Ca²⁺ is a ubiquitous second messenger in eukaryotes (Galon et al., 2010a) and acts as a signal to induce organisms to respond to environmental stress (Ikura et al., 2010; Kudla et al., 2010). In eukaryotes, some CaM binding transcription factors (Bouche et al., 2002; Yang and Poovaiah, 2002; Finkler, 2007) have been found. The N-terminal of CAMTA protein contains a CG-1DNA binding domain, a TIG domain, three anchor repeat sequences, and an IQ motif that can bind to CaM (Reddy et al., 2000; Bouche et al., 2002; Kunhua et al., 2006). CAMTA regulates gene expression by binding cis elements in the target gene promoter region (Yang and Poovaiah, 2002). CAMTA binding cis element was first found in Arabidopsis thaliana, and its sequence was (G/A/C) CGCG (C/G/T). The rice CAMTA recognition sequence is (A/C) CGTGT, which is different from Arabidopsis thaliana. Among them, (A/C) CGTGT also contains abscisic acid (ABA) response element (ABRE: ACGTGT) (Choi et al., 2005; Kaplan et al., 2007).

CAMTA also responded to auxin, ethylene, abscisic acid and salicylic acid (Yang and Poovaiah, 2002; Galon et al., 2008). In Arabidopsis thaliana, calmodulin binding transcriptional activator 1 (AtCAMTA1) plays a role in auxin signal transduction and also responds to drought stress by producing ABA (Pandey et al., 2013). Ca²⁺/calcium regulatory protein binding transcription factor AtSR1 (AtCAMTA3) acts as a regulator of salicylic acid mediated immune response by interacting with the promoter of EDS1 gene and inhibiting its expression (Du et al., 2009).

Maize is an important crop. Its growth suffers from various abiotic and biotic stresses, and the adaptive mechanism is an important basis for survival under these challenging environmental conditions (Verslues et al., 2006). In this study, we found ZmCAMTA gene in maize genome through bioinformatics methods, analyzed the physical and chemical properties of protein, phylogenetic relationship, gene structure, promoter elements, and combined with transcriptome data mining, found the expression of ZmCAMTA gene in different tissues and under stress, laying a foundation for further research on the function of ZmCAMTA gene.

1 Results and Analysis

1.1 Identification and structural analysis of maize CAMTA gene

Through bioinformatics technology, seven ZmCAMTA genes were found in maize genome and named ZmCAMTA1~7 respectively according to their positions on chromosomes. There are two ZmCAMTA genes (ZmCAMTA1-2) on chromosome 1, and one ZmCAMTA gene (ZmCAMTA3-7) on chromosomes 2, 5, 7, 9, and 10 (Figure 1A). ZmCAMTA protein 977~1034 aa, molecular weight 109.51~115.49 kD, isoelectric point 5.51~6.54. The prediction of instability index and GRAVY showed that ZmCAMTA proteins were all hydrophilic unstable proteins (Table 1). In order to further clarify the structural characteristics of ZmCAMTA, its protein conservative motifs and gene structure were analyzed. Through the analysis of Meme software, it is found that ZmCAMTA protein contains 10 conservative motifs, most of which are located at the C-terminal of the protein (Figure 1C). The motifs of the same subfamily have the same pattern. The analysis of conservative domains found that ZmCAMTA family members have similar conservative functional domains, including a CG-1 homologous DNA binding domain, a TIG domain (related to the specific DNA contact of transcription factors), an Ank domain (existing in the form of 33 amino acid repeats) and a CaM binding motif IQ motif (Figure 1B) from the N-terminal to the C-terminal.

Figure 1 1 Chromosome distribution, protein domain, conservative motifs and gene structure analysis of ZmCAMTA Note: A: Maize chromosomes are represented by vertical lines, the number on the left represents the chromosome number, and the seven ZmCAMTA genes are located on the chromosome; B: Schematic diagram of ZmCAMTA protein functional domai; C: Schematic diagram of the conserved motif of ZmCAMTA protein; D: Structural analysis of ZmCAMTA gene based on the maize genome annotation file GFF3. The scale in the figure represents the number of the amino acid or base sequence; the gene names are shown in Table 1

Table 1 Physiochemical properties of proteins encoded by ZmCAMTA

1.2 Phylogenetic analysis of ZmCAMTA gene

In Ensembl Plants (http://asia.ensembl.org/index.html), seven CAMTA protein sequences from rice and five CAMTA protein sequences from Arabidopsis thaliana were obtained on. Together with seven ZmCAMTA genes from maize, they were analyzed by using MEGA7 for phylogenetic tree analysis. The results showed (Figure 2) that CAMTA genes were divided into four subfamilies (from Ia to III). Three 1:1 orthologous gene pairs with high bootstrap value (ZmCMTA1/OsCAMTA2; ZmCAMTA5/OsCAMTA6 and ZmCAMTA6/OsCAMTA3) and two 2:1 orthologous gene pairs (ZmCAMTA2/ZmCAMTA4/OsCAMTA7 and ZmCAMTA3/ZmCAMTA7/OsCAMTA4) were found between maize and rice. Subfamily Ia is a monocotyledon specific subfamily, and no ZmCAMTA gene belongs to subfamily Ib.

