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Article

Genome-Wide Identification and Characterization of the CCT Gene Family in Foxtail Millet (Setaria italica) Response to Diurnal Rhythm and Abiotic Stress

by
Yuntong Li
†,
Shumin Yu
†,
Qiyuan Zhang
,
Ziwei Wang
,
Meiling Liu
,
Ao Zhang
,
Xiaomei Dong
,
Jinjuan Fan
,
Yanshu Zhu
,
Yanye Ruan
and
Cong Li
*
College of Bioscience and Biotechnology, Shenyang Agricultural University, Shenyang 110866, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Genes 2022, 13(10), 1829; https://doi.org/10.3390/genes13101829
Submission received: 30 August 2022 / Revised: 4 October 2022 / Accepted: 7 October 2022 / Published: 10 October 2022
(This article belongs to the Special Issue Plant Genetics and Breeding Improvement)
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Abstract

:
The CCT gene family plays important roles in diurnal rhythm and abiotic stress response, affecting crop growth and development, and thus yield. However, little information is available on the CCT family in foxtail millet (Setaria italica). In the present study, we identified 37 putative SiCCT genes from the foxtail millet genome. A phylogenetic tree was constructed from the predicted full-length SiCCT amino acid sequences, together with CCT proteins from rice and Arabidopsis as representatives of monocotyledonous and dicotyledonous plants, respectively. Based on the conserved structure and phylogenetic relationships, 13, 5, and 19 SiCCT proteins were classified in the COL, PRR, and CMF subfamilies, respectively. The gene structure and protein conserved motifs analysis exhibited highly similar compositions within the same subfamily. Whole-genome duplication analysis indicated that segmental duplication events played an important role in the expansion of the CCT gene family in foxtail millet. Analysis of transcriptome data showed that 16 SiCCT genes had significant diurnal rhythm oscillations. Under abiotic stress and exogenous hormonal treatment, the expression of many CMF subfamily genes was significantly changed. Especially after drought treatment, the expression of CMF subfamily genes except SiCCT32 was significantly up-regulated. This work provides valuable information for further study of the molecular mechanism of diurnal rhythm regulation, abiotic stress responses, and the identification of candidate genes for foxtail millet molecular breeding.

1. Introduction

Many environment factors, including abiotic stresses and photoperiodic rhythm, affect growth and development, and thus yield [1,2,3]. The molecular mechanisms by which plants adapt to environmental changes are very complex, and a number of gene families are involved in the regulatory pathways. The CCT gene family has been shown to play important roles in plant response to environmental changes, including circadian rhythm and abiotic stress [4,5,6]. Structural analysis and functional study of CCT family genes in plants will help us to explore the molecular mechanism of environment stress response, which is significant for improving the environmental adaption of plants.
The CCT family genes contain a conserved CCT domain and are primarily known in flowering plants [7]. According to the distribution of conserved domains, CCT family genes are classified into three subfamilies, comprising the CCT motif (CMF) subfamily that contains a single CCT motif [8], the CONSTANS-like (COL) subfamily that contains a CCT domain and one or two zinc-finger B-Box domains [9], and the PSEUDORESPONSE REGULATOR subfamily (PRR) that possesses a pseudo-receiver domain and a CCT motif [5]. The study of cormophyte and streptophyte lineages indicates that the CMF subfamily evolved from the COL subfamily through the loss of the B-Box domains [8]. These changes in the domains further enhanced the functional diversification of the CCT family.
To date, many members of the CCT gene family have been identified and extensively studied in certain plant species, including Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), maize (Zea mays), and wheat (Triticum aestivum L.) [4,10,11,12]. The first CCT family gene was cloned from Arabidopsis and was named CONSTANS (CO), which controls the flowering time of Arabidopsis [4]. The CO gene regulates the expression of FLOWERING LOCUS T (FT), which encodes a mobile systemic signal, to promote flowering in Arabidopsis by binding to the cis-acting elements TCTC (N2–3) ATG in the promoter of FT and to accelerate plant flowering under long days (LD) [13]. HEADING DATE 1 (HD1), which is homologous with CO and was cloned from rice by map-based cloning, promotes heading under short days (SD) and suppresses heading under LD [11]. HD1 regulates HD3A and RICE FLOWERING LOCUS T 1 (RFT1), highly homologous FT-like genes of rice, and further regulates rice heading [14,15]. Some members of the CCT family can regulate the growth and development of plants under different photoperiods [10,11,15]. The circadian clock is a central part of these photoperiod regulation processes, and members of the PRR subfamily members are key components of the circadian clock regulatory network [5]. For example, five PRR genes (PRR9, PRR7, PRR5, PRR3, and TIMING OF CAB EXPRESSION1 (TOC1)) in Arabidopsis are regulated by the circadian clock, and the expression levels of them show diurnal rhythm oscillations with light/dark cycles [16]. PRR9, PRR7, and PRR5 can regulate the circadian clock by regulating TOC1 expression, while TOC1 affect the expression of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) [7,17,18].
In addition to regulating flowering time, members of the CCT family perform several other functions. In Arabidopsis, TOC1 regulates ABA signal relative genes (ABAR/CHLH) to be involved in drought tolerance [19]. PRR7 regulates stomatal conductance and participates in oxidative stress response [20]. Arabidopsis CONSTANS-LIKE 4 (AtCOL4) is strongly induced by abscisic acid (ABA) and osmotic stress, and AtCOL4-overexpressing plants show enhanced salt stress tolerance compared with the wild type [21]. As a negative regulator, HEADING DATE 7 (GHD7), a member of the CMF subfamily member in rice, is involved in its growth, development, and regulation of abiotic stress processes. The knock-down of GHD7 enhances drought tolerance [22]. In rice, GHD2, a COL subfamily member, performs similar roles as GHD7 and interacts with OsARID3 and 14-3-3 genes to participate in the regulation of drought stress responses and leaf senescence. The knockout mutant of GHD2 has significantly enhanced drought tolerance, whereas overexpression of GHD2 increases sensitivity to drought stress [23]. In addition to Arabidopsis and rice, CCT genes with similar functions are reported in maize. The maize ZmCCT gene delays flowering time and enhances drought tolerance by repressing the expression of Vascular Plant One Zinc Finger 1 (ZmVOZ1) and Arabidopsis Response Regulator 16 (ZmARR16) [24]. Therefore, CCT family members play a particular role in plant regulating diurnal rhythm, growth and development, and resistance to abiotic stress.
Foxtail millet was domesticated and cultivated in arid and semi-arid regions 8,700 years ago [25]. Foxtail millet is an important food crop in northwestern China that shows strong drought tolerance and sensitivity to change in photoperiod [26]. In addition, it has the characteristics of a small genome, short life cycle, and abiotic stress tolerance [27,28]. The study of the function of CCT family genes in foxtail millet is helpful for the domestication and breeding of foxtail millet. In this study, we identified 37 putative SiCCT genes and classified the genes into three subfamilies. We conducted a comprehensive bioinformatic analysis of the gene location, chromosomal distribution, exon–intron structure, motif composition, regulatory sequences, phylogenetic relationships, and synteny. The expression of the CCT genes was analyzed in different tissues by semiquantitative RT-PCR; diurnal rhythm oscillations were investigated using transcriptome data, and expression levels under different treatments were assessed by quantitative real-time PCR (qRT-PCR) analysis. The results provide insights into the function and evolution of the SiCCT gene family and its potential roles in foxtail millet growth and stress responses and provide a foundation for molecular breeding to improve the stress tolerance of foxtail millet.

