Next Article in Journal
Evaluation of Fungal Volatile Organic Compounds for Control the Plant Parasitic Nematode Meloidogyne incognita
Previous Article in Journal
Beta vulgaris L.—A Source with a Great Potential in the Extraction of Natural Dyes Intended for the Sustainable Dyeing of Wool
Previous Article in Special Issue
The Effect of Stress Hormones, Ultraviolet C, and Stilbene Precursors on Expression of Calcineurin B-like Protein (CBL) and CBL-Interacting Protein Kinase (CIPK) Genes in Cell Cultures and Leaves of Vitis amurensis Rupr
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 12
Issue 10
10.3390/plants12101934
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genome-Wide Identification and Expression Analysis of Calmodulin (CaM) and Calmodulin-Like (CML) Genes in the Brown Algae Saccharina japonica

by
Nianchao Xue
1,
Minghui Sun
1,
Zihan Gai
1,
Meihan Bai
1,
Juan Sun
2,
Shan Sai
2 and
Linan Zhang
1,*
1
School of Marine Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China
2
National Engineering Science Research & Development Center of Algae and Sea Cucumbers of China, Shandong Technology Innovation Center of Algae and Sea Cucumber, Provincial Key Laboratory of Genetic Improvement & Efficient Culture of Marine Algae of Shandong, Shandong Oriental Ocean Sci-Tech Co., Ltd., Yantai 264003, China
*
Author to whom correspondence should be addressed.
Plants 2023, 12(10), 1934; https://doi.org/10.3390/plants12101934
Submission received: 9 March 2023 / Revised: 30 April 2023 / Accepted: 2 May 2023 / Published: 9 May 2023
(This article belongs to the Special Issue Calcium Signaling in Plants: Roles in Stress Tolerance and Development)
Browse Figures
Versions Notes

Abstract

:
Calmodulins (CaMs) and Calmodulin-like proteins (CMLs) are vital in plant growth, development, and stress responses. However, CaMs and CMLs have not been fully identified and characterized in brown algae, which has been evolving independently of the well-studied green plant lineage. In this study, whole-genome searches revealed one SjCaM and eight SjCMLs in Saccharina japonica, and one EsCaM and eleven EsCMLs in Ectocarpus sp. SjCaM and EsCaM encoded identical protein products and shared 88.59–89.93% amino acid identities with Arabidopsis thaliana AtCaMs, thereby indicating that brown algae CaMs retained a similar Ca2+ sensors function as in plants. The phylogenetic and gene structure analysis results showed that there was significant divergence in the gene sequences among brown algae CMLs. Furthermore, evolutionary analysis indicated that the function of brown alga CMLs was relatively conserved, which may be related to the fact that brown algae do not need to face complex environments like terrestrial plants. Regulatory elements prediction and the expression analysis revealed the probable functioning of SjCaM/CML genes in gametophyte development and the stress response in S. japonica. In addition, the SjCaM/SjCMLs interacting proteins and chemicals were preliminarily predicted, suggesting that SjCaM/SjCMLs might play putative roles in Ca2+/CaM-mediated growth and development processes and stimulus responses. Therefore, these results will facilitate our understanding of the evolution of brown algae CaMs/CMLs and the functional identification of SjCaM/SjCMLs.

1. Introduction

Calcium ions (Ca2+) are one of the most widely available and used second messengers in eukaryotic cells [1,2]. In growth and developmental processes and during abiotic and biotic stress conditions, rapid changes in Ca2+ levels in cells caused either by extracellular events or Ca2+ influx can trigger physiological changes and help coordinate adaptive responses. The Ca2+ signaling must be decoded, relayed, and amplified by Ca2+-binding proteins (Ca2+ sensors), which bind diverse downstream protein targets to carry out the appropriate responses [3,4]. Eukaryotes have evolved an arsenal of Ca2+-binding proteins, which usually contain multiple paired EF-hand motifs and a helix–loop–helix structure. They can be divided into different protein families, mainly including calmodulin (CaM), CaM-like protein (CML), calcium-dependent protein kinase (CDPK), and calcineurin B-like proteins (CBL) [5,6,7,8].
CaM is the most quintessential and ubiquitous Ca2+-sensing protein in eukaryotes [9,10]. It typically comprises 148 residues with four EF-hand motifs that change their conformation upon binding to Ca2+. Each EF-hand motif contains two alpha helices that are connected by a 12 amino acid residue loop. CaM was the most conserved during the evolution of the Ca2+-sensing protein. All known vertebrate CaMs are identical in amino acid sequence and share 91% amino acid identity with plant CaMs [11]. Similar to CaM, CML contains only the EF-hand domain and lacks any other functional domains or intrinsic activities. However, CML has only been found and characterized in plants and certain protist groups [5]. CML is more widely divergent than CaM at the structural level. It shows variations in EF-hand sequences and EF-hand motif numbers, even in a single species [12]. Over the past several years, the whole-genome sequencing of an increasing number of plant species has helped identify increasing CaM and CML family proteins in multiple plant species, including Arabidopsis thaliana [11], Oryza sativa [13], Glycine max [14], Vitis vinifera [15], Brassica napus [16], Solanum pennelllii [17], Hordeum vulgare [18], and Triticum aestivum [19], with their diverse and multiple functions being explored and analyzed worldwide. Increasing number of reports showed that CaM and CML genes are vital in plant growth and development [20]. In Arabidopsis, CaMs participate in brassinosteroid hormone biosynthesis and pollen development via their downstream targets, i.e., DWF1 and NPG1, respectively [21,22]. CMLs are also involved in multiple developmental processes, such as flowering, pollen germination and tube elongation, seedling development, and trichome branching [23,24,25,26]. Additionally, multiple studies supported the idea that CaMs, CMLs, and downstream elements are also important players in the disease and herbivore resistance and abiotic stress (e.g., salinity, temperature, and drought) response. Most Arabidopsis CaMs/CMLs are an integral part of the regulatory network of multiple signaling pathways of both abiotic and biotic stress tolerance, including CAM7 [27], CML8 [28,29], CML37 [30,31], and CML42 [32]. Recently, the evolution of CaMs and CMLs in different representative species of the green plant lineage has been published [12]. The expansion of the CaM and CML families from algae to land plants was supposed to be driven by selective pressure to enable the adaptation to varied terrestrial environments and the development of novel functions necessary for colonizing new habitats [12]. However, the identification of CaM and CML in other eukaryotic lineages except plants and animals is currently limited, and their functions are poorly understood.
Brown algae belong to the group Stramenopiles in the TSAR eukaryotic lineage, a eukaryotic branch highly divergent from animal, fungi, and green plant lineages [33]. In brown algae, the preliminary studies related to calcium and calcium-binding proteins were only found in the development process of fucoid algae. The cytoplasmic-free calcium gradient was detected in eggs and growing rhizoid cells [34,35]. Meanwhile, calmodulin and calmodulin-binding proteins were found to be involved in diverse physiological processes, including fertilization, zygotic photopolarization, and early embryonic development through biochemical analysis [36,37,38]. Recently, the whole genomes of multiple brown algae (Ectocarpus, Saccharina, Cladosiphon, and Undaria) have been reported, which facilitated the screening of the CaM and CML family of brown algae at the whole-genome scale [39,40,41,42,43]. In the previous studies, some calcium-binding-related proteins of brown algae were annotated and these showed differential expression patterns among different developmental stages and between the sexes [44,45]. However, the identification of CaMs and CMLs and their potential functions in brown algae still remain unclear.
Ectocaropus is a model brown algae system for studying developmental processes, life cycle, and evolution [46,47,48,49,50]. Saccharina japonica is the most commercially important kelp species in China [51]. In this study, CaM and CML genes were identified in Ectocarpus sp. and S. japonica, respectively. Their phylogenetic relationships, gene structures, selection pressure, cis-acting elements, and interacting proteins were analyzed subsequently. Furthermore, we conducted the expression pattern analysis of the SjCaM/SjCML genes using both RNA-seq and qRT-PCR data. Therefore, our findings not only provide a comprehensive analysis of the CaM/CMLs in brown algae but also facilitate further functional identification of SjCaM/SjCMLs in S. japonica.