Figure 2 Phylogentic tree of CAMTA gene family from Zea mays (Zm), Arabidopsis thaliana (At) and Oryza sative (Os)

1.3 Promoter specific cis acting elements of maize CAMTA family genes

In order to study the possible mechanisms of ZmCAMTA gene in response to biotic stress, abiotic stress and hormone signals, the PlantCARE database was used to analyze the cis acting elements of the 1500 bp regulatory region upstream of the translation start site of ZmCAMTA gene (Figure 3). The results showed that ZmCAMTA1 contained more abscisic acid (ABRE) response elements, ZmCAMTA2 contained more methyl jasmonate (CGTCA) response elements, ZmCAMTA6 contained the most cis acting elements (11), and ZmCAMTA7 contained the least cis acting elements (3).

Figure 3 Distribution of stress-related cis-elements in ZmCAMTA family gene promoters Note: The 1 500 bp promoter region of the ZmCAMTA gene is used to analyze the cis-acting elements associated with stress, and the cis-acting elements are indicated by the color code given. 10 homeopathic elements were used in this study: Abscisic Acid response element (ABRE), auxin response element (TGA-box), methyl jasmonate response element (CGTCA-motif; TGACG-motif), drought response element (MBS), gibberellin response Element (GARE-motif), CAMTA binding site (CG-motif), low temperature response element (LTR), hypoxia response element (ARE), salicylic acid response element (TCA-box).The scale in the figure represents the number of bases

1.4 Colinearity analysis of camta gene in maize

The collinearity relationship of CAMTA among maize, Arabidopsis thaliana and rice was analyzed (Figure 4). Only one ZmCAMTA homologous protein gene appeared in Arabidopsis thaliana chromosome, and ZmCAMTA1~7 homologous protein genes could find their collateral homologous genes on five rice chromosomes. There are five CAMTA family members in rice that have two homologous copies in maize, so CAMTA family may have genome wide polyploidy events in maize evolution.

Figure 4 New ICT based fertility management model in private dairy farm India as well as abroad

1.5 Analysis of ZmCMATA gene expression pattern

The determination of tissue specific expression pattern of ZmCAMTA gene can provide new insights into its role in different organs of maize. In this study, the expression level of ZmCAMTA gene in roots, stems, leaves and mature tassels of 7d maize seedlings in the maize transcriptome database was analyzed (Figure 5A). Except ZmCAMTA6, the expression of other ZmCAMTA genes in leaves and stems was lower than that in roots and tassels. Cold, high temperature and high salt are the main abiotic stress types that maize plants experience under various natural conditions. Gene transcription regulation related to Ca²⁺ signal is an important way for crop growth and adaptation to environmental stress. At the same time, the transcription level of ZmCAMTA gene under cold, high temperature and high salt stress was analyzed (Figure 5B). In cold treatment, the transcription levels of ZmCAMTA4 and ZmCAMTA6 increased 2 and 3 times, respectively; Under high salt treatment, most genes did not change significantly. Under heat treatment, the transcription levels of ZmCAMTA1, ZmCAMTA 2, ZmCAMTA 3, ZmCAMTA 5, and ZmCAMTA 7 genes decreased. These data indicate that ZmCAMTA gene is involved in the response to cold and high temperature stress. In heat sensitive and sheath rot resistant materials, the expression levels of ZmCMTA4 and ZmCAMTA6 increased, while other genes did not change significantly.

Figure 5 Gene expression analysis of ZmCAMTA Note: The color bar on the upper right indicates gene abundance; green indicates low expression, and red indicates high expression

2 Discussion

In this study, seven members of the ZmCAMTA gene family were identified. The high similarity between the sequence and structural pattern of ZmCAMTA protein shows that these ZmCAMTA genes can be derived from an ancestor sequence. Based on the ZmCAMTA protein sequence, a complete phylogenetic tree of rice, Arabidopsis thaliana and maize was established to analyze the relationship of CAMTA among the three species. According to phylogenetic analysis, five sister pairs between maize and rice were identified as orthologous genes, indicating that the functions of these ZmCAMTA may be similar to those of CAMTA in rice (Figure 2).

The spatial difference of CAMTA gene expression in plants is related to growth and development. During pollen development, AtCAMTA1 and AtCAMTA5 may enhance the pollen specific expression of AVP1 (Li et al., 2005). Overexpression of NtER1, a tobacco CAMTA homologous gene, shows senescent leaves and petals, which means that NtER1 participates in development regulation and shows senescence and death (Tian et al., 2000). Some CAMTA genes in tomato showed strong expression in fruit, indicating that their potential role was closely related to fruit development and maturity. The spatiotemporal expression pattern showed that most ZmCAMTA genes were highly expressed in roots. How ZmCAMTA gene plays a role in root system under various environmental stresses needs further research.