2. Materials and Methods

2.1. Gene Identification, Chromosomal Location, and Phylogenetic Relationships of CCT Family Members in Foxtail Millet

The genome sequence of millet was obtained from the Ensembl Plant database (http://plants.ensembl.org/index.html, accessed on 30 September 2021). We downloaded the hidden Markov model of the CCT domain from the Pfam database (http://pfam.sanger.ac.uk/, accessed on 30 September 2021). The SiCCT family members were extracted from the genome database with HMMER 3.1 (http://hmmer.janelia.org, accessed on 30 September 2021). The candidate genes were screened against the National Center for Biotechnology databases (https://www.ncbi.nlm.nih.gov/cdd/, accessed on 12 October 2021) to determine the 37 final genes. Information on the CCT proteins were calculated using the ExPaSy ProtParam tool (http://web.expasy.org/protparam/, accessed on 12 October 2021), including length, molecular weight, and isoelectric point. We used the subcellular localization prediction tool Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 12 October 2021) [29] to predict the likely location of the protein. The MapChart software [30] was used to map the chromosomal locations of the genes.
The protein sequences of the CCT domain-containing genes of Arabidopsis and rice were downloaded from the Ensembl Plant database on the basis of relevant research reports [5,8,9,31,32]. The full-length CCT sequences of Arabidopsis, rice, and foxtail millet were selected for comparison with MEGA X version 7 software [33]. Subsequently, a multiple sequence alignment was used to construct a maximum likelihood phylogenetic tree with the MEGA software with the following parameters: Poisson model, pairwise deletion, and 1000 bootstrap replications.

2.2. Collinearity Analysis and Gene Duplication

To explore the synteny of orthologous CCT genes obtained from foxtail millet and other selected species, we used the MCScan (Python version) tool (https://github.com/tanghaibao/jcvi/wiki/MCscan-(Python-version), accessed on 25 October 2021) to draw the synteny map. When visualizing the results, the gene filtering parameter in the small collinearity block was set to 30. Circos [34] was used to map all SiCCT genes to foxtail millet chromosomes based on physical location information from the millet genome database. A multiple collinearity scanning kit (MCScanX) [35] was used to analyze gene duplication events. KaKs_Calculator 2.0 [36] was used to calculate Ka and Ks to detect replication events.

2.3. Gene Structure and Conserved Sequence Analysis

The location of introns, exons, and untranslated regions in the genes was extracted from the gene finding format (GFF3) file. Conserved domains of millet CCT proteins were elucidated with the MEME Suite (http://meme-suite.org/, accessed on 1 November 2021) [37]. The optimized parameters were employed as follows: the maximum number of motifs was 10, and the optimum width was from 6 to 50. The map of the gene structure and motifs was drawn using TBtools [38].

2.4. Cis-Acting Element Analysis

The genomic DNA sequence 1000 bp upstream of the translation initiation codon for the SiCCT genes was used for cis-acting element analysis. The PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 7 November 2021) [39] was used to predicte the promoter elements in the sequences.

2.5. Plant Materials and Treatments

Millet ‘Yugu1’ was used in this study. Plants were grown in fields and in a greenhouse in Shenyang, Liaoning Province, China. The stress-treated plants were grown in a greenhouse in Hoagland’s nutrient solution [40]. Abscisic acid, salt stress, and drought stress were applied separately by treating plants with 100 μM ABA, 200 mM NaCl solution, or 20% PEG6000, respectively. Plant leaves were collected after treatment for 0, 4, 8, and 12 h, with three biological replicates for each treatment. For low-temperature treatment, plants were placed in a 4 °C incubator for 12 h, and samples were collected at 0, 6, and 12 h. The leaf, immature seed, spikelet, root, leaf sheath, shoot apical meristem, stem, im-mature leaf, and seed were collected separately at three developmental stages from field-grown foxtail millet plants for semiquantitative RT-PCR.

