Flowering is an important growth period in the life cycle of plants, which is used to determine the adaptability of plants to special geographical environment and ensure the yield of cereal crops (Putterill et al., 2004; Blümel et al., 2015). CCT genes family can affect the flowering time of plants through photoperiod and vernalization, and regulate the process of plant growth and development (Yan et al., 2013; Liu et al., 2018). Based on different conserved domains, CCT genes family can be divided into three categories: COL (CONSTANS-like) subfamily, PRR (Pseudo response regulator) subfamily and CMF (CCT motif) subfamily (Cockram et al., 2012). COL subfamily contains one or two B-box domains and one CCT domain, PRR subfamily contains one CCT domain and one REC domain, and CMF subfamily contains only one CCT domain (Zhang et al., 2015). Interspecific/intraspecific phylogenetic analysis of COL, CMF and PRR proteins in gramineous plants showed that they evolved for 200 million years before the divergence of dicotyledons and monocotyledons, and the continuous evolution of COL gene led to the degradation and disappearance of B-box conserved domains, the number of which was from 2 to 1, and then to none (Cockram et al., 2012).

Nowadays, CCT genes family has been widely studied in Arabidopsis thaliana and Oryza sativa. For example, CO, TOC1, COL1, COL2 and COL3 in Arabidopsis thaliana have been cloned (Wu et al., 2014), and Hd1, Ghd7, OsCO3, OsCOL4, DTH2 and Ehd4 have been found in Oryza sativa. (Doi et al., 2004). Most of the genes regulating plant flowering contain CO (CONSTANS), COL (CO-LIKE) and TOC1 motifs. CO is a COL subfamily gene and the first cloned CCT gene controlling flowering in Arabidopsis thaliana. The C-terminal of CO protein contains a motif composed of 43~45 amino acid residues, which plays an important role in flowering regulation of Arabidopsis thaliana (Robson et al., 2001; Wenkel et al., 2006). Studies in Oryza sativa have shown that genes containing CCT domain not only regulate the flowering of Oryza sativa, but also play an important role in rice heading stage. For example, as a homologous gene of CO, Hd1 not only has the dual function of regulating flowering (Yano et al., 2000), but also its expression is up-regulated under the influence of OsCOL4, which can delay heading (Lee et al., 2010). DTH2 and Ehd4 regulate heading stage through MADS-box transcription factor under different light conditions, which improves the adaptability of Oryza sativa in different regions (Zheng et al., 2017). Besides affecting the flowering of Oryza sativa through ELF (Early flowering) genes family, Ghd7 also affects the plant height and panicle length of Oryza sativa (Saito et al., 2012). Photoperiod insensitivity of PRR subfamily members, Ppd-A1, Ppd-B1 and Ppd-D1 can lead to early heading of wheat (Kiseleva et al., 2017). In addition, the CCT gene in Medicago truncatula is also involved in hormone response, and participates in abiotic stress by regulating the expression of ABA pathway gene, so as to protect plants from abiotic damage (Ma et al., 2020); These CCT family genes regulate flowering, indicating that the functions of these genes are conservative in different species, but different CCT family genes also have specific functional differentiation between species and populations.

Drought and high temperature have a significant impact on the whole growth process of wheat (Triticum aestivum L.) and threaten the yield and quality of wheat to a great extent. Due to the huge wheat genome, there are few studies on wheat CCT genes family, and there are few reports on the stress related research of wheat CCT genes family. In this study, the genome-wide identification of wheat CCT family genes was carried out by bioinformatics, and the evolutionary tree was constructed by using the CCT genes identified from Triticum aestivum, Arabidopsis thaliana and Oryza sativa. The gene structure, conserved domain, chromosome distribution, cis-elements and expression pattern under stress were further analyzed to know more about the characteristics of wheat CCT genes family, and provide a reference for the mechanism of wheat CCT genes family and the improvement of wheat quality.

