act as a negative regulator of plant immunity to modulate pathogen defense responses by activating the EDS1-mediated salicylic acid (SA) signaling [19]. A recent study showed that TaCAMTA4 may function as a negative regulator of the defense response against Puccinia triticina, since the virus-induced gene silencing (VIGS)-based knockdown of TaCAMTA4 resulted in the enhanced resistance to P. triticina race 165 [20]. This suggested that one CAMTA member usually participates in multiple signaling pathways, while multiple CAMTA members often work together to participate in one signaling pathway.

Here, we obtained 15 TaCAMTA genes from wheat genomes. Their chromosome location, physicochemical properties, subcellular localization, gene structure, protein domain, promoter cis-elements, and expression profiles in multiple tissues as well as in response to stresses were systematically analyzed. Our work has established a foundation for the further analysis of wheat CAMTA genes and provides a basic understanding of their roles in development and stress responses.

Results and discussion

Identification of the TaCAMTA gene family in wheat

Using the method described below, a total of 15 TaCAMTA genes were identified in wheat. Since the TaCAMTA genes were clustered into six homoeologous groups, these genes were designated as TaCAMTA1 to TaCAMTA6 according to their homology with rice CAMTA genes, plus a suffix corresponding to the specific wheat genome identifier (A, B, or D) for each gene name (Table 1, Fig. 1). For example, the TaCAMTA1 genes in genomes A, B, and D were named TaCAMTA1-A, TaCAMTA1-B, and TaCAMTA1-D, respectively. The results showed that TaCAMTA1, 2, 3, and 4 contained three homolog genes (TaCAMTA1-A/B/D, 2-A/B/D, 3-A/B/D, and 4-A/B/D), while TaCAMTA5 harbored two (TaCAMTA5-A/D), and TaCAMTA6 possessed one (TaCAMTA6-B). The highest number (eight genes: TaCAMTA3-A/B/D, 4-A/B/D, and 5-A/D) of TaCAMTA genes was found in homoeologous group 2, three TaCAMTA genes (TaCAMTA1-A/B/D) in homoeologous group 3, three TaCAMTA genes (TaCAMTA2-A/B/D) in homoeologous group 4, and one TaCAMTA gene (TaCAMTA6-B) in homoeologous group 5, while no TaCAMTA gene was identified in homoeologous groups 1, 6, and 7. Information relating to the 15 TaCAMTA genes, including gene names, locus IDs, open reading frame (ORF) lengths, chromosome locations, and the deduced polypeptides is provided in Table 1. The predicted TaCAMTA proteins contain 805 (TaCAMTA1-B) to 1067 (TaCAMTA2-B) amino acid residues, with molecular weights ranging from 90.82 kDa (TaCAMTA1-B) to 119.32 kDa (TaCAMTA2-A), and the isoelectric points ranged from 5.14 (TaCAMTA4-B) to 8.96 (TaCAMTA5-A) (Table 1).

Table 1 Information of the 15 CAMTA gene members in wheat
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Fig. 1
figure 1

Phylogenetic relationships of the CAMTA homologs in different species

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The size of the CAMTA gene family in wheat is similar to that of oilseed rape (B. napus) and soybean (G. max) with 18 and 15 members [12, 13], respectively, but is higher than that of A. thaliana with six members, citrus (C. sinensis and C. clementina) with nine members, maize (Z. mays) with nine members, and alfalfa (M. truncatula) with seven members [5, 11, 14, 15]. The higher number of CAMTA genes may be due to gene duplication during chromosome polyploidization, since oilseed rape and soybean are tetraploid, whereas wheat is allohexaploid (AABBDD).

The subcellular locations were predicted with Plant-mPLoc. According to the results, all 15 wheat CAMTA proteins were located in the nucleus, which corroborates recent studies where the CAMTA proteins have typically been located in the nucleus [4, 21], confirming that their main function is to regulate the expression of other genes as transcription factors.