2.6. Analysis of CCT Gene Expression in Millet and Quantitative Real-Time PCR

Total RNA was extracted from each tissue using a plant RNA extraction kit (Accurate Biotechnology Co., Ltd., Changsha, China) in accordance with the manufacturer’s instructions. The cDNA was synthesized from 2 µg total RNA with M-MLV reverse transcriptase (Accurate Biotechnology Co., Ltd.). Semiquantitative PCR was performed using GoTaq® Green Master Mix (Promega, Shanghai, China) with specific primers (synthesized by Genewiz, Suzhou, China). The GoTaq® qPCR Master Mix kit (Promega) was used for qRT-PCR reactions using a C1000 Thermal Cycler and quantified using a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). The relative transcript level was calculated using the 2−ΔΔCt method [41]. Primers used in this experiment are enlisted in Table S1. Actin gene (SETIT_010361mg) was used as internal control for this experiment. Transcriptome data used in this study were downloaded from the NCBI SRA database under accession number PRJNA684771 [42]. We processed per kilobase per million (FPKM) data for diurnal rhythm analysis. Yi et al. [42] sampled the two groups of samples at the beginning of light and the beginning of darkness, respectively, and there were two groups of data at 16 h, 20 h, and 24 h. Therefore, we draw the line graph using the average of the FPKM values of these two sets of data.

3. Results

3.1. Identification and Analysis of CCT Gene Family Members in Foxtail Millet

A total of 37 predicted CCT genes were extracted from the foxtail millet genome using a hidden Markov model (PF06203). The genes were designated as genes from SiCCT1 to SiCCT37 based on their chromosomal distribution (Table 1). The gene identifier, genomic position, predicted molecular weight, protein length, and isoelectric point of each gene are listed in Table 1. The length of the proteins ranged from 160 aa (SiCCT27) to 760 aa (SiCCT35); the molecular weight varied from 17.6 kDa (SiCCT27) to 82.7 kDa (SiCCT35), and the isoelectric point ranged from 4.37 (SiCCT32) to 10.18 (SiCCT27). All of the genes were predicted to be localized in the nucleus except SiCCT4. SiCCT4 was predicted to be localized in both the mitochondria and the nucleus.
Thirty-six SiCCT genes were mapped to eight of the nine foxtail millet chromosomes. The number of SiCCT genes varied among the chromosomes, and no genes were detected on chromosome V (Figure 1). Four chromosomes (IV, VI, VII, and VIII) contained three genes, and two chromosomes (II and III) carried four genes. Chromosome IX contained the largest number of nine genes. One gene was not mapped to a chromosome and was recategorized as unplaced (Figure 1). SiCCT14 and SiCCT15 were located on chromosome 2 and identified as a pair of tandem repeats.

3.2. Evolutionary and Synteny Analysis of CCT Genes in Foxtail Millet and Other Species

To detect the evolutionary relationships among CCT family members, a maximum likelihood phylogenetic tree was constructed based on 99 full-length CCT protein sequences, comprising 32 from Arabidopsis, 30 from rice, and 37 from foxtail millet (Figure 2 and Table S2). The CCT proteins were resolved into three main clades consisting of eight groups, which were designated A to H. Group B represented the PRR subfamily with 15 members. The CMF subfamily comprised groups A (23 members), C (4 members), E (5 members), and G (9 members). The remaining groups (D with 16 members, F with 18 members, and H with 9 members) belonged to the COL subfamily.
To enable further inference on the phylogenetic affinities of the foxtail millet CCT family, synteny analyses were performed between the CCT genes of foxtail millet and other species, comprising the dicotyledons Arabidopsis, tomato (Solanum lycopersicum), and alfalfa (Medicago truncatula) and the monocotyledons rice, sorghum (Sorghum bicolor), and maize (Figure 3). Only one pair of homologous genes between foxtail millet and Arabidopsis, and two pairs between foxtail millet and each of alfalfa and tomato, were detected. A greater number of gene pairs were detected between foxtail millet and the other monocotyledonous species, namely rice (26 orthologous gene pairs), sorghum (28 orthologous gene pairs), and maize (31 orthologous gene pairs). Among these genes, several genes comprised more than three pairs in a collinear relationship between foxtail millet and the other species (Table S3), such as for SiCCT34, SiCCT35, and SiCCT36. SiCCT34 was detected in all studied species except Arabidopsis, suggesting that this gene may be evolutionarily conserved and may have played an important role in the evolution of CCT gene family.
Collinearity within the SiCCT family was analyzed to explore segmental duplication events within the SiCCT genes. Six pairs of segmental-duplicated genes were found on foxtail millet chromosomes (Figure 4). We calculated the nonsynonymous substitution rate (Ka)/synonymous substitution rate (Ks) ratio for each gene pair to examine the evolutionary mechanism of the CCT genes (Table 2). All Ka/Ks values were less than one, except for SiCCT32/SiCCT36. To further explore the evolution of CCT genes in other Poaceae species, we also selected the CCT genes of maize and sorghum to plot the respective intraspecific collinearity relationships (Figure S1 and Figure S2). Twenty-three and 10 pairs of segmental-duplicated genes were found in the maize and sorghum genomes, respectively.

3.3. Gene Structure and Conserved Motif Analysis

The phylogenetic tree of SiCCTs was constructed and divided into 10 groups (I to X; Figure 5A). To better understand the evolution of CCT genes in foxtail millet, we analyzed the intron–exon structure of the identified CCT genes (Figure 5B). Most SiCCT genes contained one to three introns (eight genes with one intron, seven with two introns, and seven with three introns). A smaller proportion of the genes contained four to eight introns (two genes with four introns, four with five introns, three with six introns, four with seven introns, and one with eight introns). Only SiCCT20 lacked an intron.
We constructed domain diagrams of the 37 predicted SiCCT genes to study their conserved motifs using the MEME Suite of sequence analysis tools (Figure 5C). The CCT domain was composed of motifs 1 and 3, and all SiCCT genes had complete CCT institutional domains, except for SiCCT14, SiCCT15, and SiCCT24. SiCCT14 only had motif 3, and SiCCT24 and SiCCT15 only had motif 1. Motif 2 was the B-Box domain, which was specific to the COL family. The PRR domain was composed of motif 5 and motif 10. In the PRR subfamily, SiCCT9, SiCCT4, SiCCT35, and SiCCT11 had intact PRR domains, whereas SiCCT26 only had motif 10. Furthermore, we found that SiCCT29, SiCCT31, SiCCT27, and SiCCT18 had independent evolutionary branches in the phylogenetic tree and their CCT domains (motif 1 and 3) were closer to the 5′ end than other CCT genes. These results indicated that the domains of these proteins were relatively conserved and conformed with the subfamilial classification of the CCT family.