2. Results

2.1. Identification of CaM/CML Genes in Brown Algae

Using a combination of the results of BLASTP and HMMER research, we obtained a total of 26 and 35 CaM/CML proteins containing EF-hand domain in the S. japonica and E. sp. protein databases, respectively (Table S1). Then, we discarded those that contained domains other than the EF-hand domain and whose polypeptide length was significantly longer than CaMs. Finally, we designated the 9 and 12 filtered sequences as SjCaM/SjCMLs and EsCaM/EsCMLs, respectively (Table 1 and Table S2). Among them, we identified only one SjCaM (SJ01891) and one EsCaM (Ec-06_002270.1), and they shared 88.67% identity at the nucleotide level while encoding identical protein products. SjCaM/EsCaM had the same protein length (149 aa), number of EF-hands (4), and high degrees of amino acid identities (88.59–89.93%) as AtCaMs (Table S3). They also showed shared conserved amino acid residues and sequences-related functionality with plant CaMs, including lysine at position 116, six amino acids (at positions 1, 3, 5, 7, 9, and 12) serving as Ca2+-binding ligands in the Ca2+ binding loop in each EF-hand, and the distribution pattern with hydrophobic amino acids in both the E and F helices (Table 1; Figure 1). Additionally, we also detected a high proportion of methionine (~7.4%) in brown alga CaMs (Table 1).
We identified a total of 8 SjCMLs and 11 EsCMLs and named them in the order of their respective protein sequence identity to SjCaM/EsCaM (Table 1). All the CMLs shared >26% identity with SjCaM/EsCaM and contained at least two EF-hand domains. Over half the CMLs contained four EF-hand domains for each species. The protein length, pI, MW, and methionine percentage (M%) of SjCMLs ranged from 130 to 215 aa, 4.04 to 6.10, 14.60 to 23.47 kDa, and 2.2% to 6.9%, respectively. We found similar characteristics in EsCMLs. Their length, pI, MW, and M% ranged from 154 to 182 aa, 4.03 to 5.42, 17.13 to 20.39 kDa, and 2.9% to 5.5%, respectively. The subcellular location prediction indicated that the CaM proteins were localized in the cytoplasm and the CMLs proteins were localized in the cytoplasm, chloroplast, nucleus, mitochondrion, and peroxisome.

2.2. Phylogenetic and Gene Structure Analysis of the Brown Algae CaMs/CMLs

We constructed the phylogenetic tree by taking the protein sequences of CaMs/CMLs from A. thaliana, Chlamydomonas reinhardtii, S. japonica, and E. sp. (Table S4), and showed three monophyletic groups in the tree (Figure 2). Group I contained eight SjCMLs, eight EsCMLs, and all CaMs. Group III comprised twelve CMLs and three of them were EsCMLs. No brown algae CaMs/CMLs were found in Group II, which only consisted of 32 AtCMLs. In Group I, brown algae CaMs/CMLs (except SjCML7) were assigned in a clade, whereas all AtCaMs were clustered in another clade.
We further comprehensively analyzed the phylogeny, conserved motifs, and gene structures of brown algae CaMs/CMLs (Figure 3 and Table S5). In cases where no full-length cDNA was available, we used partial cDNA (SjCML3 and SjCML5). As shown in Figure 2, 18 brown algae CaMs/CMLs were clustered in one Group (I) and three EsCMLs were clustered in another Group (III). In Group I, CaMs and CMLs were divided into two distinct clades, respectively. CaM genes only contained one intron, which is the same as CaM genes in other plants. Contrastingly, CML genes were intron-rich. The CMLs clade was divided into two subclades. CML genes in one subclade (SjCML2, SjCML3, EsCML2, EsCML3, and EsCML5) and CaMs genes shared the same motif number and similar motif arrangements. CMLs genes in another subclade showed more diversity in the numbers and the structure of introns and motifs.

2.3. Chromosomal Distribution and Duplication Analysis of Brown Algae CaMs/CMLs

The nine SjCaM/CML genes were distributed on seven chromosomes in the genome. Both chromosome 0 and chromosome 12 contained two SjCMLs, and the other five chromosomes contained only one SjCaM/SjCML, respectively (Figure S1). The distance between two SjCML genes on the same chromosome was far >150 kb, which indicated that the tandem duplication region was not formed among the SjCaM/CML genes in this study. Meanwhile, the segmental duplication event was not detected using the MCScanX method with E-value < 1 × 10−10. The twelve EsCaM/EsCML genes were distributed on eight chromosomes, and we also did not detect tandem duplication and segmental duplication in E. sp.

2.4. Molecular Evolution of Brown Algae CaMs/CMLs

To assess the selection pressure of CaMs/CMLs in brown algae, we used the CODEML of the PAML package to compute the dN, dS, and ω values. We found that the ω value of all the brown algae CaMs/CMLs genes was <1 and very low, thus indicating that these genes underwent strong purifying selection (Figure S2). Although ω did not differ significantly among Groups I-CaM, I-CML, and III, the average ω and dN values in Groups I and III (CML genes) were higher than those in Group I (CaM genes), thus suggesting that CMLs may have more extensive functional diversity than CaMs in brown algae.

2.5. Cis-Element Analysis of Brown Algae CaMs/CMLs

We explored the 2 kb upstream region of the start codon ATG of the SjCaM/SjCML and EsCaM/EsCML genes obtained from the genomic sequence. We identified 41 and 38 cis-acting elements in promoter regions of SjCaM/SjCML and EsCaM/EsCML genes, respectively (Figure 4 and Table S6). Among them, the ABRE motif, G-box motif, CAAT-box motif, and TATA-box motif were ubiquitously detected in all brown algae CaM/CML genes, thereby indicating their potential critical roles in ABA responsiveness, light responsiveness, and controlling the transcription initiation. Additionally, the CGTCA-motif and TGACG-motif involved in MeJA responsiveness and the stress-related GC-motif involved in anoxic-specific inducibility were also widely presented in both the SjCaM/SjCML and EsCaM/EsCML genes.

2.6. Expression Analysis of SjCaM/SjCML Genes

We evaluated the expression profiles of all the SjCaM/SjCML genes during different life cycle stages using the RNA-sequencing data (Figure 5). In the gametophytic generation, SjCaM/SjCML genes showed variable expression patterns with the gametophyte development. In the mature gametophyte stage, the expressions of SjCaM and four SjCMLs (SjCML1, SjCML2, SjCML5, and SjCML8) were upregulated, whereas that of SjCML7 was downregulated. In contrast, the SjCML6 expression was related to the sexes of gametophyte and was higher in the mature stage than in the immature stage for male gametophytes and vice versa for female gametophytes. There was no significant difference in expression for the remaining two SjCML genes (SjCML3 and SjCML4). Additionally, two SjCML genes (SjCML3 and SjCML6) showed male-biased expression during the mature stage.
As compared to the gametophytic generation, most genes were expressed at lower levels in the sporophytic generation, except SjCaM and SjCML2. Although the expressions of five SjCML genes (SjCML1, SjCML3, SjCML4, SjCML5, and SjCML7) showed fluctuations during the sporophyte development process, the difference was not significant in pairwise comparisons between the six developmental stages. Notably, the expression of two SjCML genes (SjCML6 and SjCML8) could not be detected in any sporophyte developmental stages. Contrastingly, SjCaM was highly expressed in the SPsp1-Spsp3 stages followed by a downregulation in the SPsp4-Spsp6 stages. The SjCML2 expression pattern shared similarity with that of SjCaM, besides its upregulated expression during the SPsp6 stage.
To further verify the expression patterns of these SjCaM/SjCMLs genes, we selected six genes with significant differences during gametophyte development for qRT-PCR analysis (Table S7). The data of these genes were highly consistent with the RNA-seq data (Figure S3). Therefore, these results further suggested that SjCaM/SjCMLs were involved in gametophyte development and could be considered as candidates for further studying their function in S. japonica.

2.7. Interaction Network Analysis of SjCaM/CMLs

We constructed the protein interaction network referring to Ectocarpus orthologous proteins to speculate the relationship and synergy between SjCaM/SjCMLs and other S. japonica proteins. A total of six SjCaM/SjCMLs interacted with 57 proteins, while three SjCMLs (SjCML1, SjCML5, and SjCML8) did not interact with any proteins (Figure 6a). Among these interacting proteins, calcineurin-like phosphoesterases were the most abundant (Table S8). In addition, five proteins were predicted to interact with all six SjCaM/SjCMLs, including a protein kinase (SJ01496), a cytoskeletal adhesion protein (SJ14075), a tubulin protein (SJ22156), a SAC3/GANP protein (SJ12208), and a hypothetical protein (SJ14390) (Figure 6a). GO functional enrichment analysis showed that a total of 151 GO terms enriched for SjCaM/SjCMLs interacting proteins, including 113 (74.8%) biological process terms (BP), 25 (16.6%) molecular function terms (MF), and 13 (8.6%) cellular component terms (CC). The interacting proteins were significantly enriched in “cellular process” and “response to stimulus” in the BP category, “cell” and “cell part” in the MF category, and “non-membrane-bounded organelle” in the CC category (Table S9). The KEGG pathway analysis revealed that four pathways, including “Nucleotide excision repair”, “Phagosome”, “mRNA surveillance pathway”, and “Mismatch repair”, were significantly enriched (Figure S4).
Due to the lack of protein–chemical interaction information on brown algae in the STITCH database, we constructed the protein–chemical interaction network referring to the organism of A. thaliana. The result suggested the interaction of four SjCaM/CMLs with two different chemicals: trifluoperazine and calcium ions (Figure 6b). Among them, the SjCaM protein interacted with both chemicals. The two proteins SjCML2 and SjCML3 showed interaction with trifluoperazine, while the SjCML5 protein interacted with calcium ions.