1 Results and Analysis

1.1 Identification of wheat CCT genes family and prediction of physical and chemical properties

Through two comparative searches, 118 CCT candidate genes were screened in the wheat genome. After that, these candidate genes were submitted to SMART, Pfam, NCBI-CDD and other online websites for structural verification of the conserved domain. Finally, 80 wheat CCT genes were obtained and named according to the location of the genes on the chromosome and their homologous relationship. The physical and chemical properties of the protein encoded by wheat CCT genes were further analyzed (Table 1). The results showed that the longest CCT protein TaCCT21-D contained 520 amino acid residues, and the shortest TaCCT13-D contained 170 amino acid residues; The relative molecular weight was 19.48 (TaCCT13-D)~57.95 kD (TaCCT21-D); The theoretical isoelectric point (pI) ranged from 4.23 (TaCCT34-D) to 10.82 (TaCCT13-B). At the same time, it was found that TaCCT proteins with pI<7 reached more than 80% (65), indicating that most TaCCT proteins were rich in acidic amino acids. The CELLO online website was used for subcellular localization analysis, it was found that most wheat CCT proteins were located in the nucleus, and a few were located in chloroplast (6), cytoplasm (3) and extracellular (1).

1.2 Analysis of phylogeny, gene structure and conserved domain

In order to clarify the classification of wheat CCT family members, the protein sequences of 23 Arabidopsis thaliana CCT genes and 27 Oryza sativa CCT genes were compared with wheat TaCTT protein, and the evolutionary tree was constructed. The results showed that wheat TaCCT gene had high homology with Arabidopsis thaliana and Oryza sativa, indicating that the species differentiation of CCT gene was very conservative, and the similarity of protein sequences showed that the CCT genes of the three species may have similar biological functions. According to the classification of homologous genes of Arabidopsis thaliana and Oryza sativa, the wheat CCT family was divided into three subfamilies: COL, CMF and PRR (Figure 1; Figure 2I). The COL subfamily included four subgroups A, B, C and D, with a total of 36 CCT genes. The CMF subfamily included eight subgroups E, F, G, H, I, J, K and M, with a total of 41 CCT genes, while the PRR subfamily included subgroup L, with only 3 CCT genes. Among all subgroups, subgroup A had the largest number of TaCCT genes, with 22 members, while subgroup F had the least, with only 2 members. According to phylogenetic tree analysis, CCT gene was not only found in Triticum aestivum, Arabidopsis thaliana and Oryza sativa, but also had high homology, indicating that CCT gene was highly conservative in the process of species differentiation, and the evolution time was earlier than the differentiation time of Triticum aestivum, Arabidopsis thaliana and Oryza sativa.

MEME software was used to analyze the composition and number of conserved motifs of wheat CCT genes family. A total of 10 conserved motifs were identified and named as motif 1 to motif 10 (Figure 2Ⅲ). The conserved motifs contained in each subgroup were basically the same, indicating that the same subgroup was composed of similar conserved domains and may have similar biological functions. Conserved motif 3 appeared in all TaCCT genes, conserved motif 2 appeared in all COL subfamily genes, conserved motif 4 and motif 10 only appear in PRR subfamily at the same time, subgroup F contained no other motifs except motif 3, and conserved motif 8 only appeared in subgroup G. Motif 1 and motif 2 together constituted the B-box domain, which may have protein interaction (Cockram et al., 2012); Motif 3 constituted the conserved domain of CCT, which was speculated to have the function of regulating flowering (Zheng et al., 2017); Motif 4 and motif 10 together constituted the REC domain. The above showed that these motifs can be used as markers to identify different subfamilies, and TaCCT gene has internal differentiation in the process of evolution, which may lead to functional differentiation.

1.3 Chromosome location and gene replication analysis

According to the chromosome annotation information of wheat genome, the identified TaCCT gene was carried out chromosome location (Figure 3). The results showed that 80 TaCCT genes were unevenly located on different chromosomes, of which 7D chromosome contained the largest number of genes, with 7 TaCCT genes; 3A, 3B and 3D contained the least, and all contained only one TaCCT gene. In addition, the three chromosomal groups of wheat were also unevenly distributed, including 28 A genome, 24 B genome and 28 D genome, suggesting that TaCCT gene had been copied or lost in the process of wheat evolution. The results of gene replication showed that a total of 64 TaCCT genes formed 60 homologous gene pairs, which showed that gene replication played an important role in the evolution and member expansion of TaCCT genes family.