Environmental stress will lead to changes in gene expression (Schutzendubel et al., 2002; Aykinson et al., 2012). It is reported that CAMTA of different species can respond to a variety of environmental stresses, such as high salt, drought and heavy metal toxicity (Aykinson et al., 2012; Poovaiah et al., 2013). In addition, CAMTA gene is also involved in the relationship between environmental stress and stress related hormones (Reddy et al., 2000; Yang and Poovaiah, 2002). Cis element analysis showed that ZmCAMTA gene promoter contained several motifs related to environmental stress (Figure 3). It is worth noting that there are many elements related to environmental stress in the promoter region of ZmCAMTA4 and ZmCAMTA6, and the expression of these two genes changes most under stress treatment (Figure 5), which indicates the regulatory basis of these two stress expressions.

Through bioinformatics analysis of maize ZmCAMTA gene family, seven ZmCAMTA genes were found in maize genome, and their transcription levels under abiotic stress were analyzed, which provided a scientific basis for future research on ZmCAMTA transcription factor family.

3 Materials and Methods

3.1 Isolation and identification of maize CAMTA gene and analysis of its physical and chemical properties

In order to better identify CAMTA gene in maize genome: (https://www.arabidopsis.org) Arabidopsis thaliana CAMTA gene family protein sequence was obtained on MaizeGDB (https://www.maizegdb.org/) Obtain the B73 whole genome DNA sequence and GFF3 file on the. Use TBtools (Chen et al., 2020) to obtain the possible CAMTA gene in maize genome with Arabidopsis thaliana CAMTA gene. The obtained CAMTA gene family was further tested by blast on NCBI. By Utilizing online tool ProtParam (https://web.expasy.org/protparam/), the physicochemical properties of ZmCAMTA gene family members were analyzed.

3.2 Systematic evolution and domain analysis of ZmCAMTA protein

By using MEME (http://meme-suite.org/tools/meme), the conserved motifs of ZmCAMTA protein were analyzed by online tools. The parameters are set as follows: Select the site distribution: Zero or One Occurrence Per Sequence, Select the number of motives: 10, and the other parameters are set by default. By utilizing NCBI online tool Batch Web CD Search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi), the protein conservative domain of ZmCAMTA was analyzed, and by using TBtools, the conservative domain was visualized. The CAMTA gene family protein sequences of Arabidopsis thaliana and rice were located in Ensmbl (http://plants.ensembl.org/index.html) Database. ClusterW was used to conduct multiple alignments of protein sequences of CAMTA gene family member of three species. MEGA7 was used to construct the evolutionary tree by using the neighborhood joining (NJ) method, and 1000 bootstrap tests were conducted.

3.3 Chromosome distribution and intermediate collinearity analysis of ZmCAMTA family

One StepMCScanX module in Tbtools software was used to carry out pairwise comparison of protein sequences of maize and rice, maize and Arabidopsis thaliana, combine the whole genome chromosome position information of three species, use MCScanx to obtain interspecific collinearity relationship of ZmCAMTA family, and obtain ZmCAMTA chromosome position based on GFF3 file and display it with visual tools.

3.4 Analysis of ZmCAMTA gene structure and promoter elements

Based on the gene location information in the GFF3 file, analyze the gene structure of ZmCAMTA, and draw the gene structure map through Tbtools. Extract 1500 bp promoter sequence upstream of ZmCAMTA gene translation start site (ATG), and submit the sequence to PlantCARE (Magali et al., 2002) (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Carry out cis acting element analysis, and use Tbtools to visualize the position of cis acting elements.

3.5 ZmCAMTA gene expression analysis

Select four groups of transcriptome data of different parts of maize (root, stem, leaf, tassel) in MaizeGDB, and select transcriptome data of maize under cold, heat, salt stress and normal growth in NCBI SRA database (registration number: SRP061 276). The research team found two kinds of materials in the field, namely, heat sensitive materials and sheath rot materials. The transcriptome data were obtained through transcriptome sequencing technology to calculate the expression abundance value (TPM) of ZmCAMTA gene, respectively. The Amazing Heatmap module in TBtools software was used to draw a heat map with log2 TPM value.

Authors’ contributions

LWY was the experimental designer and executor of this research, who completed data analysis and wrote the first draft of the paper; DHW participated in the experimental design and analysis of the experimental results; HM was the designer and director of the project, guiding experimental design, data analysis, thesis writing and revision. All authors read and approved the final manuscript.

Acknowledgements

This research was jointly supported by the General Program of the National Natural Science Foundation of China (31771801; 32072069) and the Planned Project of Colleges and Universities of Hubei Province for Excellent Young and Middle Aged Science and Technology Innovation Team (T201704).

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