Phylogenetic analysis of the TaCAMTAs

To investigate the phylogenetic relationships of the CAMTA gene families, a phylogenetic tree of CAMTAs from five species, including wheat, Triticum urartu, Aegilops tauschii, A. thaliana, and rice, was constructed using the neighbor-joining (NJ) algorithm. The CAMTA gene families were highly conserved during the evolution of these species (Fig. 1). All of the 36 proteins from the five species were distinctly clustered into three groups (groups A, B, and C). Seven wheat CAMTAs (TaCAMTA2-A/−B/−D, 3-A/B/D, and 6-B), one T. urartu CAMTA (TuCAMTA3), two Ae. tauschii CAMTAs (AetCAMTA2, and 3), three rice CAMTAs (OsCAMTA2, 3, and 6), and three Arabidopsis CAMTAs (AtCAMTA1, 2, and 3) were clustered into group A. In addition, six wheat CAMTAs (TaCAMTA1-A/−B/−D, and TaCAMTA4-A/−B/−D), two T. urartu CAMTAs (TuCAMTA1, and 4), two Ae. tauschii CAMTAs (AetCAMTA1 and 4), two rice CAMTAs (OsCAMTA1, and 4), and one Arabidopsis CAMTA (AtCAMTA4) grouped into group B, while two wheat CAMTAs (TaCAMTA5-A/−D), one T. urartu CAMTA (TuCAMTA5), one Ae. tauschii CAMTA (AetCAMTA5), one rice CAMTA (OsCAMTA5), and two Arabidopsis CAMTAs (AtCAMTA5 and 6) clustered into group C.

An unrooted phylogenetic tree was constructed using MEGA-X with the NJ algorithm and 1000 bootstrap replicates. The bootstrap values are displayed next to the branches, and the wheat CAMTAs are marked in red. The CAMTA gene ID numbers are listed as follows: A. thaliana: AtCAMTA1 (AT5G09410), AtCAMTA2 (AT5G64220), AtCAMTA3 (AT2G22300), AtCAMTA4 (AT1G67310), AtCAMTA5 (AT4G16150), AtCAMTA6 (AT3G16940); rice: OsCAMTA1 (LOC_Os01g69910), OsCAMTA2 (LOC_Os03g09100), OsCAMTA3 (LOC_Os07g43030), OsCAMTA4 (LOC_Os04g31900), OsCAMTA5 (LOC_Os07g30774), OsCAMTA6 (LOC_Os10g22950); T. urartu: TuCAMTA1 (TRIUR3_22499-P1), TuCAMTA3 (TRIUR3_23792-P1), TuCAMTA4 (TRIUR3_26386-P1), TuCAMTA5 (TRIUR3_19786-P1); Ae. tauschii: AetCAMTA1 (XP_020189402), AetCAMTA1 (XP_020179695), AetCAMTA1 (XP_020196708), AetCAMTA1 (XP_020147564), and AetCAMTA1 (XP_020186933).

Gene architectures and protein domain structures of the TaCAMTA members

The number of introns in all of the 15 TaCAMTA genes varied from 10 to 13, in which three CAMTA genes (TaCAMTA1-A/D and 6-B) possessed 10 introns, four CAMTA genes (TaCAMTA1-B and 4-A/B/D) possessed 11 introns, six CAMTA genes (TaCAMTA2-A/B/D and 3-A/B/D) possessed 12 introns, and two CAMTA genes (TaCAMTA5-A/D) possessed 13 introns (Fig. 2). Similar genomic structures of the CAMTA genes have been observed in other plant species, suggesting the conservation of CAMTA genes across plant species [8, 11, 12, 21].

Fig. 2
figure 2

Gene architectures of the TaCAMTA genes

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The exon-intron structures of the TaCAMTA genes were analyzed by comparing the coding sequences and the corresponding genomic sequences using the Gene Structure Display Server (GSDS, http://gsds.cbi.pku.edu.cn/). The black box indicates exons, and the black line indicates introns.

Ten TaCAMTA proteins (TaCAMTA2-A/B/D, 3-A/B/D, 4-A/B/D, and 6-B) were predicted to contain all of the conserved domains of a typical CAMTA protein, including a CG-1 DNA-binding domain (Pfam03859), a TIG domain involved in non-specific DNA binding (Pfam01833), several ankyrin repeats (Pfam12796), an IQ motif (Pfam00612), and a calmodulin-binding domain (CaMB) (Fig. 3). Additionally, five TaCAMTA proteins (TaCAMTA1-A/B/D and TaCAMTA5-A/D) contained all of the conserved domains except for the TIG domain, which is consistent with previous studies that CAMTAs can be divided into two groups based on whether the TIG domain is present [22].

Fig. 3
figure 3

Protein domain structure of the TaCAMTAs

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It has been confirmed that the IQ motif is able to bind with CaM in a Ca2+-independent manner, while the CaMB domain interacts with CaM in a Ca2+-dependent way [5, 7, 8]. It is interesting to note that all the wheat CAMTAs contain the IQ motif and a CaMB domain, indicating that wheat CAMTAs may interact with CaM in both a Ca2+-dependent and Ca2+-independent manner.

