The CaCA superfamily genes in Saccharum: comparative analysis and their functional implications in response to biotic and abiotic stress

Abstract

Background

In plants, Calcium (Ca²⁺) acts as a universal messenger in various signal transduction pathways, including responses to biotic and abiotic stresses and regulation of cellular and developmental processes. The Ca²⁺/cation antiporter (CaCA) superfamily proteins play vital roles in the transport of Ca²⁺ and/or other cations. However, the characteristics of these superfamily members in Saccharum and their evolutionary and functional implications have remained unclear.

Results

A total of 34 CaCA genes in Saccharum spontaneum, 5 CaCA genes in Saccharum spp. R570, and 14 CaCA genes in Sorghum bicolor were identified and characterized. These genes consisted of the H⁺/cation exchanger (CAX), cation/Ca²⁺ exchanger (CCX), EF-hand / CAX (EFCAX), and Mg²⁺/H⁺ exchanger (MHX) families, among which the CCX and EFCAX could be classified into three groups while the CAX could be divided into two groups. The exon/intron structures and motif compositions suggested that the members in the same group were highly conserved. Synteny analysis of CaCAs established their orthologous and paralogous relationships among the superfamily in S. spontaneum, R570, and S. bicolor. The results of protein-protein interactions indicated that these CaCA proteins had direct or indirect interactions. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis demonstrated that most members of Saccharum CaCA genes exhibited a similar expression pattern in response to hormonal (abscisic acid, ABA) treatment but played various roles in response to biotic (Sporisorium scitamineum) and abiotic (cold) stresses. Furthermore, ScCAX4, a gene encoding a cytoplasm, plasma membrane and nucleus positioning protein, was isolated from sugarcane. This gene was constitutively expressed in different sugarcane tissues and its expression was only induced at 3 and 6 h time points after ABA treatment, however was inhibited and indued in the whole process under cold and S. scitamineum stresses, respectively.

Conclusions

This study systematically conducted comparative analyses of CaCA superfamily genes among S. spontaneum, R570, and S. bicolor, delineating their sequence and structure characteristics, classification, evolutionary history, and putative functions. These results not only provided rich gene resources for exploring the molecular mechanism of the CaCA superfamily genes but also offered guidance and reference for research on other gene families in Saccharum.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-021-07828-3.

Background

Calcium (Ca²⁺) is a universal ion that exists in all organisms as a critical element and an essential nutrient and also functions as a ubiquitous secondary messenger [1, 2]. There are several particularly important transporters that act as “gatekeepers”, mediating the movement of Ca²⁺. Previous studies showed that three classes of membrane transporters, Ca²⁺-ATPases (PMCAs), Ca²⁺ permeable channels, and Ca²⁺/cation antiporters (CaCAs), act as “gatekeepers” to mediate Ca²⁺ flux across the membrane and to regulate cytosolic Ca²⁺ levels [3–5].

CaCA superfamily proteins are widespread in archaea, bacteria, fungi, plants and animals [6, 7]. They can enhance the efflux of Ca²⁺ across membranes against the concentration gradient by exchanging the influx of monovalent cations such as H⁺, Na⁺, or K⁺ to energize the process [6–8]. As a superfamily, CaCAs consist of a number of exchanger protein families [7]. According to a study by Cai et al. [7], the CaCA superfamily can be classified into six families, i.e., the YRBG, Na⁺/Ca²⁺ exchanger (NCX), Na⁺/Ca²⁺, K⁺ exchanger (NCKX), cation/Ca²⁺ exchanger (CCX), and H⁺/cation exchanger (CAX) families.

As previous studies have shown, YRBG family proteins are present in many prokaryotes but are absent in eukaryotes [7, 9]. Regarding the NCX and NCKX families, they are primarily present in animal groups [7]. Due to the speed and high capacity for Ca²⁺ in the NCX family, NCXs are important regulators of cellular Ca²⁺ homeostasis [8]. In mammals, the NCX exchange proteins consist of three distinct types (NCX1, NCX2 and NCX3) [8]. Plants have evolved a novel CaCA group, the Mg²⁺/H⁺ exchanger (MHX) proteins, which belong to the NCX family [8, 10, 11]. The CAX protein family has been observed in various organisms including bacteria, protozoa, fungi, animals, algae, and plants [8, 12–14]. Normally, the CAX family is divided into three types: 1, 2, and 3 [12]. In addition, a novel group of EF-hand / CAX (EFCAX) proteins containing EF-hand domain which are also termed as NCX-like proteins (NCL), has been identified in the CAX family [8]. This novel group is evolutionarily closer to CAX proteins than NCX proteins [8, 15]. Furthermore, functional characterization demonstrated that AtNCL exhibited Na⁺/Ca²⁺ exchange activity [16].