3. Discussion

As Ca2+ sensors, CaM and CML genes are widely involved in plant growth and development and also the mediation of plant stress tolerance. However, whole-genome-scale studies about CaM/CML families have not been performed in brown algae, which have evolved independently of the well-studied green plant lineage. In this study, we identified one SjCaM/8 SjCMLs and one EsCaM/11 EsCMLs in S. japonica and E. sp., respectively. The size of the CaM/CML families in brown algae was smaller than those in the plant species, but showed similarity with green algae, including Klebsormidium flaccidum (one CaM and eight CMLs) and Chlamydomonas reinhardtii (one CaM and nine CMLs) [12]. In the green lineage, two major transitions in the evolution of CaM and CML gene numbers from green algae to angiosperm were likely associated with the acquisition of multicellularity, the adaption of terrestrial environments, and the development of novel functions necessary for colonizing new habitats. In brown algae, Ectocarpus is multicellular single-row filamentous, whereas the Saccharina sporophyte showed a morphological differentiation of rhizoids, stipes, and blades. Nonetheless, a limited number of CaM and CML genes were found in these two marine brown algae species, thus indicating that the acquisition of novel tissue differentiation-related functions could not be the evolutionary driving force of the expansion of CaM/CMLs in brown algae.
We constructed an ML phylogenetic tree based on amino acid sequences from brown algae (S. japonica and E. sp.) and green lineage species (C. reinhardtii and A. thaliana) to determine the phylogenetic relationship and further validate the identification of brown algae CaM/CMLs. As shown in Figure 2, all the CaMs were assigned to Group I. SjCaM and EsCaM were closer to AtCaMs than CrCaM, with CrCaM forming the basal root. This result indicates that plant and brown algae CaMs evolved from the common ancestor of basal unicellular eukaryotic lineages, which may have originated from the bacterial endosymbiotic event due to the presence of EF-hand-containing proteins in many bacteria and the widespread presence of CaMs among eukaryotes [12,52]. In Group I, most brown algae CaMs/CMLs were clustered in a different clade to AtCaMs, indicating the independent evolution of brown algae CaMs/CMLs. Additionally, most AtCMLs were clustered in Group II, in which no brown algae CMLs and green algae CMLs were found, thus illustrating that these AtCMLs might have evolved by duplication and acquired their diverse functions to respond to environmental signals as plants have adapted to their terrestrial environments.
In this study, we identified only one SjCaM and one EsCaM. They encoded identical protein products and showed high degrees of amino acid identities (88.59–89.93%) and typical gene structure characteristics with AtCaMs, including lysine at position 116, six conserved amino acids in the Ca2+ binding loop, the distribution pattern of hydrophobic amino acids in both the E and F helices, a high proportion of methionine, four EF-hand motifs, and one intron. The highly conserved structure of CaMs shared between S. japonica/E. sp. and A. thaliana indicates that brown algae CaMs may retain the similar function of being Ca2+ sensors in cell signaling, like in plants. In contrast, CMLs showed greater diversity in length, gene structure, methionine percentage, and EF-hand number in brown algae. The amino acid identity ranged from 19.88% to 61.94% among the SjCMLs (Table S10). However, the tandem duplication and segmental duplication events were not detected in brown algae CaM/CMLs. The molecular evolution analysis showed brown alga CMLs had undergone strong purifying selection during evolution, thus indicating that the function of brown alga CMLs may be relatively conserved, and the functional differentiation is limited. This may be related to the fact that brown algae do not need to face complex environments like terrestrial plants.
CaM and CML genes play diverse and multiple roles in plant growth, development, and stress responses. To explore the potential functions of brown algae CaMs/CMLs, we first analyzed the cis-acting elements due to their key role in gene expression regulation. The results showed that “light response elements” had the highest abundance of cis-acting elements in both SjCaM/SjCMLs (41.5%) and EsCaM/EsCMLs (36.8%), with all the brown algae CaMs/CMLs identified in this study containing the light-responsiveness-related element (G-box motif). This result indicates that CaM/CMLs may be vital in the growth and developmental processes of brown algae. Additionally, the ABRE motif was also ubiquitous in the brown algae CaMs/CMLs. ABRE is a key cis-regulatory element involved in ABA signaling and mediates plant response to many abiotic stresses, including osmotic, drought, heat, and cold [53,54]. Thus, we speculated that CaM/CML genes may be involved in the mediation of ABA signaling in the abiotic stress response of brown algae. Then, we analyzed the expression patterns of SjCaM/SjCML genes in different development stages of two life cycle generations. Generally, the expressions of SjCaM/SjCML genes in gametophyte generation were greater than those in sporophyte generation, thus suggesting they may be mainly involved in the regulation of gametophyte developmental. SjCaM/SjCML genes showed various expression patterns during gametophytic development. For example, one SjCaM gene and four SjCML genes were induced and one SjCML7 gene was repressed in mature gametophytes, thus indicating that these genes could participate in gametophyte growth and development by cis-regulation and trans-regulation, respectively. The expressions of SjCML3 and SjCML6 were upregulated significantly only in the mature male gametophytes. The male-biased expression in mature gametophytes indicated these two genes may be mainly involved in spermatogenesis or sperm development. Although the expression of CaM/CML genes during fertilization, zygotic, and early embryonic development was not explored in this study, CaM’s involvement in these developmental processes in brown algae has been reported in previous studies [36,37,38]. Based on the culture conditions of S. japonica gametophytes and the cis-acting element characteristics of SjCaM/SjCML genes, we discussed the factors that induced gene expression changes. S. japonica gametophytes are microscopic, filamentous, and dioicous. The gametophyte generation has a very short life cycle as it takes only ~20 days from gametophyte formation to the release of gametes through gametogenesis in natural conditions. To preserve gametophytes in the lab for a long time, we cultured the immature gametophytes under vegetable growth conditions, which is regarded as the adaptive form for stressful environments [55]. Light intensity, photoperiod, and nutrient status were important conditions for the initiation of gametogenesis and the maturation of gametophytes in brown algae, including kelp [56,57,58,59]. Therefore, we adjusted the conditions of the light intensity, photoperiod, and culture medium to obtain mature gametophytes in this study. Meanwhile, many light-responsive elements (e.g., G-box motif) were ubiquitous in the upstream region of the identified brown algae CaM/CML genes, thus indicating that these genes directly responded to light-related stress recovery to be involved in the pathway of gametophytes maturation. Additionally, hormone-responsive elements, including ABRE, CGTCA-motif, and TGACG-motif, were also widespread in the promoters of the brown algae CaM/CML genes. This indicates that these genes may be indirectly involved in stress response via the hormone regulation pathway. Under external stimuli, cytosolic Ca2+ concentration increased through either the influx of extracellular Ca2+ or the temporary release of intracellular stores. Ca2+ binding proteins decoded Ca2+ signals and regulated the corresponding physiological processes subsequently. A calcium influx induced by gamete fusion played a direct role in egg activation during the development of brown algae [60]. This indicates that the influx of calcium ions in the culture medium via the Ca2+ channel may have led to changes in the expression of CaM and CMLs during gametophyte development in this study. However, we cannot rule out the mobilization of intracellular Ca2+ from storage compartments, such as the vacuole and the endoplasmic reticulum.
The sporophytes we collected from January to July in this study should have faced variable environmental conditions. Unexpectedly, the expressions of most SjCML genes were very low and did not change significantly during the generation of sporophytes. The differential expression detected in the SjCaM and SjCML2 genes may be related to the reduced growth rate at the SJsp4 stage and the appearance of sporangia on the sporophyte blade at the SJsp6 stage [61].
We predicted a total of 57 interacting proteins using high-confidence criteria (interaction score ≥0.70) with STRING. The protein interaction network showed that calcineurin-like phosphoesterases were the most abundant. The calcineurin-like phosphoesterases family includes a diverse range of phosphoesterases, which are related to the calcium- and calmodulin-dependent serine/threonine protein phosphatase. They could be involved in signal transduction related to various stress responses in plants, such as p starvation, drought stress, and zine exposure [62,63,64]. There were five proteins predicted to interact with all six SjCaM/SjCMLs. SJ01496 encodes a protein kinase that contains an STKc_CAMK domain. It may be a key player in the Ca2+/CaM-mediated signaling pathway by catalyzing protein phosphorylationas as a calmodulin-binding protein kinase [65,66]. SJ14075 and SJ22156 encode a cytoskeletal adhesion protein and a tubulin protein, respectively. They may play a role in the regulation of microtubule cytoskeleton in growth and development as CaM-binding microtubule-associated proteins [67]. SJ12208 was predicted to be a SAC3/GANP family protein. A recent experiment demonstrated that CML19 could interact with SAC3B to form the transcription export complexes in A. thaliana [68]. These genes could be used as candidates for studying the regulatory network of CaM/CMLs. The protein–chemical interaction analysis has been used to understand the molecular and cellular function of proteins [69,70]. In this study, the interaction of SjCaM/CMLs with trifluoperazine and calcium ions indicated their probable role in Ca2+ signaling. Trifluoperazine is a well-known CaM antagonist that can bind to CaM and modulate the interactions between CaMs and their target biological processes [71,72]. The protein–protein and protein–chemical interactions found in this study need to be validated in future studies in brown algae.

