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Article

Genome-Wide Identification and Expression Analysis of CsCaM/CML Gene Family in Response to Low-Temperature and Salt Stresses in Chrysanthemum seticuspe

by
Manman Fu
,
Chao Wu
,
Xia Li
,
Xiaoyu Ding
and
Fangqi Guo
*
Institute of Horticulture, Zhejiang Academy of Agricultural Sciences, Hangzhou 310058, China
*
Author to whom correspondence should be addressed.
Plants 2022, 11(13), 1760; https://doi.org/10.3390/plants11131760
Submission received: 7 June 2022 / Revised: 27 June 2022 / Accepted: 27 June 2022 / Published: 1 July 2022
(This article belongs to the Special Issue New Insights into Plants' Defense Mechanisms against Abiotic Stresses)
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Abstract

:
Calmodulin (CaM) and calmodulin-like proteins (CML) act as significant Ca2+ sensors binding Ca2+ with EF-hand motifs and have been reported to be involved in various environmental stresses in plants. In this study, calmodulin CsCaM/CML gene family members were identified based on the genome of Chrysanthemum seticuspe published recently; a phylogenetic tree was constructed; gene structures and chromosomal locations of CsCaM/CML were depicted; cis-acting regulatory elements were predicted; collinearity and duplicate events of CaM/CML were analyzed using MCScanX software; and the expression levels of CsCaM/CML in response to abiotic stress were analyzed, based on the published RNA-seq data. We identified 86 CsCaM/CML (4 CsCaMs and 82 CsCMLs) genes in total. Promoter sequences of CsCaM/CML contained elements related to abiotic stresses (including low-temperature and anaerobic stresses) and plant hormones (including abscisic acid (ABA), MeJA, and salicylic acid). CsCaM/CML genes were distributed on nine chromosomes unevenly. Collinearity analysis indicated that recent segmental duplications significantly enlarged the scale of the CML family in C. seticuspe. Four CsCMLs (CsCML14, CsCML50, CsCML65, and CsCML79) were statistically differentially regulated under low-temperature and salt stress compared with those in the normal condition. These results indicate diverse roles of CsCaM/CML in plant development and in response to environmental stimuli in C. seticuspe.

1. Introduction

Calcium (Ca2+), one of the most significant second messengers of signal transduction, is reported to have been involved in abiotic stress responses as well as plant growth and development [1,2,3]. Ca2+ was stimulated by various abiotic stresses, including salt, low-temperature, and oxidative stress [1]. The ability of Ca2+ as a messenger, interacting with other proteins that modulate plant stress and plant development, is guaranteed by Ca2+ sensors, such as calmodulin (CaM), Ca2+ protein kinases (CPKs) or Ca2+-dependent protein kinases (CDPK), calcineurin B-like proteins (CBL), and calmodulin-like proteins (CML) [4,5,6].
CaMs are conserved Ca2+-binding proteins, typically with four EF-hand motifs and CML, usually with 1–7 EF-hand motifs. Each EF-hand motif consists of two alpha helices which are connected by a 12-amino-acid residue loop [7]. The EF-hand motif can accommodate calcium and magnesium that which have subtle differences in affinity, as well as a wide range of induced conformational changes [8]. CaM/CMLs were involved in multiple processes related to plant development, growth, and various environmental stress responses [9]. Magnan et al. found that AtCML9 was significantly stimulated under ABA and abiotic stress treatments and showed a significant role in salt stress responses in Arabidopsis thaliana [10]. AtCML20 was negatively regulated under drought stress, while overexpression of rice CML OsMSR2 could enhance the drought tolerance of A. thaliana [11,12]. Rao and his team found that overexpressing GmCaM4 in soybean could enhance resistance to pathogens and salt stress [13]. Townley et al. found that AtCaM negatively regulated the expression of COR (Cold on regulated), D29A, KIN1, and KIN2 [14].
With the development of sequencing technology, the genome-wide identification of CaM/CMLs was analyzed in monocots (including rice (Oryza Sativa L.)), multiple eudicots (including brassicaceae plants such as A. thaliana, Chinese cabbage (Brassica rapa L. ssp. Pekinensis), Carica papaya, and Brassica oleracea), solanaceae plants (including tomato (Solanum lycopersicum and Solanum pennellii)), rosaceae plants (including woodland strawberry (Fragaria vesca) and apple (Malus × domestica)), fabaceae plants (including Lotus japonicas, as well as vitales plants (including grapevine (Vitis vinifera)) [15,16,17,18,19,20,21,22,23,24,25,26,27,28]. Chrysanthemum, one of the most industrially essential cut flowers worldwide, has charmed people with various flower colors and morphologies [29], and is susceptible to long-term cold stress [30]. Previous transcriptomic analysis revealed that five CML-resembling genes in Chrysanthemum nankingense were significantly changed under cold acclimation [31]. Recently, Nakano et al. constructed a pure diploid, a model strain, and Gojo-0 (C. seticuspe) with self-compatibility. They sequenced its genome and obtained a chromosome-level reference genome with 3.05 Gb, covering 97% of the C. seticuspe genome [29]. Therefore, it is necessary to perform genome-wide identification and expression analysis of CaM/CML genes in C. Seticuspe for candidate gene selection and further functional characterization.
In this study, a whole-genome-scale identification of CaM/CML genes was performed using CaM/CML members of A. thaliana with the accessibility of the chromosomal-level assembling genome of C. seticuspe [29]. A phylogenetic tree of CsCaM/CML and AtCaM/CML was constructed. Gene structures, motifs or domains, and cis-acting regulatory elements of promoters of CsCaM/CML genes were analyzed. Collinearity analysis of CaM/CML genes between C. seticuspe and A. thaliana was performed. The expression levels of CsCaM/CML members in response to cold stress and salt stress were analyzed.