Saccharum spp. (sugarcane), an important sugar and biofuel feedstock crop, accounts for 80 % of the world’s total sugar production and provides 40 % of bio-ethanol [17, 18]. At present, various stresses, are the main factors that restrict the well development of sugar industry [19]. For example, it is manifested that salt stress cause considerable reduction in growth rate at various sugarcane growth stages [20]. Under cold and drought stresses, the photosynthetic rate of sugarcane is severely reduced [19, 21]. In order to avoid the negative effects of stresses, plants have evolved complex mechanisms, such as osmotic adjustment [22] which is mainly dependent on the regulation of inorganic ions (Na⁺, K⁺, Ca²⁺, and Cl⁻) [23]. Previous studies have demonstrated that CaCAs are essential for controlling ion concentrations to maintain cellular functions [13, 24]. However, no comprehensive and systematic research on the CaCA superfamily was previously conducted in Saccharum. Herein, two currently available Saccharum species genomes, R570 (Saccharum spp., the haploid genome of the modern sugarcane cultivar) [25] and AP85-441(Saccharum spontaneum, the sugarcane ancestor) [17] as well as the representative genome of the closest relative (Sorghum bicolor) [26] were selected to perform genome-wide identification and comprehensive characterization of CaCA proteins in Saccharum. The phylogenetic relationships, gene and protein characteristics, duplication events, and synteny relationships were further used to investigate the evolutionary relationships of CaCA genes. The interactive relationships between CaCAs and microRNAs, gene ontology annotation, and protein interactions of CaCA proteins and their expression patterns in response to hormonal (abscisic acid, ABA), biotic (Sporisorium scitamineum), and abiotic (cold) stresses were also evaluated. Furthermore, one CAX gene was isolated from sugarcane, and its expression patterns and subcellular localization were analyzed. The present study is expected to support a theoretical basis for further investigations of the clear functions of CaCA genes in Saccharum.

Results

Identification and sequence features of CaCA genes in S. spontaneum, R570 and S. bicolor genomes

Statistical results showed that 34 copies of CaCA genes were present in S. spontaneum, with 14 copies in S. bicolor, while R570 had only five CaCA genes. To reveal the taxonomic information of CaCA superfamily genes, a phylogenetic tree based on the amino acid homology among Arabidopsis, S. spontaneum, R570, and S. bicolor was constructed using the neighbor-joining (NJ) method (Fig. 1). The phylogenetic tree indicated that S. spontaneum possessed 11 CAX genes, 12 CCX genes, 7 EFCAX genes, and four MHX genes. In R570, two copies of CAX genes and only one CCX gene, one EFCAX gene, and one MHX gene were identified. In S. bicolor, there were six CAX genes, five CCX genes, two EFCAX genes, and one MHX gene.

Fig. 1.

Phylogenetic analysis of the CaCA genes from A. thaliana, S. spontaneum, R570, and S. bicolor

The physical and chemical parameters of these CaCA proteins were computed using the ExPASy ProtParam tool (Supplemental Figure S1, Supplemental Table S1 and Table S2). Comparative analysis showed that the number of amino acid residues spanned the largest range in SsCaCA proteins, from 247 in SsCCX4c to 1214 in SsEFCAX2. The number of amino acid residues ranged from 347 (ShEFCAX1) to 641 (SbCCX3) in ShCaCAs and SbCaCAs, respectively. The computed theoretical isoelectric points indicated that the acidity or alkalinity of CaCAs varied greatly in Saccharum and S. bicolor. The results also suggested that these CaCAs in S. spontaneum, R570, and S. bicolor contained at least five transmembrane domains, most of which were located in the plasma membrane.

Phylogenetic classification of the CaCA superfamily

The phylogenetic tree, which was based on comparing the amino acid sequences among algae, mosses, monocots, and dicots, was constructed using the NJ and maximum likelihood (ML) methods to unveil the CaCA superfamily functional information (Fig. 2 and Supplemental Figure S2). In generally, the topologies of the NJ and ML trees constructed in this study were highly consistent, demonstrating the reliability of our classification. In the CAX family, 19 CAX (11 SsCAXs, two ShCAXs, and six SbCAXs) proteins could be divided into two groups (Type 1A and Type 1B). The Type 1B group contained CAX members from mosses, monocots, and dicots, while the Type 1A group only contained CAX members from monocots and dicots. Within the Type 1A group, there was a clear distinction between the genes from monocot and dicot plants, though this division was not as obvious as that within the Type 1B group. In the CCX family, 18 CCXs (12 SsCCXs, one ShCCX, and five SbCCXs) could be classified into three groups (Group 1, Group 2, and Group 3). A clear distinction between the proteins from monocot and dicot plants was also observed among these three groups. Interestingly, the EFCAX family was clearly clustered into three major groups (Group 1, Group 2, and Group 3), which corresponded to mosses, monocots, and dicots, respectively. Ten EFCAXs (seven SsEFCAXs, one ShEFCAX, and two SbEFCAXs) were all sorted into the monocot group, which was also named Group 2. In the MHX family, except for the two MHX members from mosses, the other MHXs from monocots and dicots were on the same branch. It should be noted that six MHXs, i.e., four SsMHXs, one ShMHX, and one SbMHX, had closer relationships with ZmMHX.

Fig. 2.

Phylogenetic evolutionary tree of the CaCA superfamily members. (a) An NJ phylogenetic tree was constructed using the full-length sequence alignments of 47 CAX proteins identified using MUSCLE in MEGAX. (b) An NJ phylogenetic tree was constructed using the full-length sequence alignments of 43 CCX proteins identified using MUSCLE in MEGAX. (c) An NJ phylogenetic tree was constructed using the full-length sequence alignments of 28 EFCAX proteins identified using MUSCLE in MEGAX. (d) An NJ phylogenetic tree was constructed using the full-length sequence alignments of 12 MHX proteins identified using MUSCLE in MEGAX. All SsCaCA, ShCaCA, and SbCaCA proteins are highlighted in red, blue, and green, respectively. All the corresponding reference numbers are listed in Supplemental Table S1 and Table S3