4. Materials and Methods

4.1. Identification and Characterization of CaM/CML Genes in S. japonica and E. sp.

The genome and protein sequences of brown algae (S. japonica and E. siliculosus V2) were downloaded from ORCAE (https://bioinformatics.psb.ugent.be/orcae/, accessed on 10 September 2022). The protein sequences of CaMs/CMLs in A. thaliana [11,73] and O. sativa [13] were downloaded from TAIR (http://www.arabidopsis.org, accessed on 10 September 2022) and RAP-DB (https://rapdb.dna.affrc.go.jp/, accessed on 10 September 2022), respectively, and used as queries (Blastp, E-value < 1×10−5) to search against the brown algae genome database. The obtained sequences from S. japonica and E. sp. were further used to construct the hidden Markov model (HMM) profile using HMMER 3.3.2 (http://hmmer.org/download.html, accessed on 11 September 2022), which was then searched against the local protein database of each species. Based on the results of BLAST hits and HMMER, the common sequences of each species were selected and manually examined for the EF-hand domain using the Pfam/NCBI/SMART database. Those proteins containing only the EF-hand functional domain and <400 amino acids were considered putative SjCaM/SjCML and EsCaM/EsCML proteins for S. japonica and E. sp., respectively. The theoretical isoelectric point (pI) and molecular weight (Mw) of each CaM/CML protein was calculated using ExPASy (https://web.expasy.org/compute_pi/, accessed on 15 September 2022). Subcellular localization of each SjCaM/SjCML and EsCaM/EsCML protein was predicted by the WoLF PSORT tool (https://www.genscript.com/wolf-psort.html, accessed on 19 April 2023).

4.2. Alignment and Phylogenetic Analysis of Brown Algae CaM/CML Genes

The sequences of CaM/CML proteins of C. reinhardtii were obtained from Phytozome (https://phytozome-next.jgi.doe.gov/, accessed on 25 September 2022). Alignments were performed by the multiple sequence alignment program ClustalW using default settings. The alignment of CaM proteins was viewed using SeqVu1.0.1 (Garvan Institute of Medical Research, Sydney, Australia). The Maximum-Likelihood (ML) phylogenetic tree was constructed using ATGC PhyML 3.0 with 1000 bootstrap replicates (http://www.atgc-montpellier.fr/phyml/, accessed on 15 October 2022) [74] and was displayed using Evolview: Tree View (http://www.evolgenius.info/evolview/, accessed on 17 October 2022) [75].

4.3. Gene Structure and Cis-Acting Elements Analysis of Brown Algae CaM/CML Genes

Structure information on the SjCaM/SjCML and EsCaM/EsCML genes was obtained from their respective GFF files. The conserved motifs of CaMs/CMLs were analyzed using the MEME program (https://meme-suite.org/meme/tools/meme, accessed on 20 October 2022) with a motif number setting of 10 [76]. Gene structures and motifs were visualized using the TBtools software (https://github.com/CJ-Chen/TBtools/releases, accessed on 8 March 2023; Version number: v1.108; Creator: Chen Chengjie; Guangzhou, China) [77]. To assess the regulatory elements of brown algae CaM/CML genes, 2000 bp upstream of the coding sequences of SjCaM/SjCML and EsCaM/EsCML genes were extracted for the cis-acting element analysis using the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 25 October 2022) [78].

4.4. Chromosomal Distribution, Gene Duplication, and Selection Pressure of Brown Algae CaM/CML Genes

The chromosomal location information on the brown algae CaM/CML genes was also obtained from the GFF file, and the chromosomal distribution of SjCaM/SjCMLs and EsCaM/EsCMLs was presented using the TBtools software [77]. The duplication events of SjCaM/CMLs were analyzed by MCScanX program in TBtools software. To evaluate the variation in selective pressures for each CaM/CML gene or groups of CaM/CML genes, the nonsynonymous substitution values (dN), synonymous substitution values (dS), and nonsynonymous/synonymous substitution rate (ω = dN/dS) were calculated using the CODEML program in the PAML software package (http://abacus.gene.ucl.ac.uk/software/paml.html, accessed on 8 March 2023; Version number: 4.9j; Creator: Yang Ziheng; London, UK) [79].

4.5. Expression Analysis of the SjCaM/SjCMLs in Different Life Cycle Stages

To gain insight into the expression characteristics of the SjCaM/SjCML genes at different developmental stages, the transcriptome sequencing data were obtained from our previous studies under the accession numbers PRJNA656182 and PRJNA511944, which represented the gametophyte and sporophyte generation, respectively [45,61]. Two male gametophyte developmental stages (SIm and SMm), two female gametophyte developmental stages (SIf and SMf), and six sporophyte developmental stages (SJsp1–SJsp6) were subjected to RNA-sequencing. Three biological replicates were tested for each condition. The FPKM value (Fragments Per Kilobase of transcript per Million fragments mapped) of each SjCaM/SjCML gene was calculated and normalized using the log2-transformed values to generate heatmaps. Differential gene expression levels among the different samples were analyzed based on an absolute log2 (fold change) > 1 and adjusted p-value < 0.05.

4.6. qRT-PCR Analysis

The six differentially expressed SjCaM/SjCML genes involved in gametophyte generation were selected for qRT-PCR analysis. The immature gametophytes were preserved in seawater (supplemented with 12 μmol NaNO3 and 7.35 μmol KH2PO4) at 10 °C under a constant irradiance of 15–20 μmol photos m−2 s−1. The mature gametophytes were collected after being cultured at 10 °C in the Provasoli-enriched natural seawater [80] under 40–50 μmol photons m−2 s−1 with a photoperiod of 12:12 h (light/dark). Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s procedure. The cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Dalian, China). The expression profile of the SjCaM/SjCML genes was examined using SYBR Green PCR Master Mix (Takara, China). The EF1α was selected as the internal control and the 2−ΔΔCt method was used to assess the expression level of genes [81]. The expression levels were compared using t-test (p < 0.05) in the SPSS 19.0 software (https://www.ibm.com/spss, accessed on 8 March 2023; Version number: 19.0; New York, NY, USA).

4.7. Protein–Protein and Protein–Chemical Analysis

The CaMs/CMLs protein–protein and protein–chemical interactions were performed using the STRING (https://cn.string-db.org/, accessed on 10 December 2022) and STITCH (http://stitch.embl.de/, accessed on 21 April 2023) servers, respectively. Protein interaction network was constructed with the reference organism of E. siliculosus. Protein–chemical interaction network was constructed with the reference organism of Arabidopsis thaliana. Proteins or chemicals with a combined interaction score ≥ 0.70 were selected. The network was visualized using Cytoscape (version 3.9.1). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of interaction proteins were performed the TBtools software [77].

5. Conclusions

This study was the first to systematically identify CaM and CML genes in brown algae. There were one SjCaM/eight SjCMLs and one EsCaM/eleven EsCMLs found in S. japonica and E. sp., respectively. The phylogenetics, gene structures, gene duplication, and molecular evolution analysis helped investigate their evolutionary history. According to cis-acting regulatory element and expression analysis, CaM/CMLs might be involved in the development of and stress response in brown algae, and their expression patterns in gametophyte development were also validated in S. japonica by qRT-PCR. The interaction analysis suggested the involvement of SjCaM/CML genes in Ca2+/CaM-mediated developmental processes and stress responses and provided the candidates for studying the CaM/CMLs regulatory network. This study indicates that SjCaM/SjCMLs have diverse functions in gametophyte development, but the specific role of each gene needs to be further determined in future research.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/plants12101934/s1, Figure S1: Distribution of CaM/CML genes in chromosome of S. japonica (a) and E. sp. (b); Figure S2: Nonsynonymous/synonymous substitution rate (ω) of brown algae CaM/CML genes in different phylogenetic groups; Figure S3: Validation of gene expression by qRT-PCR; Figure S4: KEGG enrichment analysis of SjCaM/SjCMLs interacting proteins; Table S1: List of EF-hand-domain-containing proteins in S. japonica and E. sp.; Table S2: Protein sequences of brown algae CaMs/CMLs; Table S3: Protein (lower left) and nucleotide (upper right) sequence identity (%) of CaMs among the brown alga and green lineage; Table S4: CaMs and CMLs proteins used for phylogenetic analysis and their group information; Table S5: The amino acid sequence of conserved motifs of CaM and CML proteins; Table S6: Cis-acting elements in promoter regions of brown algae CaMs/CMLs; Table S7: List of qRT-PCR primers used in this study; Table S8: List of SjCaM/SjCMLs interacting proteins; Table S9: GO enrichment of SjCaM/SjCMLs interacting proteins; Table S10: Protein (lower left) and nucleotide (upper right) sequence identity (%) of CaM and CMLs in S. japonica.