2. Results

2.1. Identification and Phylogenetic Analysis of CaM/CML in C. seticuspe

A total of 86 CaM/CML were identified based on BLASTP and SMART analysis, containing 82 CML and 4 CaM gene family members. The phylogenetic analysis showed that 86 CsCaM/CML were divided into nine groups, from Group I to Group IX (Figure 1). Group I contained 4 CsCaM members, and 82 CsCMLs belonged to Groups II–IX.

2.2. Analysis of Gene Structure, Protein Motif, and Cis-Acting Regulatory Elements of Promoters

The gene structure of CsCaM/CML members was analyzed according to their coding and genomic sequences. Most CsCaM/CML gene family members in the same group shared similar gene structures (Figure 2a). The number of introns varied from 0 to 10. Almost all the members in Group V-VIII contained no intron except CsCML79 in Group V, CsCML11 in Group VI, CsCML37 in Group VII, and CsCML32 and CsCML82 in Group VIII. CsCaM/CML members in Group IX had the highest number of introns, generally greater than six. The motifs of CsCaM/CML were discovered by submitting the protein sequences using the online tools. The discovered motifs in the same group almost shared the same motifs (Figure 2b). The prediction of cis-acting regulatory elements of promoters showed that CsCAM/CML contained cis-acting regulatory elements associated with abiotic stresses such as low-temperature, drought, and anaerobic stress (Figure 2c); in addition, six elements involved in light response were observed. Elements in response to plant hormone and signaling molecules such as auxin, abscisic acid (ABA), MeJA, and salicylic acid were found in CsCMLs promoters (Figure 2c). CsCML63 contained 13 ABRE elements and 14 elements related to MeJA responses (Figure 2c).

2.3. Chromosomal Location and Cis-Acting Regulatory Elements of Promoters

According to the position of CsCaM/CML members on the C. seticuspe chromosome, chromosomal locations were depicted. Eighty-six CsCaM/CML gene family members distributed on nine chromosomes unevenly (Figure 3, Supplementary File S5). Chromosome 8 contained the highest number of CsCaM/CMLs, while chromosome 2 contained the lowest number of CsCaM/CMLs. CsCaM1, CsCaM2, CsCaM3, and CsCaM4 were located on chromosome 1, chromosome 2, chromosome 3, and chromosome 4, respectively.

