Systematic analysis of the OSCA family members in Vigna radiata and their involvement in osmotic resistance

Mung bean (Vigna radiata) is a warm-season legume crop and belongs to papilionoid subfamily of the Fabaceae. China is the leading producer of mung bean in the world. It has signicant economic and health benets and is a promising species with broad adaptation and high tolerance to stress environments. The OSCA family members play an important role in the modulation of hypertonic stresses, such as drought and salinity. However, genome-wide analysis of the OSCA family in mung bean is lacking. revealed functional of in promoter of VrOSCA large-scale expansion of -2.2, -2.4, -2.5, -2.6 and − 3.1 increased by a factor of more than 10-fold compared with 0 h. Under NaCl treatment, the expression of VrOSCA1.4, -1.5, -2.2, -2.4, -2.5 and − 3.1 increased by a factor of more than 10-fold compared with 0 h. Among these genes, VrOSCA1.4showed the largest folds change in relative expression levels under the three types of osmotic stress compared with normal growth conditions. These results indicated that mung bean OSCA genes responded to osmotic stress caused by ABA, PEG and NaCl treatment. The total RNA from the leaves was isolated using an RNAprep Pure Plant Kit (Tiangen, Beijing, China), and the rst-strand cDNA was synthesized using SuperScript™ III Reverse Transcriptase kit (Invitrogen, USA). Quantitative real-time PCR (qRT-PCR) was performed in ABIViiA 7 Real-Time PCR system (Applied Biosystems, USA) by SYBR Green PCR mix (QIAGEN). The PCR reaction was performed with the following conditions: 95°C for 2 min followed by 40 cycles of 94°C for 10 s and 59°C for 10 s. The relative expression level of VrOSCA genes was calculated by the 2 − ∆∆ CT method [42]. Gene-specic primers were designed using Primer Express Software v2.0 (Additional le 7) and synthesized commercially (HUADA Gene, Beijing, China). The V. radiata actin gene (GenBank: AF143208.1) was used as an endogenous control for qRT-PCR. Each experiment was repeated using different cDNAs from three biological replicates.


Results
We identi ed a total of 13 OSCA genes in the mung bean genome and named according to their homology with AtOSCAs. All the OACAs were phylogenetically splitted into four clades. Phylogenetic relationship and synteny analyses showed that the VrOSCAs in mung bean and soybean shared a relatively conserved evolutionary history. In addition, three duplicated VrOSCA gene pairs were identi ed and the duplicated VrOSCA shave mainly undergone purifying selection pressure during evolution. Protein domain, motif and transmembrane analysis indicated that most of the VrOSCAs shared similar structures with their homologs. The expression pattern showed that exception of VrOSCA2.1, the other 12VrOSCAs were up-regulated expression under treatment with ABA, PEG and NaCl, among which VrOSCA1.4 showed the largest increased expression levels. The duplicated genes VrOSCA2.1/VrOSCA2.2 showed divergence expression, which might experience functionalization during subsequent evolution. The expression pro les under ABA, PEG and NaCl stress revealed a functional divergence of VrOSCA genes, which agreed with the cis-acting elements analysis in the promoter of VrOSCA genes.

Conclusions
Collectively, the study provided a systematic analysis of the VrOSCA family in mung bean. Our results would lay an important foundation for functional and evolutionary analysis of VrOSCAs, and provide promising genes for further investigation of abiotic stress tolerance in mung bean.