1.4 Analysis of cis-elements

This study analyzed the cis-elements of TaCCT gene by using the genome sequence of 1 500 bp upstream of wheat CCT gene, and sorted out the cis-elements related to hormones and abiotic stress (Figure 4). The results showed that the types and number of elements related to hormone response are the most, with a total of 11 kinds, such as ABA related element ABRE element, gibberellin related element GARE-motif element, P-box element and TATC-box element, auxin related element AuxRR-core element, TGA-box element and TGA-element, MeJA related element CGTCA-motif element and TGACG-motif element, salicylic acid related element TCA-element and SARE element. Further study found that 86.25% of TaCCT genes had ABA related regulatory element ABRE, indicating that TaCCT family genes may be widely involved in ABA metabolic pathway. In addition, there were five elements related to abiotic stress, such as MBS in response to drought, LTR in response to low temperature, DRE in response to low temperature, dehydration and salt stress, and GC-motif in response to hypoxia. TaCCT family genes have a variety of cis-elements related to hormone and abiotic stress, suggesting that TaCCT family genes participate in the growth of plant through different physiological processes such as hormone regulation pathways and abiotic stress response.

1.5 Expression pattern analysis of wheat CCT genes family

In order to explore the potential biological function of CCT gene in the process of wheat growth, the high-throughput data of four different tissues of wheat root, stem, leaf and grain was used for expression pattern analysis in this study (Figure 5A). Most CCT genes in wheat were expressed in tissues, and the expression difference was obvious. For example, compared with other tissues, TaCCT3-B and TaCCT3-D had the highest expression in grains, TaCCT6-B, TaCCT6-D, TaCCT11-A, TaCCT11-B, TaCCT11-D, TaCCT15-A, TaCCT15-B, TaCCT15-D, TaCCT19-A and TaCCT19-D had the highest expression in leaves, and TaCCT24-A, TaCCT21-B and TaCCT21-D had the highest expression in stems. The above results suggested that different TaCCT genes may be involved in different growth processes of wheat.

In order to better understand the biological function of TaCCT gene under abiotic stress, NCBI Short Read Archive database was used to analyze the expression pattern of wheat CCT genes family under drought stress, heat stress and combined stress in this experiment (Figure 5B). The expression of most CCT family genes in wheat changed in varying degrees after drought and heat stress. For example, the expression of TaCCT15-A, TaCCT15-B and TaCCT15-D were significantly down-regulated after 6 h of heat stress and drought-heat combined stress, and the expression of TaCCT32-B and TaCCT8-A were significantly up-regulated after 6 h of drought-heat combined stress. In addition, the expression levels of TaCCT21-A, TaCCT21-B, TaCCT21-D, TaCCT24-A and TaCCT24-D were significantly up-regulated after 6 h of drought stress, heat stress and combined stress, indicating that these five genes were sensitive to drought and heat stress. Some genes with similar evolutionary relationship had similar expression patterns under abiotic stress. For example, TaCCT6-A, TaCCT6-B and TaCCT6-D were up-regulated after 1 h of drought stress and down-regulated after 6 h of drought stress.