Protein motifs and gene structure analysis

A total of 10 distinct conserved motifs found in each species are illustrated in Supplemental Figure S3. Whether in the CAX, CCX, EFCAX, or MHX family, most members belonged to the same group and shared common motif compositions. What should also be stressed here is that, even in the same classification, the motifs of some proteins were unique. For example, compared with the other CAXs, SsCAX3c contained double motifs 1, 2, 3, 4, 5, 7, and 9. ScCAX4e was the duplicated gene of ScCAX4a, and motif 4 was lost in ScCAX4e. Compared with SbCCX4, SsCCX4a, SsCCX4b, and SsCCX4c, the motifs 2, 4, 5, 6, and 10 were lost in SsCCX4c and motif 6 was lost in ScCAX4a. In the EFCAX family, SsEFCAX2 had the largest number of motifs, containing double motifs 2, 3, 4, 5, 6, 7, 8, 9, and 10, while ShEFCAX1 only had six motifs. It is interesting that all of the MHX proteins contained the same motif composition, expect for SsMHX2.

As exhibited in the pattern of exon–intron distribution and the position of all CaCA genes, the genes from the CCX family were intron-poor with < 3 introns. It was notable that those closely related genes were usually more similar in gene structure. For instance, SsEFCAX1a, SsEFCAX1b, and SsEFCAX1c all had six introns. However, some closely related genes showed significant differences in structural arrangements. For example, SsCAX3a possessed 11 introns and SsCAX3b had eight introns, while SsCAX3c, a closely related gene, had 19 introns. Intriguingly, all MHX genes contained seven introns in the three studied species (S. spontaneum, R570 and S. bicolor).

Chromosomal distribution, duplications, and synteny analysis of the CaCA superfamily

The chromosomal distribution showed that 34 SsCaCA, five ShCaCA, and 14 SbCaCA genes were unevenly distributed on 20, 4, and 7 numbers of chromosomes, respectively. Expect for ShCaCAs, there were 25 and two duplicated SsCaCA gene pairs in the S. spontaneum and S. bicolor genomes, respectively (Fig. 3a, Supplemental Table S5).

Fig. 3.

Duplication events of CaCA genes in S. spontaneum, R570, and S. bicolor. (a) Mapping of CaCA genes and the duplications among them on the S. spontaneum, R570, and S. bicolor chromosomes. Gray lines indicate all syntenic blocks in the S. spontaneum, R570, and S. bicolor genome. The red lines indicate collinear relationships among CaCA genes. The chromosome number is indicated at the top of each chromosome (b) Distribution of gene type among CaCA genes in S. spontaneum, R570, and S. bicolor. (c) Syntenic relationships of S. spontaneum, R570, and S. bicolor genes among S. spontaneum, R570, and S. bicolor

To elucidate the evolutionary genome rearrangement and duplication patterns of the CaCA protein encoding genes in S. spontaneum, R570, and S. bicolor, an analysis of gene duplication events including whole genome duplications (WGD)/segmental, dispersed duplication, proximal duplication, singleton duplication, and tandem duplication was performed (Fig. 3b, Supplemental Table S6). Duplication was observed in all predicted CaCA genes, among which WGD/segmental duplications were the main modes in SsCaCAs, while dispersed duplications were the main modes in ShCaCAs and SbCaCAs (Fig. 3b).

In order to further infer the evolutionary mechanism of CaCA superfamily genes, syntenic maps between S. bicolor, R570, and S. spontaneum were constructed (Fig. 3c). As shown in Fig. 3c, only four orthologous pairs between S. spontaneum and R570 were found. Between S. spontaneum and S. bicolor, 27 syntenic orthologous gene pairs were observed. We found that one S. bicolor gene corresponded to multiple S. spontaneum genes, such as SbCCX1 - SsCCX1a/1b/1c. A comparison of the syntenic blocks showed that 19 collinear gene pairs, 18 pairs between S. bicolor and S. spontaneum and one pair between S. bicolor and R570, were anchored to the highly conserved syntenic blocks, which spanned more than 100 genes. Only three collinear gene pairs (SbCAX3-SsCAX3b, SbCCX1-SsCCX1b, and SbCCX5-SsCCX3b) were located in syntenic blocks that possessed fewer than 30 orthologous gene pairs (Supplemental Table S7).

According to the syntenic relationships of CaCA genes from S. spontaneum, R570, and S. bicolor, the synonymous (Ks), non-synonymous (Ka), and Ka/Ks ratio values were calculated (Supplemental Table S7). The Ka/Ks ratio showed that all Ka/Ks values of the orthologous CaCA genes among S. spontaneum, R570, and S. bicolor were < 1, suggesting that these orthologous genes underwent strong purifying selection for retention.

microRNA target prediction

In order to reveal the interactions between microRNAs (miRNAs) and their CaCA gene targets, the potential networks were predicted by the psRNATarget server (Supplemental Figure S4 and Supplemental Table S8). In S. spontaneum, four SsCAXs and three SsCCXs were regulated by four miRNAs. It is worth noting that ShCAX1 has nine miRNA target sites in two miRNA families. Surprisingly, seven SbCaCA genes, i.e., two SbCAXs, four SbCCXs, and one SbMHX, were regulated by 49 miRNAs. In general, one CaCA gene might be targeted by multiple miRNAs, while several CaCA genes might be regulated by the same miRNA.