Background
In natural environmental conditions, plants are subjected to many stresses. The osmotic stress caused by drought and salinity is one of the key stress factors affecting plants growth and yield [1]. Osmotic stress usually broke the plants' osmotic balance and nally caused damage of cell membrane system [2]. Previous studies have found plant responses to stresses mainly include the perception, transmission of signals through various pathways and the regulation of stressresponsive gene expression, resulting physiological and morphological modi cations to resist the stresses [3][4][5]. During the signal perception, calcium is an important second messenger in the signal transduction pathway when plants respond to stresses [6,7]. Under Osmotic stress, plants induce a rapid increase of free calcium ion concentration in intracellular, thereby inducing the expression of many stress-related genes to regulate plant tolerance to osmotic stress [5,8,9]. The increase of calcium ion concentration in intracellular is mainly regulated by calcium transport systems such as calcium channels and calcium pumps [10]. Previous studies showed that stimuli-gated Ca 2+ -permeable channels served as osmosensors in bacteria and animals [11,12], which indicated that there might be speci c calcium permeable channels that function as osmosensors in plants.
In plants, the hyperosmolality-gated calcium-permeable channels (OSCA) is the calcium non-selective cation channel protein as well as the receptor protein for hypertonic stress [13][14][15]. Studies of the functional domain have shown OSCA family contained the calcium-dependent channel domain (DUF221) that may participate in osmotic adjustment [16,17]. In rice, the entire OSCA family is characterized by the presence of a conserved DUF221 domain, which functions as an osmotic-sensing calcium channel [18]. In Arabidopsis, OSCA1, a hyperosmolality-gated calcium-permeable channel, was characterized as an osmosensor and mediated osmotic-stress-evoked Ca 2+ concentration increases [14]. Studies have shown maize gene ZmOSCA2.4 could enhance drought tolerance in transgenic Arabidopsis [19]. It can be seen that OSCA family numbers play a crucial role in plants resistance to osmotic stress. Therefore, it is an important path to excavate and study potential genes for breeding osmotic-stress-resistant varieties. The predecessors have systematically identi ed and analyzed the OSCA family in dicotyledon such as Arabidopsis thaliana and soybean, and monocotyledon rice [14,18,20]. However, genome-wide analysis of the OSCA family in mung bean is lacking.
Mung bean (Vigna radiata (L.) R. Wilczek, 2n = 2×=22) belongs to the papilionoid subfamily of the Fabaceae and is always grown in poor-soil regions because its broad adaptation and high tolerance to stresses. Mung bean seeds are rich in proteins and contain higher levels of folic acid and iron than most other legumes [21]. With the completion of mung bean genome sequencing, an opportunity was opened to systematic research the OSCA family in mung bean [22]. In the present study, we identi ed putative OSCA family members in mung bean and analyzed their phylogeny, syntenic relationship, conserved motifs, transmembrane regions and cis-elements responsive to abiotic stresses in promoters. In addition, we studied the expression pro les of OSCAs under PEG, NaCl and ABA treatments. These ndings will facilitate further research on the biological function of this gene family and provide promising genes for cultivation of genetically modi ed osmotic-stress-resistant plants.

Genome-wide identi cation of OSCA gene family members in mung bean
The Hidden Markov Model of DUF221 domain (Pfam accession number: 02714) was used to search against the mung bean genome. Ultimately, a total of 13 VrOSCA genes were identi ed in mung bean and named according to the Arabidopsis orthologs (Table 1). Among the 13 genes, 12VrOSCA genes were distributed randomly on all the 11 chromosomes except chr2, 8 and 10, while VrOACA2.5 was located on scaffold_100. The amino acid numbers of all identi ed VrOACAs varied from 592 (VrOACA2.2) to 880 (VrOACA4.1). The molecular weight of the VrOSCA proteins varied from 67.16 (VrOACA2.2) to 99.16 kDa (VrOACA4.1) and the isoelectric points ranged from 6.28 (VrOACA4.1) to 9.44 (VrOACA2.5).  16 4 Phylogenetic analysis of the OSCA gene family in mung bean To elucidate the phylogenetic relationships of OSCA proteins in mung bean, Arabidopsis, soybean and rice, the phylogenetic tree based on the alignment of 60 full-length OSCA protein sequences was build ( Fig. 1, Additional le 1). The 60 OSCA proteins were classi ed into four major group, clade 1, 2, 3, and 4. The clade 1 and 2 contained more members than clade 3 and 4. Phylogenetic analysis result showed that the OSCA family experienced a similar evolutionary history between the mung bean, Arabidopsis, soybean and rice genomes. Moreover, OSCA proteins derived from mung bean had a higher similarity with those from soybean, demonstrating a closer phylogenetic relationship between mung bean and soybean since both of them belong to the leguminous family.
Collinearity analysis of OSCA genes in mung bean, Arabidopsis, soybean and rice Comparative genomics analyses of gene collinearity reveal homologous gene functions and phylogenetic relationships between species. Thus, we analyzed the colinearity relationship of OSCA genes with three representative species, including one monocot (rice) and two dicots (Arabidopsis, soybean) ( Fig. 2, Additional le 2). We found that the OSCA genes of mung bean had the most homologous gene pairs with the OSCA genes of Glycine ma (17), followed by Arabidopsis (7) and Oryza sativa (1), indicating that in comparison with Arabidopsis and rice, mung bean OSCAs genes show a closer phylogenetic relationship with soybean. The result was consistent with the phylogenetic analysis ( Fig. 1), a rming the accuracy of our analysis. Some VrOSCAs (VrOSCA1.1, -1.4, -1.5, -2.4, -2.5, -3.1 and − 4.1) were found to be associated with two syntenic gene pairs in mung bean and soybean. These genes may play a crucial role during evolution. Meanwhile, no collinear segments of VrOSCA1.2 and VrOSCA2.2were found in the genome of mung bean and soybean. The results indicated that large-scale expansion of OSCAs probably occurred before mung bean-soybean division, and certain VrOSCAs might originate from duplication of the mung bean genome after phylogenetic divergence of mung bean.