Analysis of the functionally conserved domains was performed using the Pfam database (http://pfam.janelia.org/) and NCBI Conserved Domains Search online tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). CaM-binding domains (CaMBD) were analyzed in the Calmodulin Target Database (http://calcium.uhnres.utoronto.ca/ctdb/ctdb/). The domain structures of the TaCAMTAs were illustrated using TBtools software. CG-1, CG-1 DNA binding domain; TIG, TIG domain involved in non-specific DNA binding, ANK, ankyrin repeats responsible for mediating protein-protein interactions; IQ, Ca2+-independent CaM-binding IQ motifs; CaMBD, Ca2+-dependent CaM binding domain.

Cis-acting regulatory elements in the promoters of the TaCAMTAs

Several stresses/stimuli response cis-acting elements in the promoter regions (2000 bp upstream of the translation start site ATG) of the 15 TaCAMTA genes were predicted. Seven cis-elements were used in this study: abscisic acid (ABA)-responsive element (ABRE: ACGTG, ACGTSSSC, or MACGYGB) [23], SA-responsive promoter element (SARE: TGACG) [24], environmental signal response element (G-box: CACGTG) [25], WRKY binding site (W-box: TTGAC, or TGACC/T) [26, 27], phosphate starvation-responsive element (P1BS: GNATATNC) [28], sulfur-responsive element (SURE: GAGAC) [29], and the CAMTA binding site (CG-box: A/C/GCGCGG/T/C) [5].

The results showed that there were various known stresses/stimuli-related cis-acting elements that existed in the promoter regions of the 15 TaCAMTA genes. ABRE, SARE, W-box, and CG-box could be found in the promoter of all the 15 TaCAMTA genes, and four TaCAMTAs (TaCAMTA1-D, 3-B, 4-A, and 4-D) contained all seven types of cis-elements in the promoter region, including ABRE, SARE, G-box, W-box, P1BS, SURE, and CG-box. Meanwhile, the remainder of the 11 TaCAMTA genes contained at least five cis-elements in their promoter region (Table 2). It has been reported that more stress-related cis-elements are located in the promoter regions of wheat CAMTA genes than other plant species [13, 14], indicating that wheat CAMTA genes may be more widely involved in the plant response to stress.

Table 2 Numbers of stress-related cis-elements in the promoter regions of the TaCAMTA genes
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Tissue-specific expression patterns of the TaCAMTA genes

To elucidate the possible functions of the TaCAMTA genes in wheat, qRT-PCR assay was performed to investigate the spatial expression patterns of the TaCAMTAs. The results showed that all of the 15 TaCAMTA genes were expressed in multiple tissues with different expression levels. TaCAMTA3-D, 5-A, and 5-D showed highest expression level in shoot during seedling stage, while highest expression level of TaCAMTA1-D and 3-B was observed in spike during reproductive stage, suggesting that various CAMTA gene members maintain different functions in wheat growth and development (Fig. 4).

Fig. 4
figure 4

Expression patterns of the TaCAMTA genes in multiple tissues

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Expression of TaCAMTAs were analyzed by qRT-PCR in root and shoot of ten-day-old seedlings, root, stem, leaf, spike at flowering in reproductive stage, and grain 15 DAA (days after athesis). The relative expression levels were normalized to 1 in roots of ten-day-old seedlings (0 h).

Expression profiles of the TaCAMTA genes during abiotic stress

Previous studies have shown that plant CAMTAs could be involved in diverse environmental stresses. AtCAMTA1 and SlSR1L played a positive function in drought stress in Arabidopsis and tomato [18, 30], while plant CAMTAs also respond to salt and cold stress [11, 16, 31]. However, to date there is no information available on wheat CAMTAs involved in abiotic stresses. In this light, the expression profiles of the TaCAMTAs were analyzed under drought, NaCl, cold and heat stress. Under drought stress, TaCAMTA1-A, 1-B, 1-D, 2-B, 4-B, 4-D, 5-A, 5-D and 6-B were significantly up-regulated, while the expressions of TaCAMTA2-A, 2-D, 3-A, 3-D were moderately down-regulated (Fig. 5a). In response to NaCl stress, the expressions of TaCAMTA1-A, 1-D, 5-A, 5-D and 6-B were enhanced, while the expressions of TaCAMTA2-A, 2-B, 2-D, 3-A, 3-B, 4-A, 4-B, 4-D were inhibited (Fig. 5b). In the cold treatment assay, the expressions of TaCAMTA1-A, 1-D, 3-A, and 3-D increased dramatically, while the expressions of TaCAMTA2-A, 4-A, 4-B, and 4-D decreased (Fig. 5c). In the heat treatment group, the expressions of TaCAMTA1-A, 1-B, 1-D, 2-A, and 4-B remarkably increased within one hour; by contrast, the expressions of TaCAMTA2-B, 2-D, 3-B, 4-A, 5-A, 5-D, and 6-B were repressed, especially in the late stage of heat treatment (Fig. 5d).