Gene ontology (GO) annotation

GO annotation was performed for all CaCA genes to determine their potential functions. As shown in Supplemental Figure S5, CaCA genes are involved in various biological processes (BP), molecular functions (MF), and cellular components (CC) (Supplemental Table S9). Under the BP category, we found that all of the CaCA genes (53) were further annotated to localization and cellular processes, while 28 were annotated to biological regulation, 10 to response to stimulus, and two to metabolic processes. In the MF category, they were annotated to transporter activity (33 genes), binding (10 genes), and catalytic activity (two genes), which agreed well with the transporter property of these CaCA genes. With respect to the CC category, the majority of CaCA genes were predicted to be involved in the cellular anatomical entity (39 genes) and intracellular (38 genes) categories. In addition, 28 CaCA genes were involved in the cell category and two CaCA genes encoded protein-containing complexes.

Interactions among CaCA proteins

Predicting the interactions among CaCA proteins is helpful for understanding their interactive relationships. As shown in Fig. 4, a total of 53 CaCA proteins were predicted to have direct or indirect interaction relationships. For example, Sb09g030750.1 was predicted to have direct interactions with Sb05g026100.1, Sb03g008600.1, Sb04g008850.1, Sb01g033220.1, or Sb08g002860.1. It is worth noting that these CaCA proteins may interact with the peroxisome biogenesis protein (Sb09g001850.1), plasma-membrane choline transporter (Sb01g013160.1), plasma membrane-type calcium-transporting ATPase 2 (Sb07g028160.1), and endoplasmic reticulum-type calcium-transporting ATPase 4 (Sb01g038990.1 and Sb09g001850.1). In general, these interactive relationships provide an important reference for identifying the true interactions of CaCA proteins in biochemical experiments.

Fig. 4.

Predicted protein–protein interactions of CaCAs according to their orthologs in S. bicolor. In the network, only the pairs with more than 60 % sequence identity between SbCaCAs, ShCaCAs, or SsCaCAs and SbCaCAs and with an interaction score > 0.4 are shown. Line and node colors indicate the different types and degrees of interactions, respectively. The filled or empty nodes represent known or unknown 3D structures, respectively. The gene names in parentheses indicate that paralogous or orthologous gene names

Expression profiles of CaCA genes in sugarcane in response to hormonal (ABA) stress

Eight CaCA genes were retained for the quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. The expression profiles of eight CaCA genes in sugarcane under ABA treatment were successfully detected (Fig. 5). In brief, all CaCA genes were induced at 6-h time points, and five CaCA genes from the CAX, CCX, and EFCAX families peaked at 6 h post-treatment. Five CaCA genes (SsCAX2a, SsCAX3c, SsCAX4a, SsCCX4b, and SsMHX2) were induced at both 3 h and 6 h.The transcript profiles of SsCAX2a, SsCAX4a, SsCCX4b, and SsMHX2 were promoted at all treated time points.

Fig. 5.

Expression dynamics of the candidate CaCA genes in sugarcane in response to ABA treatment. Error bars represent the standard error (SE) of three independent biological experimental repeats. The value on the Y-axis indicates the relative gene expression levels. The x-axis represents the time points when the samples were collected. Different lowercase letters indicate a significant difference, determined by one-way ANOVA, followed by Duncan’s new multiple range test (p < 0.05)

Expression characteristics of CaCA genes in sugarcane under biotic (Sporisorium scitamineum) stress

qRT-PCR analysis was performed to investigate the expression characteristics of eight CaCA genes in sugarcane in response to S. scitamineum (Fig. 6). In the CAX family, the expression of SsCAX1 was inhibited at all treatment time points. Three CAX genes (SsCAX2a, SsCAX3c, and SsCAX4a) had the highest expression at 48 h. In the CCX family, SsCCX4b were downregulated at all treatment time points. At 24 h, SsCCX2b had the highest expression levels. The expression of SsEFCAX2 was upregulated at 6 and 24 h, and downregulated at 120 h. The expression level of SsMHX2 was upregulated at 48 h.

Fig. 6.

Expression dynamics of the candidate CaCA genes in sugarcane after Sporisorium scitamineum infection. Error bars represent the standard error (SE) of three independent biological experimental repeats. The value on the Y-axis indicates the relative gene expression levels. The x-axis represents the time points when the samples were collected. Different lowercase letters indicate a significant difference, determined by one-way ANOVA, followed by Duncan’s new multiple range test (p < 0.05)

The abiotic (cold) stress-induced expression profiles of CaCA genes in sugarcane

The transcriptional profiles of eight CaCA genes under cold stress were monitored by qRT-PCR in this study (Fig. 7). In the CAX family, the expression of SsCAX1 was upregulated at 12 and 24 h. Under cold stress, three CAX genes were downregulated at all treatment time points. In the CCX family, SsCCX2b were downregulated at all treatment time points and the expression levels of SsCCX4b were inhibited at 6 h. SsEFCAX2 was upregulated at 12 and 24 h. The expression levels of SsMHX2 were downregulated at all treatment time points.

Fig. 7.