Author Contributions

N.X. and L.Z. conceived and designed the research. N.X., M.S. and M.B. performed the experiment; N.X. and Z.G. wrote the original draft.; J.S. and S.S. provided the samples and helped perform the experiment. L.Z. obtained the funding and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32072962); Agricultural Industrial Technology System in Shandong Province (SDAIT-26); “First Class Fishery Discipline” Programme [(2020)3] in Shandong Province; a Special Talent Programme “Yishi Yiyi” in Shandong Province, China; China Agriculture Research System (CARS-50); Shandong Province Agriculture Seed Project (2021LZGC004).

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Clapham, D.E. Calcium signaling. Cell 1995, 80, 259–268. [Google Scholar] [CrossRef]
  2. Laude, A.J.; Simpson, A.W. Compartmentalized signalling: Ca2+ compartments, microdomains and the many facets of Ca2+ signalling. FEBS J. 2009, 276, 1800–1816. [Google Scholar] [CrossRef]
  3. Niki, I.; Yokokura, H.; Sudo, T.; Kato, M.; Hidaka, H. Ca2+ signaling and intracellular Ca2+ binding proteins. J. Biochem. 1996, 120, 685–698. [Google Scholar] [CrossRef]
  4. Yáñez, M.; Gil-Longo, J.; Campos-Toimil, M. Calcium binding proteins. In Calcium Signaling; Islam, M.S., Ed.; Springer: Dordrecht, The Netherlands, 2012; pp. 461–482. [Google Scholar]
  5. DeFalco, T.A.; Bender, K.W.; Snedden, W.A. Breaking the code: Ca2+ sensors in plant signalling. Biochem. J. 2009, 425, 27–40. [Google Scholar] [CrossRef]
  6. Ranty, B.; Aldon, D.; Galaud, J.P. Plant calmodulins and calmodulin-related proteins: Multifaceted relays to decode calcium signals. Plant Signal. Behav. 2006, 1, 96–104. [Google Scholar] [CrossRef]
  7. Kaur, A.; Madhu; Upadhyay, S.K. EF-hand domain-containing proteins: Diversity and role in plants. In Cation Transporters in Plants; Upadhyay, S.K., Ed.; Academic Press: Cambridge, MA, USA, 2022; pp. 185–203. [Google Scholar]
  8. Taneja, M.; Upadhyay, S.K. An introduction to the calcium transport elements in plants. In Calcium Transport Elements in Plants; Academic Press: Cambridge, MA, USA, 2021; pp. 1–18. [Google Scholar]
  9. Chin, D.; Means, A.R. Calmodulin: A prototypical calcium sensor. Trends Cell Biol. 2000, 10, 322–328. [Google Scholar] [CrossRef]
  10. Léger, C.; Pitard, I.; Sadi, M.; Carvalho, N.; Brier, S.; Mechaly, A.; Raoux-Barbot, D.; Davi, M.; Hoos, S.; Weber, P.; et al. Dynamics and structural changes of calmodulin upon interaction with the antagonist calmidazolium. BMC Biol. 2022, 20, 176. [Google Scholar] [CrossRef]
  11. McCormack, E.; Braam, J. Calmodulins and related potential calcium sensors of Arabidopsis. New Phytol. 2003, 159, 585–598. [Google Scholar] [CrossRef]
  12. Zhu, X.; Dunand, C.; Snedden, W.; Galaud, J.P. CaM and CML emergence in the green lineage. Trends Plant Sci. 2015, 20, 483–489. [Google Scholar] [CrossRef]
  13. Boonburapong, B.; Buaboocha, T. Genome-wide identification and analyses of the rice calmodulin and related potential calcium sensor proteins. BMC Plant Biol. 2007, 7, 4. [Google Scholar] [CrossRef]
  14. Zeng, H.; Zhang, Y.; Zhang, X.; Pi, E.; Zhu, Y. Analysis of EF-hand proteins in soybean genome suggests their potential roles in environmental and nutritional stress signaling. Front. Plant Sci. 2017, 8, 877. [Google Scholar] [CrossRef] [PubMed]
  15. Vandelle, E.; Vannozzi, A.; Wong, D.; Danzi, D.; Digby, A.M.; Dal Santo, S.; Astegno, A. Identification, characterization, and expression analysis of calmodulin and calmodulin-like genes in grapevine (Vitis vinifera) reveal likely roles in stress responses. Plant Physiol. Biochem. 2018, 129, 221–237. [Google Scholar] [CrossRef] [PubMed]
  16. He, X.; Liu, W.; Li, W.; Liu, Y.; Wang, W.; Xie, P.; Kang, Y.; Liao, L.; Qian, L.; Liu, Z.; et al. Genome-wide identification and expression analysis of CaM/CML genes in Brassica napus under abiotic stress. J. Plant Physiol. 2020, 255, 153251. [Google Scholar] [CrossRef] [PubMed]
  17. Shi, J.; Du, X. Identification, characterization and expression analysis of calmodulin and calmodulin-like proteins in Solanum pennellii. Sci. Rep. 2020, 10, 7474. [Google Scholar] [CrossRef]
  18. Cai, K.; Kuang, L.; Yue, W.; Xie, S.; Xia, X.; Zhang, G.; Wang, J. Calmodulin and calmodulin-like gene family in barley: Identification, characterization and expression analyses. Front. Plant Sci. 2022, 13, 964888. [Google Scholar] [CrossRef]
  19. Liu, Y.; Chen, W.; Liu, L.; Su, Y.; Li, Y.; Jia, W.; Jiao, B.; Wang, J.; Yang, F.; Dong, F.; et al. Genome-wide identification and expression analysis of calmodulin and calmodulin-like genes in wheat (Triticum aestivum L.). Plant Signal. Behav. 2022, 17, 2013646. [Google Scholar] [CrossRef]
  20. Upadhyay, S.K. Calcium Transport Elements in Plants; Academic Press: Cambridge, MA, USA, 2021; pp. 1–464. [Google Scholar]
  21. Golovkin, M.; Reddy, A.S. A calmodulin-binding protein from Arabidopsis has an essential role in pollen germination. Proc. Natl. Acad. Sci. USA 2003, 100, 10558–10563. [Google Scholar] [CrossRef]
  22. Du, L.; Poovaiah, B.W. Ca2+/calmodulin is critical for brassinosteroid biosynthesis and plant growth. Nature 2005, 437, 741–745. [Google Scholar] [CrossRef]
  23. Tsai, Y.C.; Delk, N.A.; Chowdhury, N.I.; Braam, J. Arabidopsis potential calcium sensors regulate nitric oxide levels and the transition to flowering. Plant Signal. Behav. 2007, 2, 446–454. [Google Scholar] [CrossRef]
  24. Wang, Y.; Zhang, W.Z.; Song, L.F.; Zou, J.J.; Su, Z.; Wu, W.H. Transcriptome analyses show changes in gene expression to accompany pollen germination and tube growth in Arabidopsis. Plant Physiol. 2008, 148, 1201–1211. [Google Scholar] [CrossRef]
  25. Dobney, S.; Chiasson, D.; Lam, P.; Smith, S.P.; Snedden, W.A. The calmodulin-related calcium sensor CML42 plays a role in trichome branching. J. Biol. Chem. 2009, 284, 31647–31657. [Google Scholar] [CrossRef]
  26. Wang, S.S.; Diao, W.Z.; Yang, X.; Qiao, Z.; Wang, M.; Acharya, B.R.; Zhang, W. Arabidopsis thaliana CML25 mediates the Ca2+ regulation of K+ transmembrane trafficking during pollen germination and tube elongation. Plant Cell Environ. 2015, 38, 2372–2386. [Google Scholar] [CrossRef]
  27. Basu, R.; Dutta, S.; Pal, A.; Sengupta, M.; Chattopadhyay, S. Calmodulin7: Recent insights into emerging roles in plant development and stress. Plant Mol. Biol. 2021, 107, 1–20. [Google Scholar] [CrossRef]
  28. Zhu, X.; Perez, M.; Aldon, D.; Galaud, J.P. Respective contribution of CML8 and CML9, two Arabidopsis calmodulin-like proteins, to plant stress responses. Plant Signal. Behav. 2017, 12, e1322246. [Google Scholar] [CrossRef]
  29. Zhu, X.; Mazard, J.; Robe, E.; Pignoly, S.; Aguilar, M.; San Clemente, H.; Lauber, E.; Berthomé, R.; Galaud, J.P. The same against many: AtCML8, a Ca2+ sensor acting as a positive regulator of defense responses against several plant pathogens. Int. J. Mol. Sci. 2021, 22, 10469. [Google Scholar] [CrossRef]
  30. Scholz, S.S.; Vadassery, J.; Heyer, M.; Reichelt, M.; Bender, K.W.; Snedden, W.A.; Boland, W.; Mithöfer, A. Mutation of the Arabidopsis calmodulin-like protein CML37 deregulates the jasmonate pathway and enhances susceptibility to herbivory. Mol. Plant. 2014, 7, 1712–1726. [Google Scholar] [CrossRef]