Fig. 5
figure 5

Expression profiling of the TaCAMTA genes under abiotic stress

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It can been found that the expression of each TaCAMTA gene could respond to at least one abiotic stress, and TaCAMTA1-A and 1-D could be up-regulated by all abiotic stresses used in this study, including drought, NaCl, cold and heat stress (Fig. 5), implying different regulations and functions of TaCAMTA gene members while coping with various abiotic stresses in wheat. It can also been found that the CAMTA genes from same homoeologous group showed similar expression patterns, such as TaCAMTA1-A/B/D under drought treatment (Fig. 5a), TaCAMTA5-A/D under NaCl treatment (Fig. 5b), and TaCAMTA1-A/B/D under heat shock stress (Fig. 5d). However, several homoeologous CAMTA genes from same group showed different expression patterns under stresses. For example, TaCAMTA1-A/D and TaCAMTA3-A/D were up-regulated by cold treatment, while the expressions of TaCAMTA1-B and TaCAMTA3-B were relatively stable (Fig. 5c). These results suggest that the homoeologous CAMTA genes from the same group generally have the same regulations and functions, while functional differentiation may have occurred in some homoeologous CAMTA genes.

Expression of TaCAMTAs were analyzed by qRT-PCR in roots of ten-day-old seedlings, which had been treated with 16.1% PEG 6000 (drought), 200 mM NaCl, 4 °C (cold) and 40 °C (heat) for indicated durations. The relative expression levels were normalized to 1 in unstressed plants (0 h).

Prediction of target genes by CAMTA

It has been found that CAMTA has the specific binding activity to CGCG box in promoter of target genes [5]. In this study, a search of the data base revealed that cis-acting elements ACGCGG/CCGCGT were present in the promoter regions of about 584 genes (more than two copies) in wheat genome, which were considered as potential target genes by CAMTA (Additional file 1:Table S1). These genes are related to RNA regulation (69 genes), protein degradation (42 genes), signalling transduction (30 genes), biotic and abiotic stresses (17 genes), hormone metabolism (17 genes), and lipid metabolism (13 genes), demonstrating that CAMTA can be widely involved in plant development and growth, as well as coping with stresses.

Conclusions

In conclusion, 15 CAMTA genes were identified in wheat in the present study. Analysis of the gene structure and protein domain, physicochemical properties, and the phylogenetic relationships indicated that the CAMTA gene family was highly conserved during plant evolution. Tissue-specific expression analysis showed that all of the 15 TaCAMTA genes were expressed in multiple tissues with different expression levels, suggesting that various CAMTA gene members maintain different functions in wheat growth and development. Under abiotic stress, the expressions of all the TaCAMTA genes could respond to at least one abiotic stress, implying different regulations and functions of TaCAMTA gene members while coping with various abiotic stresses in wheat. 584 genes in wheat genome were predicted to be potential target genes by CAMTA, demonstrating that CAMTA can be widely involved in plant development and growth, as well as coping with stresses. Our findings provide new insight into the CAMTA gene family in wheat as well as a foundation for further studies on the roles of TaCAMTA genes in wheat development and growth as well as the stress response.

Methods

Genome-wide identification of the CAMTA gene family

Protein sequences of Triticum aestivum (IWGSC1.1), Triticum urartu (ASM34745v1), and Aegilops tauschii (ASM34733v1) were obtained from the Ensemble plant database (http://plants.ensembl.org) to predict the CAMTA genes [32]. The Hidden Markov Model (HMM) profile of the CG-1 domain (PF03859), the ANK repeat domain (PF00023), and the IQ domain (PF00612) sequences were downloaded from the PFAM database [33] and used to examine all wheat protein sequences using the HMMER search tool with E-value <= 0.0001 [34]. The obtained protein sequences were checked using the National Center for Biotechnology Information (NCBI) - Conserved domain database (CDD) search (https://www.ncbi.nlm.nih.gov/cdd) to identify the conserved protein domain with the default parameters. The redundant sequences containing complete CG-1, ANK repeats, and the IQ domain were further removed by alignment, and the remainder were considered as putative CAMTA genes. Finally, the biochemical parameters of the TaCAMTA proteins were calculated using the Compute pI/MW tool in the ExPASy database with the default parameters (https://web.expasy.org/compute_pi/). Subcellular localization of the TaCAMTA proteins was predicted online by Plant-mPLoc with the default parameters (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) [35].