Expression dynamics of the candidate CaCA genes in sugarcane under cold stress. Error bars represent the standard error (SE) of three independent biological experimental repeats. The value on the Y-axis indicates the relative gene expression levels. The x-axis represents the time points when the samples were collected. Different lowercase letters indicate a significant difference, determined by one-way ANOVA, followed by Duncan’s new multiple range test (p < 0.05)

Identification and sequence analysis of ScCAX4

As calcium-binding proteins, CAXs play vital roles in regulating the concentration of Ca²⁺ in cell compartments [27]. In this study, through reverse transcription-polymerase chain reaction (RT-PCR), one CAX gene (GenBank Acc No. MW206380) that encoded a polypeptide of 417 amino acids, was isolated from ROC22, a Saccharum spp. hybrid (Fig. 8a). Because ScCAX had a high homology with SsCAX4a (99.21 %) and SsCAX4e (99.13 %), both of which belong to the CAX family Type 1B (Table S10), this ScCAX was renamed ScCAX4. Analysis of the primary protein structure indicated that ScCAX4 is an acid unstable basic hydrophobic protein (Table S11). The phylogenetic tree showed that ScCAX4 belongs to the Type 1B group, among which all members have a broad substrate range, and modulation of this transporter may be an important component of future strategies to improve plant ion tolerance. Pfam and transmembrane analyses showed that ScCAX4 contained two “Na_Ca_ex” domains (Pfam ID: PF01699). The “Na_Ca_ex” domain in the C-terminal denoted five core consensus transmembrane hydrophobic regions, and the one in the N-terminal represented another five core consensus transmembrane hydrophobic regions (Fig. 8b).

Fig. 8.

The sequence of the ScCAX4 gene and the characteristics of its encoded protein. (a) Complete cDNA and deduced amino acid sequences of the ScCAX4 gene. The full length cDNA was 1251 bp with an open reading frame encoding 417 amino acids. The sequences marked with the rectangle show the specific amplification primer pair for ScCAX4. * represents the stop codon. (b) The schematic diagram of the ScCAX4 protein conserved domains and transmembrane domains

Expression of ScCAX4 in different tissues and under ABA, Sporisorium scitamineum, and cold stresses

The expression of ScCAX4 in four different sugarcane tissues, and under ABA, S. scitamineum, and cold (4 °C) stresses was detected by qRT-PCR (Fig. 9). ScCAX4 was constitutively expressed in various tissues with different levels, and the expression in root was the highest. Under ABA treatment, the expression of ScCAX4 was induced at 3 and 6 h. Under 4 °C stress, the transcript of ScCAX4 was inhibited at all treatment time points. However, in response to S. scitamineum stress, the expression of ScCAX4 was induced at all treatment time points.

Fig. 9.

Expression patterns of the ScCAX4 gene in sugarcane under ABA, Sporisorium scitamineum, and cold stresses. Error bars represent the standard error (SE) of three independent biological experimental repeats. The value on the Y-axis indicates the relative gene expression levels. The x-axis represents the time points when the samples were collected. Different lowercase letters indicate a significant difference, determined by one-way ANOVA, followed by Duncan’s new multiple range test (p < 0.05)

Subcellular localization of ScCAX4

The subcellular localization of the ScCAX4::GFP fusion protein was detected by laser scanning confocal microscopy after infiltration for two days. As indicated in Fig. 10, the ScCAX4::GFP fusion protein was localized in the cytoplasm, plasma membrane, and nucleus.

Fig. 10.

Subcellular localization analysis of ScCAX4 in N. benthamiana leaves. White arrow a, b, and c indicates the nucleus, plasma membrane, and cytoplasm, respectively. Epidermal cells were imaged using visible light, and green fluorescence, and merged images are shown. The scale bar is on the bottom right

Discussion

Members of the Ca²⁺/cation antiporter (CaCA) superfamily, as important “gatekeepers”, function in cellular ion homeostasis [13, 24]. At present, the CaCA superfamily genes have been identified and characterized in many plant species such as A. thaliana [15], Zea mays [8], and Oryza sativa [15]. However, virtually no systematic and comprehensive analyses of the CaCA superfamily genes in S. spontaneum, R570, or S. bicolor have been undertaken. In the present study, a multi-level analysis of CaCA genes from S. spontaneum, R570, and S. bicolor was performed to investigate their evolutionary relationships and functional divergence. The systematic characterization of the CaCA superfamily genes in the present study should provide a better foundation for further functional verification of this gene superfamily in the future.

Evolution, conservation, and expansion of the CaCA superfamily

As phylogenetic analysis illustrated, these 53 CaCA superfamily genes from S. spontaneum, R570, and S. bicolor consisted of the CAX, CCX, EFCAX, and MHX families. Commonly, the CAX family genes are classified into two groups (Type 1A and Type 1B) [8]. The phylogenetic tree showed that 11 CAXs (eight SsCAXs and three SbCAXs) were clustered into Type 1A and eight CAXs (three SsCAXs, two ShCAXs, and three SbCAXs) were sorted into Type 1B. Type 1A CAX proteins, such as AtCAX1 and AtCAX3, are usually considered to be specific for Ca²⁺ [28, 29]. However, Type 1B CAX proteins, such as AtCAX2 and AtCAX5, can promote the transportation of various ions such as Ca²⁺, Cd²⁺, and Mn²⁺ [28, 30, 31]. In this study, we also divided 12 SsCCXs, one ShCCXs, and five SbCCXs into three categories according to the classification methods for AtCCXs [8]. In general, Group 1 and Group 2, which include CCXs from monocot and dicot plants, also contain CCXs from mosses. Group 3 contains solely angiosperm CCXs. Except for R570, within Group 3, there were usually two CCXs for each dicot species (A. thaliana and Vitis vinifera), three CCXs for each monocot species (Brachypodium distachyon, Z. mays, and S. bicolor), and nine CCXs for S. spontaneum, which clearly demonstrated recent gene duplication within Group 3.