3.4. Analysis of Cis-Acting Elements in the SiCCT Gene Promoter Region

To predict the function and regulatory mechanism of the SiCCT genes, we analyzed the cis-acting elements in the promoter region of the genes. Based on the results of a PlantCARE analysis, the cis-acting elements were divided into three categories: plant growth and development, abiotic and biotic stresses, and phytohormone responsive (Table S4). We selected 27 cis-acting elements from the promoter of the SiCCT genes and divided them into the three categories (Figure 6 and Figure S3). In the growth and development group, the G-Box element involved in light response was most numerous and was detected in 24 of the 37 genes. In addition to light-response elements, the CAT-Box element involved in the regulation of meristem expression was present in 14 genes. An O2-site element involved in zein metabolism regulation was detected in nine genes. In the abiotic and biotic stresses group, multiple response elements were observed, such as oxidation, (ARE and GC-motif), wounding (box-S and WUN-motif), drought (MBS), high temperature (STRE), low temperature (LRE), and defense (CCAAT-box). The most numerous elements in the stress-response group were MYB and MYC binding sites, which are general stress-responsive elements. In the phytohormone-responsive group, we detected many elements involved in hormone response, including elements responsive to ABA (ABRE), salicylic acid (as-1), indoleacetic acid (AuxRR-core and TGA-element), gibberellins (GARE-motif and P-box), and methyl jasmonate (TGACG-motif). Among them, the number of ABRE elements was 92, which was the largest number of elements related to hormone regulation.

3.5. Expression Analysis of SiCCT Genes in Different Tissues and under Abiotic Stress and Exogenous Hormone Treatments

We used semiquantitative RT-PCR to investigate the expression of the SiCCT genes in various tissues, comprising the leaf, immature seed, spikelet, root, leaf sheath, shoot apical meristem, stem, immature leaf, and seed (Figure S4). Four genes (SiCCT17, SiCCT22, SiCCT26, and SiCCT31) were highly expressed; three genes (SiCCT18, SiCCT24, and SiCCT37) were weakly expressed, and two genes (SiCCT14 and SiCCT16) were not expressed in all tissues. The RT-PCR results showed that the SiCCT genes were expressed in various tissues and differed in expression patterns.
In the cis-element analysis, a large number of elements related to light response and diurnal rhythm were detected in the promoter region of CCT genes. To further explore the CCT genes response to diurnal rhythm, an expression analysis of CCT genes under diurnal rhythm was performed. The transcriptome data (PRJNA684771) used for diurnal rhythm analysis were downloaded from NCBI database according to the research of Yi et al. [42]. The expression pattern of 16/37 CCT genes shoedn diurnal rhythm oscillations, which includes all PRR members, 6 COL members, and 5 CMF members (Figure 7 and Table S5). The expression of SiCCT4 and SiCCT9 reached the highest level at 12 h, and the expression of SiCCT11 and SiCCT35 reached the highest at 8 h, while SiCCT26 showed different rhythm oscillation than other PRR members. In addition to the PRR family genes, the COL subfamily members (SiCCT2, 5, 7, 8, 16, and 17) and CMF subfamily members (SiCCT19, 28, 31, 32, and 33) also showed diurnal rhythms, and the expression peak of these genes appeared in different times. Among them, the expression peak of SiCCT5, 8, 16, 19, and 31 reached the highest level at dark.
Many genes in the CMF subfamily have been reported to be involved in abiotic stress and hormone regulation in plants [22,24]. To clarify whether foxtail millet CMF subfamily genes responded to abiotic stresses and ABA treatment, we analyzed their expression patterns under different treatments (Figure 8). Overall, the transcript abundance of many genes was significantly increased, especially under ABA and drought treatment. After ABA treatment, all genes were up-regulated except SiCCT3 and SiCCT12. Among the up-regulated genes, the expression of SiCCT13 was the highest, significantly up-regulated by more than 29 times, and its expression peaked at 8 h. Under drought induction, the expression levels of all genes were induced except SiCCT22, SiCCT29, and SiCCT32. The expression level of SiCCT3 was significantly up-regulated by 40-fold at 12 h. The expression levels of half of the genes were induced by salt treatment, and the expression of most genes was induced by more than three times. Eight genes responded to low-temperature treatment, of which the expression levels of five genes were induced and three genes were inhibited. SiCCT31 was most significantly induced by low temperature, and the expression level of SiCCT31 was up-regulated by more than eight times at 8 h.
In general, most of the CMF subfamily genes showed a trend for up-regulated expression in response to abiotic stress and hormone treatment (Figure S5A). After ABA treatment, 10 genes were up-regulated, followed by drought treatment. The expression of SiCCT37 was up-regulated under the four treatments. The expressions of SiCCT21, SiCCT28, and SiCCT18 was induced by exogenous ABA, salt, and drought. The number of genes down-regulated by salt treatment were the most among all treatments (Figure S5B). The expressions of SiCCT22 and SiCCT29 was inhibited by salt and drought.