Gene duplication of VrOSCAs in mung bean
To better understand the evolutionary relationship, gene duplication events were analyzed to elucidate the expansion patterns of the OSCA genes in mung bean. Three segmental duplication events with ve OSCAs were identi ed, which are located on duplicated segments on chromosomes 1, 4, 5, 6 and scaffold_100 (Fig. 3). Moreover, the Ka/Ks ratio of the duplicated VrOSCA gene pairs was calculated to evaluated the molecular evolutionary ( Table 2). All of the Ka/Ks ratios were less than 1. To further explore potential function of VrOSCAs, we detected additional conserved motifs using the MEME tool and a total of 20 conserved motifs were detected (Fig. 4b, Additional le 4). It is noteworthy that all clades contained motif1, 2 and 4, indicating that all genes perform the three functions. Among them, motif1 and motif2 located in the calciumdependent channel domain and motif4 located in the late exocytosis domain. Some conserved domains were restricted to speci c clades. For example, motif 16 and motif 12 were only detected in clade1 and clade2 respectively, which indicated functional difference between clade1 and clade2. We also observed different motifs within the same clade, suggesting that there were different action mechanisms within each clade. Cis-acting elements analysis in the promoter of VrOSCA genes The cis-acting elements in promoter regions of genes participate in various pathways, for example, the ABA and abiotic stress response signal transduction pathway [23]. Therefore, we analyzed the cis-acting elements involved in ABA and abiotic stress response in -1.5 kb promoter region of VrOSCA genes, including ABRE, DRE, MBS, TC-rich and LTR elements (Fig. 5, Additional le 5). We found all the VrOSCA genes, except VrOSCA1.5, contained at least one of these cis-acting elements. Moreover, the cis-acting elements of the VrOSCAs among clades were different. For example, clade1 and clade2 contained DER and MBS elements associated with drought stress, but genes in clade3 and clade4 did not. Clade1, clade2 and clade3 contained LTR element associated with low temperature stress, while gene in clade4 did not. It indicated that VrOSCA genes in different clades might response to stress collectively. In the clade2, only VrOSCA2.2 and VrOSCA2.4 contained MBS element, and only VrOSCA2.1 contained TC-rich element, and only VrOSCA2.2 contained LTR element. This phenomenon was also observed in clade 1. These results suggested that VrOSCAs in the same clade may have different action mechanisms.
Expression of VrOSCAs under ABA and abiotic stresses PEG and NaCl stresses may cause similar cellular damage and lead to osmotic stress [24]. Plants adapt and respond to drought and salt stress by inducing a range of gene expression. ABA is an important plant hormone that regulates the expression of stress-responsive genes in plants [25]. We studied the expression pro les of the 13 VrOSCA genes under ABA, PEG and NaCl treatments for 4h, 12h and 24h. Analysis of expression pro les showed that the exception of VrOSCA2.1, the other12 VrOSCA genes were up-regulated expression by ABA, PEG and NaCl treatments. VrOSCA2.1 was signi cantly down-regulated under ABA, PEG and NaCl treatments ( 1 increased by a factor of more than 10-fold compared with 0 h. Among these genes, VrOSCA1.4showed the largest folds change in relative expression levels under the three types of osmotic stress compared with normal growth conditions. These results indicated that mung bean OSCA genes responded to osmotic stress caused by ABA, PEG and NaCl treatment.