2.4. Collinearity of CMLs in C. seticuspe

We obtained 926 collinearity blocks containing 14,449 genes in C. seticuspe and A. thaliana using collinearity analysis, which included 24 CMLs of C. seticuspe and 15 CMLs of A. thaliana (Figure 4a). In total, 469 blocks containing 6970 genes were observed among chromosomes of C. seticuspe (Figure 4b). Duplicate events analysis showed that all 4 CaMs and 45 CMLs in C. seticuspe were considered as dispersed duplications; 14 CMLs were proximal events; 5 CMLs were tandem duplications (Supplementary File S5); and 18 CMLs were recognized as whole genome (or segmental) duplications (Figure 4). Enrichment analysis indicated that whole genome duplications (WGD) of CMLs were significant with a p value of 0.0065.

2.5. Expression Analysis of CsCaM/CML in Response to Abiotic Stress

To explore the response of CsCaM/CML under abiotic stress, the RNA-seq data were downloaded from the ENA database. The expression levels of CsCaM/CML showed variety under cold stress compared with normal conditions (Figure 5A). Fifteen CsCMLs were statistically significantly regulated under cold treatments including four significantly up-regulated CMLs (CsCML2/3/9/80) and eleven significantly down-regulated CMLs (CsCML5/14/15/16/18/23/40/50/65/75/79) (Figure 5B). We found that most of CaM/CML genes in C. seticuspe were up-regulated under salt stress compared with normal conditions (Figure 6A). Expression-level analysis revealed that 13 CsCMLs were statistically significantly regulated in response to salt stress, containing 12 significantly up-regulated CMLs (CsCML14/34/38/45/50/60/65/67/69/70/79/81) and one significantly down-regulated CML (CsCML56) (Figure 6B).

3. Discussion

A total of 86 CsCML/CaM genes, including 82 CML members and 4 CaM members, were identified using BLASTP with 57 CML/CaM from A. thaliana as the query. The phylogenetic analysis showed that those CML/CaM can be divided into nine groups, including one CaM group containing seven AtCaMs and four CsCaMs (Figure 1), which is similar with the finding in previous studies [16,32]. Furthermore, 82 CML members in C. seticuspe were divided into eight groups unevenly (Figure 1). Protein motifs of CMLs showed diversity among different groups (Figure 2b). Cis-acting regulatory elements represented variety in CML genes promoters in C. seticuspe, which indicates the diverse functions of CMLs in plants [27]. EF-hand motifs were considered the only motif in CML and played crucial roles in interacting with binding calcium ions [9]. All CsCML genes identified in this study contained two or more EF-hands (Figure 2b), indicating they have necessities in binding calcium ions. The identity of amino acid sequences of CsCML and CaM in Arabidopsis ranged from 16% to 59% (except CsCML60 with identity from 78% to 92%); the identity of amino acid sequences of CsCML and predicted CaM in Chrysanthemum nankingense ranged from 19% to 58% (except CsCML60 with identity larger than 90%). Though CsCML60 was considered as one CML in this study based on the phylogenetic analysis, it might be a CaM considering that it has high identity with CaMs and it contains four typical EF-hand motifs. Moreover, though CsCaM3 has higher identity (> 96%) than CaM in Arabidopsis, more work needs to be carried out to explore whether it is a true CaM since it contains two EF-hand motifs and the amino acid sequence is much shorter than others. Despite CsCML60, the identity result was higher than that in Arabidopsis (16%) and relatively lower than that found in Solanum lycopersicum (24%~79%) [9,21].
A previous study identified recent segmental duplication in C. seticuspe [29]. In the present study, we observed 18 CsCMLs with whole genome (or segmental) duplications based on collinearity analysis (Figure 4b). Enrichment analysis indicated that WGD or segmental duplications played a significant role (with p value of 0.0065) in the expansion of CML genes in C. seticuspe, which supports the segmental duplication events in C. seticuspe. Additionally, collinearity analysis revealed 926 collinearity blocks between C. seticuspe and A. thaliana involving 24 CMLs in C. seticuspe and 15 CMLs in A. thaliana (Figure 4a), which is consistent with previous studies [27,32]. These results indicate the conservativeness and potential role of CML genes in plants adapting to the environment.
CMLs were reported to be involved in the response to environmental stresses. Knocking out AtCML9 could enhance the tolerance to drought and salinity in A. thaliana [10]. Vanderbeld and his co-workers analyzed the expression of CML in A. thaliana with promoters, and observed the different stimulations of AtCML37 and AtCML38 under salt, oxidative, and drought stress, as well as hormonal treatments [33]. In this study, we analyzed cis-acting regulatory elements of upstream sequences (2000 bp) of CaM/CML in C. seticuspe and observed various elements involved in abiotic stress (such as low-temperature, drought, and anaerobic stress) and plant hormone response elements such as auxin, ABA, MeJA, and salicylic acid (Figure 2c). Several CMLs contained multiple cis-acting regulatory elements in response to stress, for example, CsCML63 which contained 13 ABA-related elements (ABRE) and 7 elements associated with MeJA responsiveness (TGACG-motif) simultaneously (Figure 2c). This indicated their diverse functions in response to multiple environmental stress conditions in C. seticuspe. Expression analysis showed that 15 CMLs were statistically differently regulated under cold stress, containing 11 down-regulated CMLs (CsCML5/14/15/16/18/23/40/50/65/75/79) and 4 up-regulated CMLs (CsCML2/3/9/80); 13 CML genes (including 12 up-regulated genes (CsCML14/34/38/45/50/60/65/67/69/70/79/81) and a down-regulated gene (CsCML56)) showed statistically different expressions in C. seticuspe under salt stress compared with normal conditions (Figure 5 and Figure 6). Previous publications provided evidence of CML genes in response to diverse stimuli. Delk’s team found that AtCML24 showed responses to ABA, salt treatments, different daylengths, and long-day-induced transition to flowering [34]. The expression of SlCML26 in tomato (Solanum lycopersicum) was significantly regulated under cold, drought, and salt stress [21]. Expressions of four CsCMLs (CsCML14, CsCML50, CsCML65, and CsCML79) were statistically regulated under cold and salt stress (Figure 5 and Figure 6), which indicates diverse functions of these CsCMLs under diverse environmental stimuli.