  31. Scholz, S.S.; Reichelt, M.; Vadassery, J.; Mithöfer, A. Calmodulin-like protein CML37 is a positive regulator of ABA during drought stress in Arabidopsis. Plant Signal. Behav. 2015, 10, e1011951. [Google Scholar] [CrossRef]
  32. Vadassery, J.; Reichelt, M.; Hause, B.; Gershenzon, J.; Boland, W.; Mithöfer, A. CML42-mediated calcium signaling coordinates responses to Spodoptera herbivory and abiotic stresses in Arabidopsis. Plant Physiol. 2012, 159, 1159–1175. [Google Scholar] [CrossRef]
  33. Baldauf, S. An overview of the phylogeny and diversity of eukaryotes. J. Syst. Evol. 2008, 46, 263–273. [Google Scholar] [CrossRef]
  34. Brownlee, C.; Wood, J.W. A gradient of cytoplasmic free calcium a in growing rhizoid cells of Fucus serratus. Nature 1986, 320, 624–626. [Google Scholar] [CrossRef]
  35. Roberts, S.; Gillot, I.; Brownlee, C. Cytoplasmic calcium and Fucus activation. Development 1994, 120, 155–163. [Google Scholar] [CrossRef]
  36. Brawley, S.H.; Roberts, D.M. Calmodulin-binding proteins are developmentally regulated in gametes and embryos of fucoid algae. Dev. Biol. 1989, 131, 313–320. [Google Scholar] [CrossRef] [PubMed]
  37. Love, J.; Brownlee, C.; Trewavas, A.J. Ca2+ and Calmodulin Dynamics during photopolarization in Fucus serratus zygotes. Plant Physiol. 1997, 115, 249–261. [Google Scholar] [CrossRef] [PubMed]
  38. Pu, R.; Robinson, K.R. Cytoplasmic calcium gradients and calmodulin in the early development of the fucoid alga Pelvetia compressa. J. Cell Sci. 1998, 111 Pt 21, 3197–3207. [Google Scholar] [CrossRef]
  39. Cock, J.M.; Sterck, L.; Rouzé, P.; Scornet, D.; Allen, A.E.; Amoutzias, G.; Anthouard, V.; Artiguenave, F.; Aury, J.M.; Badger, J.H.; et al. The Ectocarpus genome and the independent evolution of multicellularity in brown algae. Nature 2010, 465, 617–621. [Google Scholar] [CrossRef]
  40. Ye, N.; Zhang, X.; Miao, M.; Fan, X.; Zheng, Y.; Xu, D.; Wang, J.; Zhou, L.; Wang, D.; Gao, Y.; et al. Saccharina genomes provide novel insight into kelp biology. Nat. Commun. 2015, 6, 6986. [Google Scholar] [CrossRef]
  41. Nishitsuji, K.; Arimoto, A.; Iwai, K.; Sudo, Y.; Hisata, K.; Fujie, M.; Arakaki, N.; Kushiro, T.; Konishi, T.; Shinzato, C.; et al. A draft genome of the brown alga, Cladosiphon okamuranus, S-strain: A platform for future studies of ‘mozuku’ biology. DNA Res. 2016, 23, 561–570. [Google Scholar] [CrossRef]
  42. Cormier, A.; Avia, K.; Sterck, L.; Derrien, T.; Wucher, V.; Andres, G.; Monsoor, M.; Godfroy, O.; Lipinska, A.; Perrineau, M.M.; et al. Re-annotation, improved large-scale assembly and establishment of a catalogue of noncoding loci for the genome of the model brown alga Ectocarpus. New Phytol. 2017, 214, 219–232. [Google Scholar] [CrossRef]
  43. Shan, T.; Yuan, J.; Su, L.; Li, J.; Leng, X.; Zhang, Y.; Gao, H.; Pang, S. First genome of the brown alga Undaria pinnatifida: Chromosome-level assembly using Pacbio and Hi-C technologies. Front. Genet. 2020, 11, 140. [Google Scholar] [CrossRef]
  44. Lipinska, A.P.; D’Hondt, S.; Van Damme, E.J.; De Clerck, O. Uncovering the genetic basis for early isogamete differentiation: A case study of Ectocarpus siliculosus. BMC Genom. 2013, 14, 909. [Google Scholar] [CrossRef]
  45. Zhang, J.; Li, Y.; Luo, S.; Cao, M.; Zhang, L.; Li, X. Differential gene expression patterns during gametophyte development provide insights into sex differentiation in the dioicous kelp Saccharina japonica. BMC Plant Biol. 2021, 21, 335. [Google Scholar] [CrossRef]
  46. Peters, A.F.; Marie, D.; Scornet, D.; Kloareg, B.; Mark Cock, J. Proposal of Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) as a model organism for brown algal genetics and genomics. J. Phycol. 2004, 40, 1079–1088. [Google Scholar] [CrossRef]
  47. Peters, A.F.; Scornet, D.; Ratin, M.; Charrier, B.; Monnier, A.; Merrien, Y.; Corre, E.; Coelho, S.M.; Cock, J.M. Life-cycle-generation-specific developmental processes are modified in the immediate upright mutant of the brown alga Ectocarpus siliculosus. Development 2008, 135, 1503–1512. [Google Scholar] [CrossRef]
  48. Coelho, S.M.; Scornet, D.; Rousvoal, S.; Peters, N.T.; Dartevelle, L.; Peters, A.F.; Cock, J.M. Ectocarpus: A model organism for the brown algae. Cold Spring Harb. Protoc. 2012, 2012, 193–198. [Google Scholar] [CrossRef]
  49. Mignerot, L.; Avia, K.; Luthringer, R.; Lipinska, A.P.; Peters, A.F.; Cock, J.M.; Coelho, S.M. A key role for sex chromosomes in the regulation of parthenogenesis in the brown alga Ectocarpus. PLoS Genet. 2019, 15, e1008211. [Google Scholar] [CrossRef]
  50. Rabillé, H.; Billoud, B.; Tesson, B.; Le Panse, S.; Rolland, É.; Charrier, B. The brown algal mode of tip growth: Keeping stress under control. PLoS Biol. 2019, 17, e2005258. [Google Scholar] [CrossRef] [PubMed]
  51. Hu, Z.-M.; Shan, T.-F.; Zhang, J.; Zhang, Q.-S.; Critchley, A.T.; Choi, H.-G.; Yotsukura, N.; Liu, F.-L.; Duan, D.-L. Kelp aquaculture in China: A retrospective and future prospects. Rev. Aquacult. 2021, 13, 1324–1351. [Google Scholar] [CrossRef]
  52. Domínguez, D.C.; Guragain, M.; Patrauchan, M. Calcium binding proteins and calcium signaling in prokaryotes. Cell Calcium. 2015, 57, 151–165. [Google Scholar] [CrossRef]
  53. Kim, J.S.; Mizoi, J.; Yoshida, T.; Fujita, Y.; Nakajima, J.; Ohori, T.; Todaka, D.; Nakashima, K.; Hirayama, T.; Shinozaki, K.; et al. An ABRE promoter sequence is involved in osmotic stress-responsive expression of the DREB2A gene, which encodes a transcription factor regulating drought-inducible genes in Arabidopsis. Plant Cell Physiol. 2011, 52, 2136–2146. [Google Scholar] [CrossRef]
  54. Nakashima, K.; Yamaguchi-Shinozaki, K.; Shinozaki, K. The transcriptional regulatory network in the drought response and its crosstalk in abiotic stress responses including drought, cold, and heat. Front. Plant Sci. 2014, 5, 170. [Google Scholar] [CrossRef]
  55. Dieck, I.t. Temperature tolerance and survival in darkness of kelp gametophyles (Laminariales, Phaeophyta): Ecological and biogeographical implications. Mar. Ecol. Prog. Ser. 1993, 100, 253–264. [Google Scholar] [CrossRef]
  56. Hsiao, S.I.C.; Druehl, L.D. Environmental control of gametogenesis in Laminaria saccharina. I. The effects of light and culture media. Can. J. Bot. 1971, 49, 1503–1508. [Google Scholar] [CrossRef]
  57. Lüning, K. Critical levels of light and temperature regulating the gametogenesis of three laminaria species (Phaeophyceae). J. Phyol. 1980, 16, 1–15. [Google Scholar] [CrossRef]
  58. Martins, N.; Tanttu, H.; Pearson, G.A.; Serrão, E.A.; Bartsch, I. Interactions of daylength, temperature and nutrients affect thresholds for life stage transitions in the kelp Laminaria digitata (Phaeophyceae). Bot. Mar. 2017, 60, 109–121. [Google Scholar] [CrossRef]
  59. Ebbing, A.; Pierik, R.; Bouma, T.; Kromkamp, J.C.; Timmermans, K. How light and biomass density influence the reproduction of delayed Saccharina latissima gametophytes (Phaeophyceae). J. Phycol. 2020, 56, 709–718. [Google Scholar] [CrossRef]
  60. Roberts, S.; Brownlee, C. Calcium influx, fertilisation potential and egg activation in Fucus serratus. Zygote 1995, 3, 191–197. [Google Scholar] [CrossRef]