Discussion
With mung bean being a broadly adapted and highly stress-tolerant crop, the whole genome sequencing of mung bean is conducive to identi cation of resistance genes and genetic improvement of crops. In the present study, we performed a genome-wide analysis of the OSCA genes in mung bean and identi ed a total of 13 VrOSCA genes. The VrOSCA proteins varied substantially in the sequence and physicochemical properties (Table 1), which were comparable with OSCA genes from other plant species [14,18,20,26]. Phylogenetic tree (Fig. 1) analysis showed that OSCAs can be divided into four clades, which was consistent with evolutionary analysis of Arabidopsis, soybean and rice [14,18,20]. Each clade included OSCA members from mung bean, Arabidopsis, soybean and rice, indicated that the OSCA family originated and diversi ed prior to divergence of mung bean, Arabidopsis, soybean and rice. The clade3 and clade4 contained fewer members but conserved during species, indicating that OSCA member in clade3 and clade4 may perform an indispensable role in biological processes. The different number of OSCAs within the mung bean, Arabidopsis, soybean and rice indicates that the majority of OSCAs in mung bean, Arabidopsis, soybean and rice genomes undergo greater genetic variation after their divergence.
On the basis of OSCA family members phylogenetic relationships (Fig. 1), we systematically analyzed the synteny relationship of OSCAs in mung bean, Arabidopsis, soybean and rice. (Fig. 2, Additional le 2). Large-scale expansion of OSCAs probably occurred after monocots and dicots division. Despite VrOSCA2.2 and Glyma.04G048800 were clustered together (Fig. 1), VrOSCA2.2 was absent from the synteny analysis. We did not nd the synteny blocks related to VrOSCA1.2 and VrOSCA2.2. To elucidate the expanded mechanism of the OSCA gene family in mung bean, gene duplication events were investigated (Fig. 3 (Table 2). As the purifying selection restricts the gene divergences, the duplicated VrOSCA genes might have retained some similar functions [27]. Our results also showed that the expression pattern of VrOSCA2.3, -2.4 and − 2.5was similar under ABA-, PEG-and NaCl-induced osmotic stresses.
Previous studies have shown that OSCA protein contained 11 transmembrane helices (TMs)in Arabidopsis and 6-10TMs in rice [18,28]. In contrast, VrOSCAs contained 8-10 TMs, which indicates that VrOSCAs had experienced greater genetic variation during evolution. To investigate the structure features of VrOSCAs, conserved domain was analyzed. The results showed that the structural domain was highly conserved (Fig. 3a) and the distribution of protein domain pfam13967, pfam14703 and pfam02714 in VrOSCAs is consistent with that in OSCA proteins in maize [26]. Meanwhile, all the TMs were located in protein domain pfam 02714 and pfam13967. In this study, 20 distinct conserved motifs were identi ed.
The motifs of VrOSCAs in clade1, clade2 and clade3 were highly conserved and the composition patterns of the conserved motifs in these three clades were similar. However, VrOSCA4.1 in clade 4 contained fewer conserved motifs than other VrOSCAs. Moreover, the expression level of VrOSCA4.1 showed little difference under ABA-, PEG-and NaClinduced osmotic stresses, suggesting that VrOSCA4.1 may have a indirect function in osmotic stress response [19].
Analysis of promoter components of the 13 VrOSCA genes showed they contained variable types of core components associated with ABA responsiveness (ABRE) and stress responsiveness (DRE, MBS, LTR and TC-rich). For example, all the genes contained ABRE core components that play a crucial role in ABA-dependent gene expression, except VrOSCA1.5 (Fig. 5, Additional le 5). Similarly, the dehydration response elements DRE is involved in ABA-independent gene expression in response to osmotic stress [10]. Only ve VrOSCA genes, VrOSCA1.1, -1.2, -2.1, -1.2 and-2.6, contained DRE. Furthermore, the promoters of VrOSCA genes classi ed in the same clade also contained different types and numbers of response elements, so different genes classi ed in the same clade may show functional diversity and have different action mechanisms. Moreover, genes in different clades may exist synergies [29,30].
In this study, the dynamic osmotic stress responsive expression patterns of VrOSCAs were analyzed. Expression pro les analysis of VrOSCAs can help us to understand their possible functions in osmotic stress and offer crucial clues for functional assessment. As members of the OSCA hyperosmotic calcium channels proteins family, the VrOSCA genes responded to ABA-, PEG-and NaCl-induced osmotic stresses, consistent with the OSCA members in Arabidopsis and rice [18,31]. However, VrOSCAs exhibited differential expression under the osmotic stresses, not only amongst clades but also amongst members within the same clade, suggesting that these VrOSCAs might have diverse functions. The present results showed that VrOSCA2.1 was signi cantly down-regulated under ABA, PEG and NaCl treatments, whereas the other 12 VrOSCA genes were signi cantly up-regulated under the three osmotic stresses (Fig. 6, Additional le 6), suggesting that the 12 VrOSCAs might be crucial mediators of osmotic stress response and contribute to the establishment of complex signaling networks in mung bean. Up-regulation of the 8 VrOSCA genes (except VrOSCA1.1, -1.2, -1.3 and − 4.1) ranged from 10 to 70-fold, which indicated that these genes responded positively to osmotic stress. VrOSCA2.2 and − 2.4 responded strongly to ABA, PEG and NaCl stress, and showed more than 20-fold expression levels than the control (0 h).
Thus, VrOSCA2.2 and − 2.4 may simultaneously response to ABA, PEG and NaCl stress-response pathways and there may be interaction in the pathways responsive to the three stresses. Regardless, these genes played an important role in drought and high-salinity tolerance. Moreover, the expression of duplicated genes showed two pairs of duplicated genes shared similar expression patterns, suggesting they might retain some essential functions during subsequent evolution.
However, the duplicated genes VrOSCA2.1/ VrOSCA2.2showed divergence expression, which might experience functionalization after the duplication events [32]. Our results provided promising genes for further characterization in their functional involvement in osmotic stress.