Regarding their substrate specificity, it is tempting to speculate that members of Group 2 may share the function of AtCCX3 as an endomembrane H⁺-dependent K⁺ transporter [32], while plant members of Group 1 such as AtCCX5 may be more likely to have Ca²⁺ transport activity due to their closer relationship to NCKX6/NCLX [8]. Interestingly, the EFCAXs were divided into three groups (Group 1, Group 2, and Group 3). In addition, seven SsEFCAXs, one ShEFCAXs, and two SbEFCAXs were all clustered into Group 2, which only contained monocot EFCAXs, suggesting a diversification of EFCAX genes within land plants. With respect to the MHX family, there were some long branch lengths within the MHXs between angiosperms and mosses, indicating a degree of divergence between these MHXs. From the topology of the MHX phylogenetic tree, we can reasonably speculate that these MHXs in monocot and dicot plants evolved from the same ancestor.

Potential functional roles of CaCA superfamily genes

In plants, miRNAs are involved in various crucial biological processes including plant development [33], biotic stress responses [34], abiotic stress responses [35], and signal transduction [36]. In our study, seven SsCaCA genes had four miRNA family target sites in S. spontaneum, while in S. bicolor, ShCAX1 had two miRNA family target sites, and seven SbCaCA genes had 12 miRNA family target sites. Due to the more complete miRNA data for S. bicolor, the SbCaCAs matched more miRNAs. Interestingly, SsCAX5a, SsCAX5b, SsCAX5c, ShCAX1, SbCAX4, and SbCCX4 all had the miR437 family target sites. Since members of miR437 can be found in O. sativa, Z. may, S. bicolor, and Saccharum spp., but not in Arabidopsis or Populus, miR437 is considered to be a monocot-specific miRNA [37, 38]. Several drought-related proteins, such as aquaporin, were found to be targeted by miR437 [39]. The results suggested that these CaCA genes may be involved in the drought stress response. Abdel-Ghany and Pilon et al. found that miR408 responds to copper deficiency in Arabidopsis [40]. ShCAX1 and SbCAX4 were both found to be targeted by miR408, which means that they may play the same role in the response to copper stress.

qRT-PCR analysis is commonly used to understand the function, especially the expression characteristics, of certain genes. The CaCA superfamily genes have been reported to play a role in various stresses in many plant species [9, 41, 42]. In the present study, the expression levels of CaCA genes under hormonal (ABA), biotic (S. scitamineum), and abiotic (cold) stresses were analyzed by qRT-PCR. As an important phytohormone, ABA plays a vital role in regulating plant growth, development, and stress responses [43]. Previous studies showed that Ca²⁺ channels were ABA-induced [44, 45]. In the present study, most of the candidate CaCA genes showed similar expression patterns and were upregulated at 3 or 6 h post ABA treatment. The results indicated that CaCA genes may play similar roles and function at the early stage of signal transduction after ABA treatment.

Expression characteristics of the ScCAX4 gene and the subcellular localization of the ScCAX4 protein

ScCAX4 is a member of the Type 1B group in the CAX family, suggesting that it may have a broad substrate range (including Ca²⁺, Cd²⁺, and Mn²⁺) [9]. ScCAX4 was constitutively expressed in various tissues, with the highest expression in root. Under ABA treatment, the expression of ScCAX4 was induced at 3 and 6 h. These results were similar to those observed for most of the CaCA genes in Saccharum, indicating their conserved function under ABA stress. Under S. scitamineum and 4 °C stress, ScCAX4 exhibited two completely opposite expression trends and was inhibited and induced at all treatment time points under cold and S. scitamineum stresses, respectively. These results implied that ScCAX4 may play contrasting roles in response to biotic and abiotic stresses. The subcellular localization showed that the ScCAX4::GFP fusion protein was localized in the cytoplasm, plasma membrane, and nucleus. Previous studies showed that most plant CAX proteins were determined to be localized to vacuolar membranes [30, 46, 47]. However, Ca²⁺/H⁺ exchange activity had been detected in plasma and plastid membranes [48, 49]. Furthermore, ScCAX4 contained 11 transmembrane domains. Based on the protein interactions among CaCA proteins, two proteins (SsCAX4a, and SsCAX4e), which had a high homology with ScCAX4, interacted with one plasma membrane-type calcium-transporting ATPase (Sb07g028160.1) and two endoplasmic reticulum-type calcium-transporting ATPases (Sb01g038990.1 and Sb09g001850.1). The results indicated that ScCAX4 may function as a calcium transporter (Fig. 11).

Fig. 11.

 A hypothetical ion transport model of ScCAX4. PM-ATPase indicates plasma membrane-type calcium-transporting ATPase; ER-ATPase indicates endoplasmic reticulum-type calcium-transporting ATPase

Conclusions

In this study, 53 CaCA genes were identified from S. spontaneum, R570, and S. bicolor. These genes could be divided into four gene families: the CAX, CCX, EFCAX, and MHX families. The divergent biochemical characteristics of CaCA proteins were analyzed. Based on the phylogenetic tree, these CAX, CCX, EFCAX, and MHX family proteins had different classification statuses. The similar motif compositions of these proteins and the exon/intron structures of the CaCA genes within the families further supported the classification predicted by the phylogenetic tree. Synteny analysis and gene duplicated types of CaCA genes from S. spontaneum, R570, and S. bicolor provided valuable clues about their evolutionary characteristics. GO annotation and protein–protein interaction analysis revealed multiple functions for the CaCA proteins. The qRT-PCR analyses of the SsCaCA and ShCaCA genes confirmed that CaCA genes play a similar part under ABA treatment, but various roles in response to S. scitamineum and cold stresses. ScCAX4, a member of the Type 1B group in the CAX family, may function as a “transporter” and has a broad substrate range. Taken together, although the functions of these CaCA genes remain unknown and further research is still needed, our findings should lay a foundation for unraveling the biological roles and functions of CaCA genes in Saccharum.

