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).
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].
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 .
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) , SA-responsive promoter element (SARE: TGACG) , environmental signal response element (G-box: CACGTG) , WRKY binding site (W-box: TTGAC, or TGACC/T) [26, 27], phosphate starvation-responsive element (P1BS: GNATATNC) , sulfur-responsive element (SURE: GAGAC) , and the CAMTA binding site (CG-box: A/C/GCGCGG/T/C) .
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.
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).
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).
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 . 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.