4. Discussion

Stress tolerance and photoperiod regulation are important factors for plant adaption to growth in different regions. The CCT gene family has been reported to be involved in the regulation of photoperiod and stress responses in plants [13,43,44]. CCT genes have been identified in many crop species at the whole-genome level. For example, 41 and 53 CCT family genes have been identified in rice [45] and maize [44], respectively. Foxtail millet exhibits the characteristics of strong tolerance to abiotic stress and sensitivity to photoperiod. Therefore, we identified possible CCT genes from the foxtail millet genome. In this study, 37 putative CCT family genes were identified in foxtail millet (Table 1). A phylogenetic analysis of the CCT family genes of foxtail millet, Arabidopsis, and rice resolved the genes into eight groups within three clades (Figure 2). Several pairs of SiCCT genes homologous to the reported OsCCT and AtCCT genes were found in the phylogenetic tree. For example, SiCCT6 was homologous to HD1 and CO, SiCCT5 and SiCCT8 were homologous to DTH2, SiCCT35 was homologs to HD2, and SiCCT4 and SiCCT26 were homologous to APRR1. The high homologies among the genes may imply that the proteins perform similar functions.
Whole-genome duplication (WGD) and nested chromosome fusions (NCFs) are important driving forces in the evolution of flowering plants [46,47]. WGD can provide a plant with the opportunity for diversification in gene functions, and NCFs can change the number of chromosomes. These processes are important for speciation and the evolution of novel gene functions. Flowering plants have experienced many WGD events [48], of which three WGD events have occurred in the genomes of cereal grasses [49]. To explore the evolution of SiCCT genes in cereals, we constructed a collinearity map for maize and sorghum (Figure S1 and Figure S2) and calculated the Ka and Ks values for foxtail millet, sorghum, and maize (Table 2). We then estimated the timing of the doubling event of the SiCCT genes. The doubling time points for sorghum and maize were also calculated as controls. Foxtail millet separated from maize and sorghum approximately 27 million years ago (Mya), and maize and sorghum diverged approximately 13 Mya [25]. Based on the estimated dates for these two nodes and the respective estimated divergence dates for the Poaceae (98.2 Mya) [50] and the subfamily Panicoideae (48 Mya) [25], we divided the calculated WGD events into time periods comprising an intermediate ancestor period (~98.2 Mya), Poaceae ancestor period (~98.2 Mya to ~48 Mya), Panicoideae ancestor period (foxtail millet: ~48 Mya to ~27 Mya; maize and sorghum: ~48 Mya to ~13 Mya), and post-speciation period (foxtail millet: ~27 Mya; maize and sorghum: 13 Mya). Based on the calculations, the CCT genes of foxtail millet and sorghum diverged in relatively ancient times, with the diversification of most of the genes concentrated between the intermediate ancestor period and the Poaceae ancestor period. These findings suggest that SiCCT genes have hardly changed after speciation. From an evolutionary point of view, SiCCT genes have been essentially stable since entering the Panicoideae ancestor period. These results indicate that the functions of SiCCT genes were determined before the divergence of foxtail millet, which further implies that the functions of the genes are conserved.
Tandem repeats and fragment repeats gave rise to 14 genes, which were important for CCT family amplification in foxtail millet (Figure 1 and Figure 4). We speculate that the generation of these new genes might be caused by changes in the domains. The generation of the CMF subfamily resulted from the loss of the B-Box domain of the COL subfamily members [5]. Therefore, we investigated the domains of SiCCT proteins and observed that most proteins had complete CCT domains except SiCCT14, SiCCT15, and SiCCT24 (Figure 5). The CCT domain was composed of motifs 1 and 3. SiCCT15 and SiCCT14 had no other domain differences except in the CCT domain (SiCCT15 only had motif 1 and SiCCT14 only had motif 3). Both SiCCT14 and SiCCT15 were tandem repeats. Therefore, we consider that SiCCT14 and SiCCT15 were generated during the process of tandem duplication, owing to the separation of motif 1 and motif 3 in the CCT domain. The differences of SiCCT14 and SiCCT15 were not detected in SiCCT24. However, SiCCT24 formed a collinear pair with SiCCT25. The motif 3 in SiCCT24 may have been lost when segment duplication caused by the WGD occurred. In addition, the protein structure of six pairs of collinear genes were compared. Three pairs were observed to be structurally different (SiCCT24/SiCCT25, SiCCT32/SiCCT36, and SiCCT2/SiCCT17). In addition to the loss of motif 3 in SiCCT25 to form SiCCT24, SiCCT36 lost three domains (motifs 4, 7, and 9), and SiCCT17 lost the B-Box (motif 2) to form SiCCT32 and SiCCT2, respectively, during the WGD event. Previous studies have shown that the deletion of the B-Box domain affects grain vernalization and response to LD [8]. Therefore, after domain deletion, these proteins are likely to have assumed novel functions, which are worthy of further exploration. Furthermore, we found that SiCCT29, SiCCT31, SiCCT27, and SiCCT18 had independent evolutionary branches in the phylogenetic tree and their CCT domains were closer to the C terminus of protein than other CCT genes. This suggested that they may had different functions with other SiCCT genes.
It had been found that CCT genes responded to photoperiod signal and are involved in diurnal rhythm regulation, therefore affecting the flowering time of plants [44,51]. A number of elements related to light response and diurnal rhythm regulation were found on the promoter of SiCCT, including G-BOX, I-box, Sp1, and so on. Therefore, we further analyzed the expression rhythmicity of the CCT gene according to the transcriptome data. PRR subfamily genes are key factors in the regulation of plant diurnal rhythm. Figure 7 showed that the expression of SiCCT4, SiCCT9, SiCCT11, and SiCCT35 had strong diurnal rhythm. Among them, the expression of SiCCT11 and SiCCT35 reached the highest value at 8 h of light, and they had similar expression patterns in diurnal rhythm. In addition, the phylogenetic tree analysis showed that both SiCCT35 and SiCCT11 were closely related to Hd2. Hd2 is a gene related to rice heading date regulation, which can inhibit heading date under long-day condition [52]. Therefore, we speculated that SiCCT35 and SiCCT11 might play an important role in regulating the heading time of foxtail millet. In addition to the genes in the PRR subfamily, some genes in the COL and CMF subfamilies show strong diurnal rhythms. These results suggested that many genes in the SiCCT family contributed an important function in the regulation of foxtail millet diurnal rhythm.
In addition to photoperiod regulation, the function of some CCT genes is also associated with abiotic stresses. Among such genes, many members of the CMF subfamily have been reported, such as ZmCCT and GHD7 [22,24]. In the present study, many of the CMF subfamily genes showed sensitivity to ABA and stress treatment (Figure 8). In response to ABA treatment, all genes except SiCCT3 and SiCCT12 were up-regulated. Analysis of cis-acting elements (Figure 6) revealed that no ABA-responsive element was detected in SiCCT3 and SiCTT12, which might be the reason why their expression was not induced by exogenous ABA. Most CMF family members were induced by drought stress except SiCCT32. They also had different degrees of response to low temperature and salt stress. Interestingly, SiCCT37 expression was up-regulated in response to all treatments. Thus, this gene may have multiple functions, and the further study of the expression pattern and functions of this gene may be of importance for elucidating abiotic stress responses.
The foxtail millet CCT gene family was analyzed in this study. Thirty-seven full-length SiCCT genes were characterized and classified into three subfamilies according to their domains. The collinearity analysis and phylogenetic relationships of SiCCT genes provide valuable clues for dissecting the evolutionary characteristics of the SiCCT genes. The expression analysis of SiCCT genes responding to diurnal rhythm and abiotic stresses provide insights into their potential functions in the growth and development of foxtail millet. The present research will be helpful for further functional research into SiCCT genes and, potentially, for the improvement of the environmental adaptability and stress tolerance of foxtail millet.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes13101829/s1, Figure S1. Interchromosomal relationships of sorghum CCT genes; Figure S2. Interchromosomal relationships of maize CCT genes; Figure S3. Distribution map of cis-acting elements on each gene; Figure S4. Expression profiles of SiCCT genes in different tissues of millet. The site of expression is shown at the top, and the gene name is shown on the right; Figure S5. The detailed numbers of simultaneously up-regulated (A) and down-regulated (B) SiCCT genes by ABA (abscisic acid), NaCl, drought, and low temperature; Table S1 Primer name and sequence information of SiCCT; Table S2: List of CCT genes in Arabidopsis, rice, and foxtail millet.; Table S3: List of homology genes of the CCT genes of foxtail millet with 6 representative plant species; Table S4: The number and function statistics of promoter elements predicted in this study.; Table S5 FPKM values of SiCCT genes under long day.