Methods

Plant materials and treatments

The sugarcane cultivar ROC22 (Saccharum spp. hybrid) was provided by the Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture (Fuzhou, China).

For ABA treatment, sugarcane seedlings with 3–4 fully expanded leaves were treated with 100 µM ABA [50]. After treatment, the leaves were sampled at 0-, 3-, 6-, and 24-h time points. For S. scitamineum stress, the sugarcane cultivar ROC22 was inoculated with 0.5 µL of a 0.01 % (v/v) Tween-20 suspension containing 5 × 10⁶ smut spores·mL^− 1; the control was inoculated with 0.01 % (v/v) Tween-20 in sterile distilled water. One mixed sample was collected for every three individual buds. Buds were randomly selected at 0 h, 6 h, 24 h, 48 h, and 120 h after inoculation [51]. For cold (4℃) treatments, the leaves were sampled at 0-, 6-, 24-, and 48-h time points [52–54]. Three biological replicates were prepared for each treatment. All the buds were frozen in liquid nitrogen and then stored at -80 °C until total RNA extraction.

Total RNA extraction and first-strand cDNA synthesis

Total RNA was isolated using TRIzol reagent (Invitrogen, Shanghai, China) following the manufacturer’s instructions. The quantity and quality of RNA were assessed by a multifunction microplate reader Synergy H1 (Bio-Tek, Winooski, VT, USA) and 1.5 % agarose gel electrophoresis. The cDNA of RT-PCR was generated using 500 ng RNA with a HiScript III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The cDNA of qRT-PCR was synthesized using 500 ng RNA with the HiScript Q RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, Nanjing, China).

Identification of CaCA superfamily genes

In this study, protein sequences and the genomic annotation of S. spontaneum were downloaded from the following link: http://www.life.illinois.edu/ming/downloads/Spontaneum_genome/ [17]. The monoploid reference R570 genome data (a single tiling path version) were obtained from the Sugarcane Genome Hub (http://sugarcane-genome.cirad.fr/) [25]. Phytozome (https://phytozome.jgi.doe.gov/) [55] was used to download S. bicolor data and the protein sequences of the other species.

To identify CaCA superfamily genes, the hidden Markov model (HMM) profile of Na_Ca_ex (PF01699) was downloaded from the Pfam protein family database (Pfam 32.0; http://pfam.sanger.ac.uk/) [56]. Hmmsearch (HMMer package version 3.1b1) was used to search Na_Ca_ex.hmm against the protein sequences from each plant genome [57]. If two or more transcripts were annotated for the same gene from alternative splicing, the first transcript isoform was selected for further study. The candidate CaCA proteins were then screened by hydropathy analysis using the TMHMM v2.0 program (http://www.cbs.dtu.dk/services/TMHMM/) to remove all non-TM proteins. Then, the remaining CaCA genes were further verified by the NCBI Conserved Domain Database (CDD: https://www.ncbi.nlm.nih.gov/cdd) [58] and Pfam (http://pfam.sanger.ac.uk/) .

Multiple sequence alignment and phylogenetic analysis

Protein multiple sequence alignment (MSA) was analyzed by MUSCLE v3.7 with default parameters [59]. MEGA X was applied to infer the phylogenetic tree using NJ method with the following parameters: Poisson model, pairwise deletion, and 1000 bootstrap replications. IQ-TREE multicore version 1.6.12 was employed to construct ML tree with 1000 ultrafast bootstraps [60]. The resulting treefile was visualized with EvolView (https://www.evolgenius.info/evolview/#login) [61].

Protein properties and sequence analyses

The basic properties (isoelectric point, molecular weight, grand average of hydropathicity, and instability index) of these CaCA proteins were predicted by ExPASy (http://web.expasy.org/protparam/). WoLF PSORT (https://wolfpsort.hgc.jp/) was used to predict the subcellular localizations of these CaCA proteins.

The protein motifs were analyzed by the MEME suite (http://meme-suite.org/tools/meme) with the following parameters: maximum number of motifs was 10, the optimum width of motifs was set between 10 and 50, and the distribution of motif occurrences was zero or one per sequence. The protein motifs and gene structures were illustrated by TBtools [62]. psRNATarget (http://plantgrn.noble.org/psRNATarget/) was applied to predict whether miRNA interacted with the CaCA genes [63].

Synteny analysis and chromosome localization

The multiple Collinearity Scan toolkit (MCScanX) was used to detect the syntenic blocks and gene duplication events with the default parameters [64]. The syntenic blocks were used for constructing a synteny analysis map within and between genomes. Diagrams were generated using the Circos program (http://circos.ca/) and MCScanX [64]. Based on the comparative synteny map between the S. spontaneum, R570 or S. bicolor genomes, the Ks and Ka nucleotide substitutions between orthologous gene pairs were calculated by TBtools [62].