Phylogenetic tree construction and sequence analysis

Protein sequences from Arabidopsis and rice were obtained from NCBI (http://www.ncbi.nlm.nih.gov/) and Ensembl Plants (http://plants.ensembl.org/index.html). The amino acid sequences of all CAMTAs were aligned using the ClustalX program with the default parameters, and a phylogenetic tree was constructed in MEGA-X using the neighbor-joining method with 1000 bootstrap replicates [36]. The display of the phylogenetic tree was optimized using the Interactive Tree Of Life (iTOL) v4 [37].

The schematic structures of the TaCAMTA genes were analyzed online using the Gene Structure Display Server 2.0 based on exon/intron data (GSDS 2.0, http://gsds.cbi.pku.edu.cn/) [38]. The domain structures of the TaCAMTA proteins were analyzed in the Pfam database (http://pfam.janelia.org/) and NCBI Conserved Domains Search online tool against database CDD v3.18 with E-value threshold <= 0.01 (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) [39]. The CaMB domain was specifically analyzed using Calmodulin Binding Site Search in the Calmodulin Target Database (http://calcium.uhnres.utoronto.ca/ctdb/ctdb/).

Prediction of cis-acting elements in the TaCAMTA genes

To investigate the cis-elements in the promoter sequences of the TaCAMTA genes, 2-kb sequences upstream of the initiation codon (ATG) were collected from the Ensembl plant database (http://plants.ensembl.org) and subjected to the PLACE database with the default parameters (https://www.dna.affrc.go.jp/PLACE) [32, 40].

Plant materials and treatments

Jinhe 9123, a wheat variety cultivated by our lab, was used in this study. For stress treatment experiments, seeds were surface-sterilized in 1% NaOCl and repeatedly rinsed with tap water, then seeded in 1/2 Hoagland nutrient solution after immersion and imbibition for 12 h [41], and hydroponically cultivated in the incubator with a 16/8-h photoperiod at 25 °C. Four ten-day-old homogeneous seedlings groups, each of which included 30 seedlings from three biological replicates, were subjected to different treatments, including 16.1% PEG6000, 200 mM NaCl and cold (4 °C) for 1, 3, 6, 12, 24 and 48 h, and heat (40 °C) for 10, 20, 30, 60, 120, 180 min. The shoot tissue was sampled for the experiment. For analyses of tissue-specific expression patterns, root and shoot from ten-day-old seedlings, root, stem, leaf, spike at flowering in reproductive stage, and grain 15 DAA were collected from wheat plants. Collected samples were immediately frozen in liquid nitrogen and stored at − 80 °C for RNA extraction.

RNA isolation and gene expression analysis

Total RNA of collected samples was isolated using the EasyPure® Plant RNA Kit (ER301–01, Transgen, China). For reverse transcription, the first-strand cDNA was synthesized using a PrimeScript™ RT reagent Kit (RR047A, TaKaRa, Japan). Quantitative real-time PCR (qRT-PCR) for examination of the TaCAMTAs expression patterns were performed using the TB Green™ Premix Ex Taq™ II (RR820A, TaKaRa, Japan) with 7500 Real-Time PCR System (Applied Biosystem, USA). Gene specific and internal reference gene TaActin primers were listed in Additional file 2: Table S2. The qRT-PCR program was carried out as follows: predenaturation at 95 °C for 30 s; denaturation at 95 °C for 5 s, annealing at 58 °C for 30 s, extension at 60 °C for 34 s, 45 cycles. 2−ΔΔCt method was used to analyze the data [42]. All experiments were performed with three technical replicates and three biological replicates, and the data were represented by mean value of three biological replicates.

Prediction of target genes by CAMTA

Prediction of the target genes by CAMTA were performed as described by Yang and Poovaiah (2002) with some modifications [5]. 1-kb sequences upstream of the initiation codon (ATG) of all genes in wheat genome were collected as promoter sequences, and a search of cis-acting elements ACGCGG/CCGCGT (CGCG box) were conducted. The genes with more than two copies of CGCG box were considered as potential target genes by CAMTA. The MapMan tool was used to facilitate the assignment of different gene sets into functional categories (BINs). A MapMan mapping file that mapped the genes into BINs via hierarchical ontologies through the searching of a variety of reference databases was generated using the Mercator tool (http://mapman.gabipd.org/web/guest/app/mercator) [43].