2 Discussion

So far, CCT genes family has been identified in many species. Among them, there were 41 in Oryza sativa, 23 in Arabidopsis thaliana (Zheng et al., 2017), 53 in Zea mays (Jin et al., 2018) and 36 in Medicago sativa (Ma et al., 2020). In this study, a total of 80 wheat CCT genes were identified at the genome-wide level, which was far more than that of other species. This may be that wheat is a heterohexaploid, which has experienced two rounds of natural doubling of three diploid ancestral species in the process of evolution, doubling the number of wheat CCT genes. The subcellular localization results of TaCCT gene showed that most wheat CCT proteins were located in the nucleus and a small part was located in chloroplast, cytoplasm and extracellular, indicating that wheat CCT proteins may perform different functions in the process of growth. In addition, the isoelectric point range of TaCCT gene was 4.23~10.82. More than 80% of wheat CCT proteins were mainly acidic proteins, which may play a role in acidic subcellular environment, which was basically consistent with the protein properties of ZmCCT members in Zea mays (Guo et al., 2019). Phylogenetic analysis divided 80 wheat CCT genes into three subfamilies, among which CMF subfamily was the largest subfamily, including 41 CCT members. PRR subfamily was the smallest subfamily, with only 3 CCT members, which was consistent with the distribution characteristics of TaCCT genes in different subfamilies in plants (Zheng et al., 2017; Ma et al., 2020). Gene structure and conserved motif analysis showed that there were great differences among CCT genes of each subgroup, but the structure of the same subgroup was relatively conserved. Conserved motif analysis showed that motif 1 and motif 2 together formed the B-box domain, motif 3 formed the CCT conserved domain, and motif 4 and motif 10 together formed the REC domain. Among them, the B-box domain belonged to a kind of zinc finger protein, which may participate in protein interaction. This structural diversity may lead to functional differentiation to better adapt to the changes of the external environment (Liu et al., 2018). In addition, the number of wheat CCT genes in three different subgroups A, B and D was different, and the number of CCT genes in wheat D genome was also different from that in wheat D genome donors - Aegilops tauschii (26) identified by predecessors (Zheng et al., 2017), indicating that the CCT gene had been retained and lost in wheat evolution. The gene replication results showed that the CCT gene of wheat had replicated between different chromosomes in the process of wheat evolution. Combined with (Figure 1; Figure 2I), it was found that these homologous gene pairs had a good genetic relationship in the evolutionary relationship.

CCT family genes are also widely involved in plant growth. For example, ZmCOL3 can inhibit the flowering of Zea mays by interfering circadian rhythm and activating the expression of other CCT genes (Jin et al., 2018); VERNALIZATION2 (VRN2) is a homologue of Ghd7 in wheat, which can delay the flowering time of wheat and protect winter wheat from freezing injury (Yan et al., 2004). At the same time, CCT family genes can also regulate plant photosynthesis by regulating chlorophyll biosynthesis (Liu et al., 2020). In this study, expression pattern analysis found that wheat TaCCT gene showed tissue specificity. For example, TaCCT3-B and TaCCT3-D were highly expressed in grains, while TaCCT21-B, TaCCT21-D and TaCCT24-A were most expressed in stems, indicating that different TaCCT genes may play a role in different tissues of wheat. In addition, CCT family genes also respond to abiotic stress. For example, the CCT family genes of Aegilops tauschii were widely involved in hormone responses such as gibberellin, auxin and methyl jasmonate (Zheng et al., 2017); COL4 in Arabidopsis thaliana participated in salt stress response through abscisic acid depending on signal transduction pathway (Min et al., 2015); OsGhd2 in Oryza sativa can not only control the number of grains, heading date and plant height, but also endow it with drought tolerance by accelerating premature aging caused by drought (Liu et al., 2016). The analysis of abiotic stress expression pattern showed that wheat TaCCT gene was also involved in the response to drought, high temperature and drought-heat combined stress. For example, TaCCT15-A, TaCCT15-B and TaCCT15-D were down-regulated after 6 h of drought-heat combined stress, while TaCCT8-A and TaCCT32-B were up-regulated after 6 h of drought-heat combined stress. Cis-element analysis showed that there were cis-elements related to hormone and abiotic stress response in the promoter region of TaCCT gene. For example, 86.25% of TaCCT genes had elements ABRE related to ABA regulation, MBS related to drought response, LTR related to low temperature response and so on. These results suggested that TaCCT was widely involved in the growth of wheat and in response to abiotic stress.

Wheat is one of the most important food crops in China and even the world. Strengthening the basic research of wheat is of far-reaching significance to ensure food security and plant research. Abiotic stresses such as drought and high temperature have seriously affected the growth of wheat, resulting in a sharp decline in wheat yield and quality (Zheng et al., 2019). The research results in recent years showed that (Guo et al., 2019) plant CCT genes play an important regulatory role in the flowering process and multiple hormone responses. However, many regulatory mechanisms in CCT gene family are still unclear, especially there are few reports about CCT genes family in wheat. Through the analysis of the gene structure, cis-elements and the expression under drought and high temperature stress of wheat CCT genes family, this study provides a reference for further understanding the gene characteristics of TaCCT gene involved in plant growth and abiotic stress response. However, the specific mechanism of wheat CCT gene response to hormones and other stress is not clear, and its principle needs to be further explored.