Gene ontology annotation

The gene ontology terms for the CaCA proteins were identified using default parameters in the Blast2GO v5 tool [65]. Initially, the sequences were screened using BLASTP, followed by mapping, InterProScan analysis and annotation. Furthermore, the biological processes, cellular components and metabolic pathways were predicted using identified GO terms.

Prediction of the protein–protein interaction network

The interaction network of CaCA proteins in S. spontaneum, R570, and S. bicolor was predicted using the STRING (version 11.0, http://string-db.org) website, which contains the known and predicted protein-protein interactions of different organisms [66]. In this study, all the CaCA protein sequences were submitted to the STRING website as queries, and S. bicolor was chosen as the reference organism for blasting because sugarcane has no protein–protein interaction database and is not included in STRING. After blasting, the matched homologs of S. bicolor with the highest scores (Bitscore) and more than 60 % identity were used to construct the network. The predicted interactions with scores > 0.4 (medium confidence level was set in STRING) are shown in the network.

Expression patterns under ABA, Sporisorium scitamineum, and cold stresses

qRT-PCR was used to detect the relative expression levels of nine CaCA genes (eight SsCaCAs and ScCAX4) under ABA, S. scitamineum, and cold stresses. The qRT-PCR primers of these CaCA genes were designed by Beacon Designer 8.14 software. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH (GenBank Acc No. CA254672)) gene was chosen as the reference gene [67]. According to the ChamQ™ Universal SYBR^@ qPCR Master Mix manual (Vazyme, Nanjing, China), the qRT-PCR reaction system was set as follows: SYBR Green Master Mix: 10 µL, 10 µM forward primers: 0.4 µL, 10 µM reverse primers: 0.4 µL, 10×diluted cDNA template: 1.0 µL, and sterile distilled water: 8.2 µL. The reaction procedure was as follows: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. The melting curves were analyzed after 40 cycles. The relative expression level of qRT-PCR data was calculated by standard curves (Supplementary Table S12) [68]. All primers used in qRT-PCR are listed in Supplementary Table S12. All sequences used in qRT-PCR are listed in Supplementary Table S13.

Gene cloning and protein analysis

Based on our previous RNA sequencing data [69], the sequence of ScCAX4 was obtained. The NCBI primer designing tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) was used to design the cloning primers (Supplementary Table S12). RT-PCR with LA Taq (TaKaRa, Dalian, China) was used to amplify the ScCAX4 from ROC22 [70]. The percentage identity between 11 SsCAXs, two ShCAXs, six SbCAXs and one ScCAX was calculated by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). ExPASy (http://web.expasy.org/protparam/), the TMHMM v2.0 program (http://www.cbs.dtu.dk/services/TMHMM/), and Pfam protein family database (Pfam 32.0; http://pfam.sanger.ac.uk/) [56] were used to predict the primary protein sturctures, transmembrane domains and conserved domains, respectively.

Subcellular localization analysis

The pFAST-R05-ScCAX4-GFP vector was constructed according to the gateway technology protocol. The induction medium (10 mM MES, 10 mM MgCl₂, 200 µM acetosyringone, pH 5.0–5.4) was used to centrifuge and resuspend the Agrobacterium tumefaciens GV3101 cells, which contained pFAST-R05-ScCAX4-GFP, at an OD₆₀₀ of 0.8. The A. tumefaciens GV3101 cells, which contained pFAST-R05-GFP, were used as a control. The induction medium was infiltrated into Nicotiana benthamiana leaves using a syringe without a needle. The subcellular localization results were visualized by laser scanning confocal microscopy (Leica TCS SP5, Wetzlar, Germany) after infiltration for two days.

Authors’ contributions

Conceived and designed the experiments: WHS and YXQ. Performed the experiments: WHS, YJR, DJW and TTS. Analyzed the data: WHS, JFF and CZ. Wrote the paper: WHS. Revised the final version of the paper: LPX, YCS, MTS and YXQ. All authors read and approved the final manuscript.

Funding

This work was funded by National Key R&D Program of China (2019YFD1000500 and 2018YFD1000503), National Natural Science Foundation of China (31871688, 31671752, 31101196 and 31340060), Natural Science Foundation of Fujian Province, China (2015J06006), and China Agriculture Research System of MOF and MARA (CARS-17). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials

The sequences of ScCAX4 genes from sugarcane analysed during the current study are available in the NCBI repository with the Accession Numbers of MW206380. The data of Saccharum spontaneum genome can be downloaded from the following link: http://www.life.illinois.edu/ming/downloads/Spontaneum_genome/. The monoploid reference R570 genome can be downloaded from the following link: http://sugarcane-genome.cirad.fr/. The Sorghum bicolor genome can be downloaded from the following link: https://phytozome.jgi.doe.gov/. the protein sequences of the other species can be downloaded from the following links: https://phytozome.jgi.doe.gov/, https://www.ncbi.nlm.nih.gov/, and http://bioinformatics.psb.ugent.be/webtools/bogas/overview/Ectsi. The accession numbers are included in the supplemental data. All the other data supporting the conclusions of this article are within the paper.

Declarations

Ethics approval and consent to participate

Not applicable. This is to confirm that no specific permits were needed for the described experiments. Besides,experimental research and field studies on plants (either cultivated or wild), including the collection of plant material, comply with relevant institutional, national, and international guidelines and legislation. And this study did not involve any endangered or protected species.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.