Conclusions
In conclusion, a total of 13 OSCA genes were identi ed in the mung bean. The comprehensive analysis of the VrOSCA gene family provided important information such as phylogenetic relationship, duplication events and expansion pro le.
These ndings lay an important foundation for understanding the molecular evolution of the OSCA family in mung bean and providing candidate genes for further study of abiotic stress tolerance in mung bean.

Methods
Identi cation of OSCA family numbers in the Mungbean Genome

Interspecies Synteny Analysis And Gene Duplication
In order to analysis the relationships of orthologous OSCA genes in different species, multiple sequence alignment was used to detect sequences of mung bean and soybean with the similarity of more than 70%. Then, Multiple Collinearity Scan toolkit (MCScanX) was adopted to analysis the collinear block with the default parameters. Finally, the linear analysis map was illustrated using Dual Synteny Plotter software (https://github.com/CJ-Chen/TBtools).Duplicated gene pairs were analyzed using MCScanX program with the default parameters and plotted with the Circos software [38]. The Ka (non-synonymous substitution rate) and Ks (synonymous substitution rate) were investigated by DnaSP v5.0 software [39], and the selection pressure was calculated by the Ka/Ks ratio.
Abiotic stress responsive cis-elements analysis in the promoter regions of VrOSCA genes The PLACE website (http://www.dna.affrc.go.jp/PLACE/signalscan.html) [40] was used to identify putative cis-elements involved in ABA and abiotic stress response in the 1.5 kb upstream promoter region from the transcription start site.

Plant materials and stress treatments
In this study, the mung bean cultivar VC1973A was used to analyze gene expression pro les under drought, salt and ABA treatments. The seeds of cultivars VC1973A were obtained from the Chinese Academy of Agricultural Sciences. The VC1973A were grown in a growth chamber at 24 ℃ with a photoperiod of 16 h. When the appearance of the rst trifoliolate leaf, seedling was treated with 20% polyethylene glycol-6000(PEG-6000), NaCl (100mM) and abscisic acid Bean Industry Technology System Special Project (CARS-08-Z5), Research Project Supported by Shanxi Scholarship Council of China (2020-136) and Beijing Academy of Agriculture and Forestry Fund for Young Scholars (QNJJ201925).
The funding bodies only provided the nancial support and had no role in the design of the experiment, collection and analysis of data and in drafting the manuscript.

Availability of data and materials
The Arabidopsis OSCA protein sequences were collected from the Arabidopsis information source (TAIR) database (http://www.arabidopsis.org). The genome sequences of mung bean, soybean and rice were downloaded from EnsemblPlants (http://plants.ensembl.org/index.html). The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.
Competing interests