Author Contributions

Conceptualization, C.L. and S.Y.; data collection, Y.L., Q.Z., M.L. and Z.W.; formal analysis, S.Y. and M.L.; funding acquisition, C.L.; investigation, J.F., Y.Z. and Y.R.; methodology, C.L. and S.Y.; supervision, C.L. and A.Z.; visualization, S.Y. and X.D.; writing-original draft, Y.L., C.L. and S.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by National Nature Science Foundation of China (No. 31601233).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data and materials obtained in this study are available from the corresponding author on reasonable request.

Acknowledgments

We thank Robert McKenzie for editing the English text of a draft of this manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Chromosomal location of putative SiCCT genes. Thirty-six genes were mapped to the millet chromosomes, and one predicted gene was not mapped. The chromosome number is shown above each chromosome, and the length of each chromosome is indicated on the left in megabases (MB).
Figure 1. Chromosomal location of putative SiCCT genes. Thirty-six genes were mapped to the millet chromosomes, and one predicted gene was not mapped. The chromosome number is shown above each chromosome, and the length of each chromosome is indicated on the left in megabases (MB).
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Figure 2. Phylogenetic relationships of CCT proteins. The phylogenetic tree was constructed from 100 protein sequences from millet, rice, and Arabidopsis using the maximum likelihood method with 1000 bootstrap replicates. The lines of different colors indicate different groups. Three subfamilies of CCT proteins are represented by different colored lines. Hollow circles, black stars, and red triangles represent the CCT proteins of Arabidopsis, rice, and millet, respectively.
Figure 2. Phylogenetic relationships of CCT proteins. The phylogenetic tree was constructed from 100 protein sequences from millet, rice, and Arabidopsis using the maximum likelihood method with 1000 bootstrap replicates. The lines of different colors indicate different groups. Three subfamilies of CCT proteins are represented by different colored lines. Hollow circles, black stars, and red triangles represent the CCT proteins of Arabidopsis, rice, and millet, respectively.
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Figure 3. Homology of CCT genes between foxtail millet and six representative plant species. Gray lines in the background represent collinear blocks in the genomes of millet and the other plant species, and red lines highlight collinear CCT gene pairs.
Figure 3. Homology of CCT genes between foxtail millet and six representative plant species. Gray lines in the background represent collinear blocks in the genomes of millet and the other plant species, and red lines highlight collinear CCT gene pairs.
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Figure 4. Interchromosomal relationships of millet CCT genes. Gray lines represent all common blocks in the millet genome, and red lines represent replicated CCT gene pairs.
Figure 4. Interchromosomal relationships of millet CCT genes. Gray lines represent all common blocks in the millet genome, and red lines represent replicated CCT gene pairs.
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Figure 5. Phylogenetic relationships, gene structure, and conserved motifs of SiCCT proteins. (A) Phylogenetic tree constructed based on the domains of the foxtail millet CCT proteins. In the phylogenetic tree, blue areas are members of the COL family, green areas are members of the CMF family, and yellow areas are members of the PRR family. (B) Exon–intron structure of foxtail millet CCT genes. Blue boxes indicate the 5′ and 3′ untranslated regions; a red box represents an exon, and black lines represent introns. (C) Motif composition of foxtail millet CCT proteins. The motifs, numbered 1–10, are indicated by different colored boxes.
Figure 5. Phylogenetic relationships, gene structure, and conserved motifs of SiCCT proteins. (A) Phylogenetic tree constructed based on the domains of the foxtail millet CCT proteins. In the phylogenetic tree, blue areas are members of the COL family, green areas are members of the CMF family, and yellow areas are members of the PRR family. (B) Exon–intron structure of foxtail millet CCT genes. Blue boxes indicate the 5′ and 3′ untranslated regions; a red box represents an exon, and black lines represent introns. (C) Motif composition of foxtail millet CCT proteins. The motifs, numbered 1–10, are indicated by different colored boxes.
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Figure 6. Cis-acting element analysis of the CCT gene family in foxtail millet. (A) Number of different promoter elements in the CCT genes indicated by different colors and numbers. (B) Number of cis-acting elements in three functional categories.
Figure 6. Cis-acting element analysis of the CCT gene family in foxtail millet. (A) Number of different promoter elements in the CCT genes indicated by different colors and numbers. (B) Number of cis-acting elements in three functional categories.
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Figure 7. Expression pattern of SiCCT proteins under long days. White and black bars represent light and dark periods, respectively. The gene expression level was the FPKM value calculated.
Figure 7. Expression pattern of SiCCT proteins under long days. White and black bars represent light and dark periods, respectively. The gene expression level was the FPKM value calculated.
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Figure 8. Expression profile of SiCCT genes under different treatments. Red boxes indicate up-regulation; blue boxes indicate down-regulation, and the intensity of the color indicates the intensity of expression. The color intensity of each color block is compared separately for each gene. The numbers on the graph were the expression levels of genes calculated by the 2−ΔΔCt method.