4. Materials and Methods

4.1. Identification of CML/CaM Genes in C. seticuspe

To identify CML/CaM gene family members in C. seticuspe, 57 CML/CaMs from Arabidopsis thaliana were collected as query sequences to search the C. seticuspe genome using BLASTP (v2.6.0+) with a cut-off e-value of <10−15 [27,29]. Candidate CML/CaM genes were collected. Genes were mapped to a SMART database (http://smart.embl-heidelberg.de/) (accessed on 7 September 2021) and genes without EF-hand domains were removed.

4.2. Phylogenetic Analysis of CsCaM/CML

To explore the evolution of CsCaM/CML, an alignment of multiple sequences of CsCaM/CMLs from A. thaliana and C. seticuspe was performed using MAFFT software (v7) [35]. The phylogenetic tree was constructed using an approximately maximum likelihood method via FastTree (v2.1.11) software [36]. The protein sequences of CaM/CML from A. thaliana and C. seticuspe were provided in Supplementary File S1.

4.3. Gene Structure Construction and Protein Motif Prediction of CsCaM/CML

The coding sequences and the genomic sequences of CsCaM/CML were used to explore the gene structures of the CsCaM/CML gene family. The sequences were provided in Supplementary Files S2 and S3. The online website Gene Structure Display Server 2.0 (http://gsds.gao-lab.org/) (accessed on 15 September 2021) was used to depict the gene structure of gene family members based on the sequences submitted. The motif sites of CsCaM/CML protein were discovered using the online tool Multiple Em for motif elicitation (https://meme-suite.org/meme/tools/meme) (accessed on 20 September 2021) according to the CsCaM/CML protein sequences submitted [37].

4.4. Cis-Acting Regulatory Elements Analysis and Chromosomal Location

Upstream sequences (2000 bp) of CsCaM/CML gene family members were obtained from the C. seticuspe genome published recently [29]. The sequences were provided in Supplementary File 4. The online database PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 23 September 2021) was used to predict the cis-acting regulatory elements of CsCaM/CML promoter sequences. Based on the position information of CsCaM/CML members in the genome, the chromosomal location of CsCaM/CML was physically mapped to each chromosome and depicted with MapChart tools (v2.3.2) [38].