  61. Zhang, L.; Cao, Z.; Liang, G.; Li, X.; Wu, H.; Yang, G. Comparative transcriptome analysis reveals candidate genes related to structural and storage carbohydrate biosynthesis in kelp Saccharina japonica (Laminariales, Phaeophyceae). J. Phycol. 2020, 56, 1168–1183. [Google Scholar] [CrossRef]
  62. Van de Mortel, J.E.; Almar Villanueva, L.; Schat, H.; Kwekkeboom, J.; Coughlan, S.; Moerland, P.D.; Ver Loren van Themaat, E.; Koornneef, M.; Aarts, M.G. Large expression differences in genes for iron and zinc homeostasis, stress response, and lignin biosynthesis distinguish roots of Arabidopsis thaliana and the related metal hyperaccumulator Thlaspi caerulescens. Plant Physiol. 2006, 142, 1127–1147. [Google Scholar] [CrossRef]
  63. Müller, R.; Morant, M.; Jarmer, H.; Nilsson, L.; Nielsen, T.H. Genome-wide analysis of the Arabidopsis leaf transcriptome reveals interaction of phosphate and sugar metabolism. Plant Physiol. 2007, 143, 156–171. [Google Scholar] [CrossRef]
  64. Gugger, P.F.; Peñaloza-Ramírez, J.M.; Wright, J.W.; Sork, V.L. Whole-transcriptome response to water stress in a California endemic oak, Quercus lobata. Tree Physiol. 2017, 37, 632–644. [Google Scholar] [CrossRef]
  65. Zhang, L.; Lu, Y.T. Calmodulin-binding protein kinases in plants. Trends Plant Sci. 2003, 8, 123–127. [Google Scholar] [CrossRef] [PubMed]
  66. Liu, H.T.; Gao, F.; Li, G.L.; Han, J.L.; Liu, D.L.; Sun, D.Y.; Zhou, R.G. The calmodulin-binding protein kinase 3 is part of heat-shock signal transduction in Arabidopsis thaliana. Plant J. 2008, 55, 760–773. [Google Scholar] [CrossRef] [PubMed]
  67. Kölling, M.; Kumari, P.; Bürstenbinder, K. Calcium- and calmodulin-regulated microtubule-associated proteins as signal-integration hubs at the plasma membrane-cytoskeleton nexus. J. Exp. Bot. 2019, 70, 387–396. [Google Scholar] [CrossRef]
  68. Pedretti, M.; Conter, C.; Dominici, P.; Astegno, A. SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana. Biochem. J. 2020, 477, 173–189. [Google Scholar] [CrossRef]
  69. Kaur, A.; Sharma, A.; Madhu; Dixit, S.; Singh, K.; Upadhyay, S.K. OSCA genes in bread wheat: Molecular characterization, expression profiling, and interaction analyses indicated their diverse roles during development and stress response. Int. J. Mol. Sci. 2022, 23, 14867. [Google Scholar] [CrossRef]
  70. Rathour, M.; Shumayla; Alok, A.; Upadhyay, S.K. Investigation of roles of TaTALE genes during development and stress response in bread wheat. Plants 2022, 11, 587. [Google Scholar] [CrossRef]
  71. Lado, P.; Cerana, R.; Bonetti, A.; Marrè, M.T.; Marrè, E. Effects of calmodulin inhibitors in plants. I. Synergism with fusicoccin in the stimulation of growth and H+ secretion and in the hyperpolarization of the transmembrane electric potential. Plant Sci. 1981, 23, 253–262. [Google Scholar] [CrossRef]
  72. Vandonselaar, M.; Hickie, R.A.; Quail, W.; Delbaere, L.T.J. Trifluoperazine-induced conformational change in Ca2+-calmodulin. Nat. Struct. Mol. Biol. 1994, 1, 795–801. [Google Scholar] [CrossRef]
  73. McCormack, E.; Tsai, Y.C.; Braam, J. Handling calcium signaling: Arabidopsis CaMs and CMLs. Trends Plant Sci. 2005, 10, 383–389. [Google Scholar] [CrossRef]
  74. Guindon, S.; Dufayard, J.F.; Lefort, V.; Anisimova, M.; Hordijk, W.; Gascuel, O. New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Syst. Biol. 2010, 59, 307–321. [Google Scholar] [CrossRef]
  75. Subramanian, B.; Gao, S.; Lercher, M.J.; Hu, S.; Chen, W.H. Evolview v3: A webserver for visualization, annotation, and management of phylogenetic trees. Nucleic Acids Res. 2019, 47, W270–W275. [Google Scholar] [CrossRef] [PubMed]
  76. Bailey, T.L.; Boden, M.; Buske, F.A.; Frith, M.; Grant, C.E.; Clementi, L.; Ren, J.; Li, W.W.; Noble, W.S. MEME SUITE: Tools for motif discovery and searching. Nucleic Acids Res. 2009, 37, W202–W208. [Google Scholar] [CrossRef]
  77. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An integrative toolkit developed for interactive analyses of big biological data. Mol. Plant. 2020, 13, 1194–1202. [Google Scholar] [CrossRef]
  78. Lescot, M.; Déhais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouzé, P.; Rombauts, S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic. Acids. Res. 2002, 30, 325–327. [Google Scholar] [CrossRef]
  79. Yang, Z. PAML 4: Phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 2007, 24, 1586–1591. [Google Scholar] [CrossRef]
  80. Starr, R.C.; Zeikus, J.A. Utex-the culture collection of algae at the University of Texas at Austin 1993 list of cultures. J. Phycol. 1993, 29, 1–106. [Google Scholar] [CrossRef]
  81. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
Plants 12 01934 g001 550
Figure 1. Alignments of amino acid sequences of the brown algae CaM family proteins with the green lineage. The regions corresponding to the E helices, the Ca2+-binding loops, and the F helices are indicated by the black, gray, and black bars, respectively. The consensus sequences for these regions are indicated beneath the relevant sequences. “E” stands for glutamic acid, “h” for hydrophobic amino acid, and “*” for any amino acid. “X, Y, Z, G, #, I, -X, -Z” are defined as follows: The X position is almost exclusively filled with aspartate (D); Y is usually aspartate (D) or asparagine (N); Z is aspartate (D), asparagine (N), or serine (S); the # position tolerates a variety of amino acids; -X also varies, but is usually aspartate (D), asparagine (N), or serine (S); -Z, which contributes two coordination sites, is nearly invariably glutamate ©. Glycine (G) at position 6 is highly conserved and is thought to provide the ability for a sharp turn within the loop. Position 8 is most often isoleucine (I), which can form hydrogen bonds with the other EF loop in a pair. The cystei©(C) residue in position 7 of the first EF hand is common among plant CaMs. Amino acid sequence identities are shaded. At, Arabidopsis thaliana; Cr, Chlamydomonas reinhardtii; Sj, Saccharina japonica; Es, Ectocarpus sp.
Figure 1. Alignments of amino acid sequences of the brown algae CaM family proteins with the green lineage. The regions corresponding to the E helices, the Ca2+-binding loops, and the F helices are indicated by the black, gray, and black bars, respectively. The consensus sequences for these regions are indicated beneath the relevant sequences. “E” stands for glutamic acid, “h” for hydrophobic amino acid, and “*” for any amino acid. “X, Y, Z, G, #, I, -X, -Z” are defined as follows: The X position is almost exclusively filled with aspartate (D); Y is usually aspartate (D) or asparagine (N); Z is aspartate (D), asparagine (N), or serine (S); the # position tolerates a variety of amino acids; -X also varies, but is usually aspartate (D), asparagine (N), or serine (S); -Z, which contributes two coordination sites, is nearly invariably glutamate ©. Glycine (G) at position 6 is highly conserved and is thought to provide the ability for a sharp turn within the loop. Position 8 is most often isoleucine (I), which can form hydrogen bonds with the other EF loop in a pair. The cystei©(C) residue in position 7 of the first EF hand is common among plant CaMs. Amino acid sequence identities are shaded. At, Arabidopsis thaliana; Cr, Chlamydomonas reinhardtii; Sj, Saccharina japonica; Es, Ectocarpus sp.