Figure 8. Expression profile of SiCCT genes under different treatments. Red boxes indicate up-regulation; blue boxes indicate down-regulation, and the intensity of the color indicates the intensity of expression. The color intensity of each color block is compared separately for each gene. The numbers on the graph were the expression levels of genes calculated by the 2−ΔΔCt method.
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Table
Table 1. List of SiCCT genes identified in this study.
Table 1. List of SiCCT genes identified in this study.
Gene NameTranscript IDAliasChrProtein Length (aa)MW (D)pILocation
SETIT_019213mgKQL27760SiCCT1Ⅰ43246,243.275.60Nucleus
SETIT_017487mgKQL28473SiCCT2Ⅰ38639,459.156.32Nucleus
SETIT_017125mgKQL28750SiCCT3Ⅰ45347,725.858.14Nucleus
SETIT_016922mgKQL30521SiCCT4Ⅰ51857,741.595.59Nucleus/Mitochondrion
SETIT_017374mgKQL31286SiCCT5Ⅰ40744,610.574.99Nucleus
SETIT_019803mgKQL31326SiCCT6Ⅰ35539,793.638.00Nucleus
SETIT_017124mgKQL31374SiCCT7Ⅰ44047,328.186.54Nucleus
SETIT_030034mgKQL24119SiCCT8Ⅱ40643,630.405.20Nucleus
SETIT_029202mgKQL25444SiCCT9Ⅱ63070,287.206.22Nucleus
SETIT_030140mgKQL27073SiCCT10Ⅱ38440,363.076.10Nucleus
SETIT_033274mgKQL27318SiCCT11Ⅱ66171,442.898.74Nucleus
SETIT_022659mgKQL13967SiCCT12Ⅲ32535,522.484.93Nucleus
SETIT_024852mgKQL15112SiCCT13Ⅲ31935,116.485.75Nucleus
SETIT_024038mgKQL16011SiCCT14Ⅲ33136,436.764.58Nucleus
SETIT_024806mgKQL16012SiCCT15Ⅲ26829,552.184.78Nucleus
SETIT_006432mgKQL10228SiCCT16Ⅳ44547,590.035.09Nucleus
SETIT_006690mgKQL11132SiCCT17Ⅳ37239,574.386.13Nucleus
SETIT_006750mgKQL11783SiCCT18Ⅳ35737,898.915.01Nucleus
SETIT_014122mgKQL01011SiCCT19Ⅵ31333,098.767.65Nucleus
SETIT_014224mgKQL01176SiCCT20Ⅵ28128,425.626.42Nucleus
SETIT_014037mgKQL02690SiCCT21Ⅵ23526,147.149.58Nucleus
SETIT_011920mgKQK96183SiCCT22Ⅶ21723,959.736.75Nucleus
SETIT_010592mgKQK97912SiCCT23Ⅶ32634,393.375.15Nucleus
SETIT_011862mgKQL00040SiCCT24Ⅶ20221,587.855.61Nucleus
SETIT_027626mgKQK93230SiCCT25Ⅷ23625,887.706.50Nucleus
SETIT_026170mgKQK93667SiCCT26Ⅷ56662,003.237.40Nucleus
SETIT_027518mgKQK95022SiCCT27Ⅷ16017,635.3010.18Nucleus
SETIT_039184mgKQK86275SiCCT28Ⅸ24827,292.516.86Nucleus
SETIT_036747mgKQK87242SiCCT29Ⅸ30832,998.378.66Nucleus
SETIT_035937mgKQK87465SiCCT30Ⅸ40644,451.226.65Nucleus
SETIT_036492mgKQK87704SiCCT31Ⅸ33935,998.104.77Nucleus
SETIT_035901mgKQK88797SiCCT32Ⅸ41243,116.934.37Nucleus
SETIT_036910mgKQK89929SiCCT33Ⅸ28629,324.747.14Nucleus
SETIT_034611mgKQK90887SiCCT34Ⅸ64869,184.428.49Nucleus
SETIT_034368mgKQK91381SiCCT35Ⅸ76082,668.806.07Nucleus
SETIT_039219mgKQK92647SiCCT36Ⅸ36539,857.304.67Nucleus
SETIT_020907mgKQK85264SiCCT37Unplaced35338,888.614.75Nucleus
Chr: chromosome, aa: number of amino acids, MW: molecular weight, Da: Dalton, pI: theoretical isoelectric point.
Table
Table 2. Evolutionary analysis of SiCCT genes.
Table 2. Evolutionary analysis of SiCCT genes.
TypeLocus 1Locus 2KaKsKa/KsT (MYA)Period
MilletSiCCT32SiCCT361.048420.8796761.1918367.67Intermediate ancestor
MilletSiCCT2SiCCT170.2425981.711380.141756131.64Poaceae ancestor
MilletSiCCT7SiCCT160.2185291.396260.15651107.40Poaceae ancestor
MilletSiCCT10SiCCT340.3323222.147940.154716165.23Poaceae ancestor
MilletSiCCT24SiCCT250.009524990.02616840.3639882.01Millet
MilletSiCCT11SiCCT350.9740771.108460.87876685.27Intermediate ancestor
SorghumEES15491EES078340.05499480.121690.4519259.36Sorghum
SorghumKXG22181EES038440.3624992.544050.142489195.70Poaceae ancestor
SorghumEES15491EES038440.8793460.7711431.1403259.32Intermediate ancestor
SorghumKXG40217EER941140.9527851.159260.82188989.17Intermediate ancestor
SorghumEES05406EES124460.1446351.204790.1200592.68Intermediate ancestor
SorghumEES05581EER881910.9973471.006590.99081377.43Intermediate ancestor
SorghumOQU84486EER901451.019350.9605861.0611873.89Intermediate ancestor
SorghumEES07342EER896330.1482720.9001180.16472669.24Intermediate ancestor
SorghumOQU85473EER882270.9967341.011330.98556677.79Intermediate ancestor
SorghumEER94860EER998730.3478081.797930.19345138.30Poaceae ancestor
MaizeZm00001d025770Zm00001d0031620.02619310.2399010.10918318.45Intermediate ancestor
MaizeZm00001d021291Zm00001d0062120.06259710.1295610.4831479.97Maize
MaizeZm00001d022500Zm00001d0071070.1766570.3331170.53031425.62Panicoideae ancestor
MaizeZm00001d029149Zm00001d0071070.6414550.9394750.6827872.27Intermediate ancestor
MaizeZm00001d042958Zm00001d0124410.07816790.3965180.19713630.50Panicoideae ancestor
MaizeZm00001d013443Zm00001d0337190.06171020.381830.16161729.37Panicoideae ancestor
MaizeZm00001d027598Zm00001d0140741.011320.9713781.0411274.72Intermediate ancestor
MaizeZm00001d032768Zm00001d0140741.105140.7956331.3890161.20Intermediate ancestor
MaizeZm00001d048369Zm00001d0140740.57820.6872730.84129652.87Intermediate ancestor
MaizeZm00001d036494Zm00001d0146561.020090.9461211.0781872.78Intermediate ancestor
MaizeZm00001d015468Zm00001d0469250.5104841.316130.387869101.24Poaceae ancestor
MaizeZm00001d025770Zm00001d0171760.1579931.592280.0992244122.48Poaceae ancestor
MaizeZm00001d051047Zm00001d0171760.08023830.4924350.16294237.88Panicoideae ancestor
MaizeZm00001d017241Zm00001d0511140.05472880.2097980.26086416.14Panicoideae ancestor
MaizeZm00001d051684Zm00001d0178850.9953961.014650.98102878.05Intermediate ancestor
MaizeZm00001d037327Zm00001d0179390.8575761.419780.604022109.21Poaceae ancestor
MaizeZm00001d045661Zm00001d0179390.2891211.63960.176336126.12Poaceae ancestor
MaizeZm00001d021291Zm00001d0527811.014460.9446871.0738672.67Intermediate ancestor
MaizeZm00001d025770Zm00001d0510470.1690241.433930.117874110.30Poaceae ancestor
MaizeZm00001d027598Zm00001d0483691.0070.968741.039574.52Intermediate ancestor
MaizeZm00001d035134Zm00001d0496510.9854931.033460.95358679.50Intermediate ancestor
MaizeZm00001d045661Zm00001d0373270.07578180.4306230.17598233.12Poaceae ancestor
MaizeZm00001d029149Zm00001d0225000.8383860.5876641.4266445.20Poaceae ancestor
Ka: non-synonymous rate, Ks: synonymous rate, T: divergent time, MYA: millions of years ago, T = Ks/(2 × 6.5 × 10−9) × 10−6 million years ago (Mya).
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MDPI and ACS Style

Li, Y.; Yu, S.; Zhang, Q.; Wang, Z.; Liu, M.; Zhang, A.; Dong, X.; Fan, J.; Zhu, Y.; Ruan, Y.; et al. Genome-Wide Identification and Characterization of the CCT Gene Family in Foxtail Millet (Setaria italica) Response to Diurnal Rhythm and Abiotic Stress. Genes 2022, 13, 1829. https://doi.org/10.3390/genes13101829

AMA Style

Li Y, Yu S, Zhang Q, Wang Z, Liu M, Zhang A, Dong X, Fan J, Zhu Y, Ruan Y, et al. Genome-Wide Identification and Characterization of the CCT Gene Family in Foxtail Millet (Setaria italica) Response to Diurnal Rhythm and Abiotic Stress. Genes. 2022; 13(10):1829. https://doi.org/10.3390/genes13101829

Chicago/Turabian Style

Li, Yuntong, Shumin Yu, Qiyuan Zhang, Ziwei Wang, Meiling Liu, Ao Zhang, Xiaomei Dong, Jinjuan Fan, Yanshu Zhu, Yanye Ruan, and et al. 2022. "Genome-Wide Identification and Characterization of the CCT Gene Family in Foxtail Millet (Setaria italica) Response to Diurnal Rhythm and Abiotic Stress" Genes 13, no. 10: 1829. https://doi.org/10.3390/genes13101829

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