4.5. Collinearity and Duplicate Events Analysis of CaM/CML

Protein sequences of A. thaliana and C. seticuspe were used to perform collinearity analysis; for genes with multiple transcripts, the longest transcripts were used. The proteins of C. seticuspe were mapped to A. thaliana and C. seticuspe using BLASTP with cutoff of e-value < 10−5 (hits were restricted to top 5). Collinearity blocks between A. thaliana and C. seticuspe and in C. seticuspe were analyzed using MCScanX software with default parameters [39]. Duplicate events of CMLs in C. seticuspe were analyzed using the ‘duplicate_gene_classifier’ function in the MCScanX software and the results were visualized using circos (http://circos.ca/) (accessed on 27 September 2021) [39]. To examine potential origins of duplications of CMLs, enrichment analysis was performed using the “origin_enrichment_analysis” function in the MCScanX package.

4.6. Expression Analysis of CsCaM/CML in Response to Abiotic Stress

To explore the response of CsCaM/CML under cold and salt stress, the RNA-seq data were downloaded from the ENA database. The buds were raised for 30 days under controlled conditions. For salt stress (PRJNA472473), samples were watered with 100 mM salt water; the same amount of purified water was used as control. Root samples were collected after 24 h of treatment. For low-temperature stress (PRJNA481579), the seedlings were treated with 4 °C for 24 h and then treated with −4 °C for 4 h. Plants in normal conditions were used as the control; leaves were collected for sequencing. Each experiment contained 6 samples—3 biological replicates in each condition (cold acclimation, salt treatment, and normal growth). The trimming tool Trimmomatic (v0.39) was used to trim the sequences in the sequencing files [40]. The reference genome of C. seticuspe (Gojo-0) was downloaded from PlantGarden (https://plantgarden.jp/) (accessed on 30 September 2021). Trimmed files were mapped to the reference genome using HISAT2 (v2.2.1) software with default parameters [41], and the gene expression of each sample was calculated using HTSeq (v0.11.5) software [42]. Files containing read counts of genes were merged. Different statuses of genes between two conditions were calculated with the edgeR package (v3.30.3) [43]. Genes with |logFC| of >1.5 and FDR of <0.05 were considered differentially expressed genes (DEGs); DEGs were depicted with volcano plots and a heatmap using the R software.

5. Conclusions

Based on the published genome of C. seticuspe, 86 CsCaM/CML genes were identified and classified into nine subgroups. The size of the CML family in C. seticuspe was significantly enlarged via whole-genome (or segmental) duplications. CsCMLs represented significant responses to cold and salt stress. Four significantly up-regulated CMLs (CsCML2/3/9/80) and 11 significantly down-regulated CMLs (CsCML5/14/15/16/18/23/40/50/65/75/79) were identified under cold treatment. Thirteen CsCMLs (CsCML14/34/38/45/50/60/65/67/69/70/79/81/56) were statistically significantly regulated in response to salt stress. Our findings provide a basis and foundation for exploring the roles of CaM/CML genes in the development of C. seticuspe and their functions in response to diverse environmental stimuli and stresses.

Supplementary Materials

The following information is available online at https://www.mdpi.com/article/10.3390/plants11131760/s1. Supplementary File S1: Protein sequences of CaM:CML; Supplementary File S2: Coding sequences of CsCaM:CML; Supplementary File S3: Genomic sequences; Supplementary File S4: Promoter sequences of CsCaM:CML; Supplementary File S5: Summary information of CsCaM:CML family members.

Author Contributions

Conceptualization, M.F.; formal analysis, M.F. and C.W.; data curation, X.L. and X.D.; writing—original draft preparation, M.F.; writing—review and editing, F.G.; supervision, F.G.; funding acquisition, F.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding (grant number: 2021C02071-06) and Zhejiang Province Science and Technology Plan Project Provincial Key R&D Program (grant number: 2019C02025).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare that they have no conflicts of interest to report regarding the present study.

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Figure 1. The phylogenetic tree of CsCaM/CML and AtCaM/CML. The phylogenetic analysis was constructed using FastTree with 57 CaM/CML in Arabidopsis thaliana and 86 CaM/CML in Chrysanthemum seticuspe; different groups were color-coded.
Figure 1. The phylogenetic tree of CsCaM/CML and AtCaM/CML. The phylogenetic analysis was constructed using FastTree with 57 CaM/CML in Arabidopsis thaliana and 86 CaM/CML in Chrysanthemum seticuspe; different groups were color-coded.
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Figure 2. Sequences analysis of CsCaM/CML genes. Gene structures (a), motifs (b), and cis-acting regulatory elements (c) of CsCaM/CML gene family members were analyzed and depicted.
Figure 2. Sequences analysis of CsCaM/CML genes. Gene structures (a), motifs (b), and cis-acting regulatory elements (c) of CsCaM/CML gene family members were analyzed and depicted.
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Figure 3. Chromosomal locations of CsCaM/CML members. Distributions of CsCaM/CML genes on Chrysanthemum seticuspe chromosomes were depicted.
Figure 3. Chromosomal locations of CsCaM/CML members. Distributions of CsCaM/CML genes on Chrysanthemum seticuspe chromosomes were depicted.
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Figure 4. Collinearity analysis of CsCaM/CML genes. Circos plots displayed collinearity blocks between Arabidopsis thaliana and Chrysanthemum seticuspe (a) as well as within Chrysanthemum seticuspe (b). Links containing CsCaM/CML were colored with red; CaM/CML genes involved in collinearity blocks were labeled.
Figure 4. Collinearity analysis of CsCaM/CML genes. Circos plots displayed collinearity blocks between Arabidopsis thaliana and Chrysanthemum seticuspe (a) as well as within Chrysanthemum seticuspe (b). Links containing CsCaM/CML were colored with red; CaM/CML genes involved in collinearity blocks were labeled.
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Figure 5. Expression of CsCaM/CML under low-temperature stress. (A) The heatmap showed the expression of CsCaM/CML gene family members in response to cold stress; gene expressions were scaled in the row. (B) Significantly differentially (*) expressed genes were depicted with boxplots using transcripts per million (TPM). Significance level symbols with one, two, three, and four asterisks represent p values less than 0.05, 0.01, 0.001 and 0.0001, respectively.
Figure 5. Expression of CsCaM/CML under low-temperature stress. (A) The heatmap showed the expression of CsCaM/CML gene family members in response to cold stress; gene expressions were scaled in the row. (B) Significantly differentially (*) expressed genes were depicted with boxplots using transcripts per million (TPM). Significance level symbols with one, two, three, and four asterisks represent p values less than 0.05, 0.01, 0.001 and 0.0001, respectively.
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Figure 6. Expression of CsCaM/CML genes under salt stress. (A) Gene expression levels of CsCaM/CML gene family member response to salt stress were depicted with the heatmap; gene expressions were scaled in the row. (B) Significantly differentially expressed genes under salt treatment were depicted with boxplots using TPM. Significance level symbols with one, two, three, and four asterisks represent p values less than 0.05, 0.01, 0.001 and 0.0001, respectively.
Figure 6. Expression of CsCaM/CML genes under salt stress. (A) Gene expression levels of CsCaM/CML gene family member response to salt stress were depicted with the heatmap; gene expressions were scaled in the row. (B) Significantly differentially expressed genes under salt treatment were depicted with boxplots using TPM. Significance level symbols with one, two, three, and four asterisks represent p values less than 0.05, 0.01, 0.001 and 0.0001, respectively.
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Fu, M.; Wu, C.; Li, X.; Ding, X.; Guo, F. Genome-Wide Identification and Expression Analysis of CsCaM/CML Gene Family in Response to Low-Temperature and Salt Stresses in Chrysanthemum seticuspe. Plants 2022, 11, 1760. https://doi.org/10.3390/plants11131760

AMA Style

Fu M, Wu C, Li X, Ding X, Guo F. Genome-Wide Identification and Expression Analysis of CsCaM/CML Gene Family in Response to Low-Temperature and Salt Stresses in Chrysanthemum seticuspe. Plants. 2022; 11(13):1760. https://doi.org/10.3390/plants11131760

Chicago/Turabian Style

Fu, Manman, Chao Wu, Xia Li, Xiaoyu Ding, and Fangqi Guo. 2022. "Genome-Wide Identification and Expression Analysis of CsCaM/CML Gene Family in Response to Low-Temperature and Salt Stresses in Chrysanthemum seticuspe" Plants 11, no. 13: 1760. https://doi.org/10.3390/plants11131760

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