3 Materials and Methods

3.1 Screening of wheat CCT family genes

The genome-wide data, DNA, CDs, promoter sequence and chromosome location information of Triticum aestivum, Oryza sativa and Arabidopsis thaliana were downloaded and obtained from Ensembl Plants database(http://plants.ensembl.org/index.html). First, download the unique conservative domain model of CCT gene (PF06203) from Pfam (http://pfam.xfam.org), use the model to conduct BLASTP comparison in the wheat protein database, set the threshold as E<1e^-5, and obtain the hypothetical wheat CCT gene. Then, use the first identified CCT gene sequence of hypothetical wheat to reconstruct the Hidden Markov Model of CCT gene about wheat for the second search and identification, and the hypothetical wheat CCT gene was obtained again. The protein sequence of hypothetical wheat CCT gene obtained for the second time was submitted to NCBI-CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), SMART (http://smart.embl-heidelberg.de/) and Pfam (http://pfam.xfam.org/) for identification of conserved domain, and the redundant sequence was eliminated to finally obtain wheat CCT gene. The molecular weight, isoelectric point, amino acid length and chromosome location of wheat CCT protein were analyzed by perl script in HMM 3.0 software, and CELLO V.2.5 (http://cello.life.nctu.edu.tw/) online website was used for subcellular localization analysis.

3.2 Analysis of phylogenetic tree, gene structure and protein conserved motif

The CCT protein sequences of Triticum aestivum, Oryza sativa and Arabidopsis thaliana were compared by Clustal W software. The neighbor joining (NJ) phylogenetic tree was constructed by using MEGA 7.0 software, and the Bootstrap value was set to 1 000 cycles. TaCCT genomic DNA sequence and CDS sequence were submitted to GSDS 2.0 (http://gsds.cbi.pku.edu.cn/) for gene structure analysis. The conservative domain was analyzed through MEME (http://meme-suite.org/), and the number of motifs was set to 10 and the width range of motifs was set to 6~50 aa.

3.3 Analysis of chromosome distribution, gene replication and cis-element

The perl script in HMM 3.0 software was used to obtain the starting position information of TaCCT gene on chromosome from the genome annotation information of wheat. According to the BLASTP comparison results between two TaCCT genes, the gene homologous replication events were obtained. Finally, the chromosome localization and comparison results were used by Circos v0.67 tools for visualization, and the homologous or replicated genes were connected by curves; The 1500 bp gene sequence of the upstream of TaCCT gene was intercepted in Ensembl Plants database, and the cis-elements were analyzed through PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/), and finally visualized by GSDS 2.0.

3.4 Expression pattern analysis

The data numbered E-MTAB-4484 were obtained from EMBL-EBI (https://www.ebi.ac.uk/) for analyzing the expression patterns of wheat genes in four different tissues (root, stem, leaf and grain). The transcriptome data numbered SRP045409 was downloaded from NCBI Short Read Archive (https://www.ncbi.nlm.nih.gov/sra/) database to analyze the expression pattern of TaCCT gene under drought, high temperature and drought-heat combined stress. TopHat and Cufflinks were used for the genome-wide Mapping of transcriptome reads. The FPKM value of each TaCCT gene was calculated, the differentially expressed genes were identified (Xing et al., 2017), and the expression map was drawn by TBtools.

Authors’ Contributions

TSJ and WSS were the experimental designers and executors of this study. TSJ and ZYL completed the data analysis and wrote the first draft of the manuscript; DSS and XM participated in the experimental design and data analysis; WSS was the conceiver and person in charge of the project, guiding experimental design, data analysis, manuscript writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This study was funded by Biological Mechanism of Saving Water and Increasing Yield of Crops in Typical Agricultural Areas of Northwest China (K3010216023).

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