Plants 12 01934 g001
Plants 12 01934 g002 550
Figure 2. Phylogenetic analysis of the CaM/CML family of proteins amongst diverse species. The phylogenetic tree was constructed based on the sequence alignments by the ML method with bootstrapping analysis (1000 replicates) of the CaMs/CMLs proteins. The ML phylogenetic tree was created using ATGC PhyML 3.0. The proteins have been divided into three groups, each filled with a different color. Different colors and shapes represent different species. Black triangle, Saccharina japonica; green checkmark, Ectocarpus sp.; yellow box, Chlamydomonas reinhardtii; blue star, Arabidopsis thaliana. Different colored circles represent different bootstrap values.
Figure 2. Phylogenetic analysis of the CaM/CML family of proteins amongst diverse species. The phylogenetic tree was constructed based on the sequence alignments by the ML method with bootstrapping analysis (1000 replicates) of the CaMs/CMLs proteins. The ML phylogenetic tree was created using ATGC PhyML 3.0. The proteins have been divided into three groups, each filled with a different color. Different colors and shapes represent different species. Black triangle, Saccharina japonica; green checkmark, Ectocarpus sp.; yellow box, Chlamydomonas reinhardtii; blue star, Arabidopsis thaliana. Different colored circles represent different bootstrap values.
Plants 12 01934 g002
Plants 12 01934 g003 550
Figure 3. Phylogenetic relationship (a), conserved motifs (b), and gene structure (c) of the brown algae CaMs/CMLs. a: Groups I and III indicate the classification of the brown algae CaMs/CMLs according to the phylogenetic relationship by ML method. b: Motif analysis was performed by the MEME program online. Different colors of boxes represent different motifs in the corresponding position of each CaMs/CMLs protein. EF-hands are marked with pink border rectangle. c: exons and introns are represented by green boxes and the black lines, respectively.
Figure 3. Phylogenetic relationship (a), conserved motifs (b), and gene structure (c) of the brown algae CaMs/CMLs. a: Groups I and III indicate the classification of the brown algae CaMs/CMLs according to the phylogenetic relationship by ML method. b: Motif analysis was performed by the MEME program online. Different colors of boxes represent different motifs in the corresponding position of each CaMs/CMLs protein. EF-hands are marked with pink border rectangle. c: exons and introns are represented by green boxes and the black lines, respectively.
Plants 12 01934 g003
Plants 12 01934 g004 550
Figure 4. Analysis of the cis-acting elements of SjCaM/SjCMLs (a) and EsCaM/EsCMLs (b). The gradient colors in the orange grid represent the number of cis-acting elements in CaMs/CMLs.
Figure 4. Analysis of the cis-acting elements of SjCaM/SjCMLs (a) and EsCaM/EsCMLs (b). The gradient colors in the orange grid represent the number of cis-acting elements in CaMs/CMLs.
Plants 12 01934 g004
Plants 12 01934 g005 550
Figure 5. Expression patterns of SjCaM/SjCMLs during different life cycle stages. Log2-transformed values to generate heatmaps. SIf: immature female gametophytes; SIm: immature male gametophytes; SMf: mature female gametophytes; SMm: mature male gametophytes. SPsp1-SPsp6: six sporophyte developmental stages.
Figure 5. Expression patterns of SjCaM/SjCMLs during different life cycle stages. Log2-transformed values to generate heatmaps. SIf: immature female gametophytes; SIm: immature male gametophytes; SMf: mature female gametophytes; SMm: mature male gametophytes. SPsp1-SPsp6: six sporophyte developmental stages.
Plants 12 01934 g005
Plants 12 01934 g006 550
Figure 6. Interaction analysis of SjCaM/SjCMLs using the STRING and STITCH servers. The networks (a) show the protein–protein interactions. The red circle identifies SjCaM/SjCMLs, the blue circle identifies SjCaM/SjCMLs interacting proteins, the pink circle identifies calcineurin-like phosphoesterases, the green circle identifies five proteins interacting with all six SjCaM/SjCMLs. The networks (b) show the protein–chemical interactions. The red circle identifies SjCaM/SjCMLs and the bule circle identifies predicted interacting chemicals.
Figure 6. Interaction analysis of SjCaM/SjCMLs using the STRING and STITCH servers. The networks (a) show the protein–protein interactions. The red circle identifies SjCaM/SjCMLs, the blue circle identifies SjCaM/SjCMLs interacting proteins, the pink circle identifies calcineurin-like phosphoesterases, the green circle identifies five proteins interacting with all six SjCaM/SjCMLs. The networks (b) show the protein–chemical interactions. The red circle identifies SjCaM/SjCMLs and the bule circle identifies predicted interacting chemicals.
Plants 12 01934 g006
Table
Table 1. Characteristics of CaM/CML proteins in S. japonica and E. sp.
Table 1. Characteristics of CaM/CML proteins in S. japonica and E. sp.
NameGene IDLength (aa)EF HandsMet (%)Lys (116)pIMW (kDa)Identity to
SjCaM/EsCaM		IntronSubcellular Location
SjCaMSJ0189114947.4+4.0916.771001Cytoplasmic
SjCML1SJ0297816344.9 4.6618.7054.363Cytoplasmic
SjCML2SJ0282215545.2 4.0417.2850.683Chloroplast
SjCML3SJ0278415946.9 4.1917.8945.273Nuclear
SjCML4SJ1530918743.7 4.54 20.9642.283Chloroplast
SjCML5SJ1637413025.4 4.85 14.6038.792Nuclear
SjCML6SJ1675518632.2 4.9420.7436.915Cytoplasmic
SjCML7SJ1252817134.1 6.10 19.5128.005Nuclear
SjCML8SJ1736321526 4.6223.4727.525Chloroplast
EsCaMEc-06_002270.114947.4+4.0916.771001Cytoplasmic
EsCML1Ec-15_000530.116445.5 4.66 18.8654.364Cytoplasmic
EsCML2Ec-24_000510.115545.2 4.19 17.4052.034Cytoplasmic
EsCML3Ec-23_000190.115445.2 4.0317.1350.682Nuclear
EsCML4Ec-04_003890.116645.4 4.50 18.9346.313Mitochondrial
EsCML5Ec-23_001960.116443.7 4.3718.2744.34Cytoplasmic
EsCML6Ec-11_005890.117634 4.6819.7442.393Peroxisomal
EsCML7Ec-06_007660.118243.8 4.4820.3941.614Mitochondrial
EsCML8Ec-16_003140.115635.1 4.0617.4438.265Cytoplasmic
EsCML9Ec-06_003120.117543.4 4.5819.6136.242Chloroplast
EsCML10Ec-26_003440.117532.9 5.4219.8235.575Cytoplasmic
EsCML11Ec-16_003330.117724.5 4.4719.1226.032Mitochondrial
aa, amino acids; Met, methionine; Lys, lysine; pI, isoelectric point; MW, molecular weight.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Xue, N.; Sun, M.; Gai, Z.; Bai, M.; Sun, J.; Sai, S.; Zhang, L. Genome-Wide Identification and Expression Analysis of Calmodulin (CaM) and Calmodulin-Like (CML) Genes in the Brown Algae Saccharina japonica. Plants 2023, 12, 1934. https://doi.org/10.3390/plants12101934

AMA Style

Xue N, Sun M, Gai Z, Bai M, Sun J, Sai S, Zhang L. Genome-Wide Identification and Expression Analysis of Calmodulin (CaM) and Calmodulin-Like (CML) Genes in the Brown Algae Saccharina japonica. Plants. 2023; 12(10):1934. https://doi.org/10.3390/plants12101934

Chicago/Turabian Style

Xue, Nianchao, Minghui Sun, Zihan Gai, Meihan Bai, Juan Sun, Shan Sai, and Linan Zhang. 2023. "Genome-Wide Identification and Expression Analysis of Calmodulin (CaM) and Calmodulin-Like (CML) Genes in the Brown Algae Saccharina japonica" Plants 12, no. 10: 1934. https://doi.org/10.3390/plants12101934

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop