The common bean (Phaseolus vulgaris) SULTR gene family: genome-wide identification, phylogeny, evolutionary expansion and expression patterns

Abstract Sulfate transporters (SULTRs) are responsible for the absorption and transport of sulfate in higher plants. They also play a key role in plant growth, development and abiotic stress responses. However, the SULTR gene family in common bean (Phaseolus vulgaris L.) is still not well studied. In this study, 15 PvSULTRs were identified. Phylogenetic analysis divided the PvSULTR genes into four subgroups. Cis-regulatory element analysis of the promoter sequence of the PvSULTR genes showed that many cis-regulatory elements were related to hormone and environmental stresses. Collinearity analysis showed that two PvSULTR gene pairs are duplicated in common bean. Tissue-specific expression analysis of the 15 PvSULTR genes showed different expression patterns in various tissues. Furthermore, real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) analysis showed that PvSULTR1/2/7 genes displayed specific responses to CdCl2 and HgCl2 stresses, whereas PvSULTR11/12 were explicitly induced by NaCl and HgCl2 stresses. These results suggest that PvSULTR family members might function under different mechanisms in response to specific metal stress. This study provides the basis for elucidating the functional diversity of PvSULTR gene family and reveals its role in metal stress response in common bean.


Introduction
Sulfur (S) is a component of the amino acids (cysteine and methionine) and secondary metabolites, including vitamins and defensive compounds like glucosinolates [1]. Sulfur is essential for plant growth and development and plays a crucial role in plant resistance to biotic and abiotic stress [2,3]. Sulfur content in crops accounts for about 0.1-0.5% of the dry weight of plants [4]. Sulfur deficiency affects the vegetative and reproductive growth of plants. Specifically, sulfur deficiency inhibits the absorption of nutrients such as carbon and nitrogen, resulting in insufficient protein synthesis. It also lowers the level of sulfur-containing defense compounds such as glutathione and hydrogen sulfide (H 2 S), resulting in reduced plant resistance to adverse environmental conditions [5].
The sulfate transporter (SULTR) was the first gene to be cloned among sulfate transport genes [6]. SULTRs are integral membrane proteins that control the sulfate (SO 4 2− ) flux into cells and subcellular compartments via the membrane lipid bilayer [7]. Inorganic S taken up by plants is assimilated into a variety of S-containing organic compounds, such as glutathione (GSH), homoGSH (h-GSH) and phytochelin (PCs), through a series of metabolic reactions [8]. These S-containing compounds play vital roles in plant responses to oxidative stress and various abiotic stresses [9]. SULTR proteins contain 12 transmembrane domains (TMDs) and a sulfate transporter anti-sigma factor antagonist (STAS) domain at the C-terminal region [10]. The evolution and identification of the SULTR gene family have been studied in Arabidopsis thaliana [11], rice (Oryza sativa L.) [12], Populus spp. L. [13], wheat (Triticum aestivum L.) [2], soybean (Glycine max L.) [14] and tea (Camellia sinensis) [15]. Based on sequence homology and phylogeny, SULTR gene family is categorized into four subgroups (I-IV). Studies have shown that SULTR genes belonging to different subpopulations have different specificity of sulfur transport [16,17]. The high-affinity sulfate transporters belong to Clade I, whereas the low-affinity transporters belong to Clade II. Plastid membrane and symbiotic membrane localization transporters with known or unknown functions belong to Clade III. The vacuolar sulfate exporters belong to Clade IV.
The SULTR gene family has been well-studied in Arabidopsis. High-affinity transporters AtSULTR1;1 and AtSULTR1;2 co-localize in roots, while double mutants AtSULTR1;1 and AtSULTR1;2 show a complete loss of sulfate absorption capacity and growth defects [11,18]. Another high-affinity AtSULTR1;3 is localized in the phloem and participates in the transport of sulfate from source to sink [19]. AtSULTR2;1 and AtSULTR2;2 are low-affinity SULTRs that mediate long-distance transport of sulfate in vascular tissues [20,21]. So far, the roles of Clade III of SULTR in A. thaliana have not been fully elucidated. Previous studies have shown that AtSULTR3;5 and AtSULTR2;1 act synergistically to enhance the ability of sulfate transport from root to stem in Arabidopsis under low S stress [22]. AtSULTR4;1 and AtSULTR4;2 plays a critical role in mediating the efflux of sulfate from the vacuole lumen into the cytoplasm in Arabidopsis roots [23].
The sulfur status of plants and abiotic stress affect the expression of sulfate transporters [24,25]. Ding et al. [14] identified 28 putative GmSULTRs and revealed that GmSULTR1;2b functions as an important sulfur uptake transporter that can alter the sulfur status of plants. Vatansever et al. [26] identified 12 putative SULTR genes in potato, most of which were shown to participate in abiotic stress responses. In a recent study, OsiSULTR3 and OsiSULTR12 were significantly up-regulated under high temperature, low-selenium and drought stresses, whereas OsiSULTR10 was up-regulated under salt stress [25].
Common bean (Phaseolus vulgaris L.) is an edible leguminous plant widely cultivated globally. Its seeds are rich in protein and sulfur-containing amino acids [27]. Although the role of the SULTR gene family has been explored in many plant species, the function of PvSULTR genes, specifically in response to abiotic stress in common bean has not been fully uncovered. In this study, 15 PvSULTR genes in common bean were characterized based on their gene structure, chromosome distribution, evolutionary relationship, cis-regulatory elements, gene replication, collinearity and spatiotemporal expression patterns. In addition, the transcriptional profiles of PvSULTR genes were examined under abiotic stress conditions. This study provides important information for elucidating the evolutionary function of PvSULTR in common bean and lays a foundation for further understanding the functional diversity of the SULTR gene family.

Gene structure, conserved motifs and promoter predictions of the PvSULTR genes
The structure of PvSULTR genes was obtained from the GSDS platform (≤seurld≥http://gsds.cbi.pku.edu.cn/≤/ seurld≥) [34]. The coordinates corresponding to DNA (containing exon and intron) and protein sequences were determined using GeneWise [35]. An in-house Perl script was then used to convert the Sulfate_transp and STAS domain coordinates in the protein sequence to those in the nucleotide sequence. The MeMe tool (≤seurld≥http://meme.nbcr.net/meme/≤/seurld≥) [36] was used to predict the conserved motifs. The length of characteristic motifs was between 10 and 50 amino acids, and the e value was less than 1e −20 . The upstream 1.5 kilobases (kb) genomic DNA sequences of PvSULTRs were extracted and submitted to the PlantCare database (http://bioinformatics.psb.ugent.be/ webtools/plantcare/) to identify possible cis-regulatory elements in the promoter region.

Collinearity analysis
The synteny and collinearity of SULTR genes were detected using Multiple Collinearity Scan toolkit (MCScanX) [37]. Circos software (version 0.69), a visualization tool, was then used to express the collinearity of the duplicated genes [38].

Plant materials and treatments
Common bean (P. vulgaris L; variety Longjiang Ziyun) seeds were surface-sterilized and then placed on Petri dishes (9 cm) with two layers of medium-speed qualitative filter papers. Then, 11 mL of distilled water was added to each Petri dish and incubated in an incubator at 28 °C in the dark until germination. A seed was considered to have germinated when the radicle broke through the seed coat. After 5 days of germination, the cotyledons, radicles and hypocotyls were harvested and flash-frozen in liquid nitrogen for 5 min, then stored at −80 °C until further analysis.
To evaluate the roles of PvSULTRs in the abiotic stress response of common bean, the 5-day uniformly germinated seeds were selected and transferred into a 150 mL flask containing 150 mL of various treatment solutions, including distilled water (CK), 70 mmol/L NaCl [39], 0.5 mg/L CdCl 2 [40] and 60 mg/L HgCl 2 [41] and incubated at 28 ± 1 °C with a light regime of 16 h light/8 h dark. Radicle samples were harvested after 24, 48 and 72 h of treatment, and stored at −80 °C. Seeds in the same flask were considered as one experimental unit, and each experiment was repeated three times.

Gene expression analysis
The frozen common bean samples (cotyledons, radicles and hypocotyls) were triturated in liquid nitrogen. Total RNA was isolated from 100 mg of triturated samples using Total RNA extraction Reagent (Vazyme Biotech Co., Ltd., Nanjing, China) following the manufacturer's instructions. RNA quality and integrity were determined based on their absorbance at 260 nm and via 1.0% agarose gel electrophoresis. First-strand cDNA was synthesized using the evo M-MLV RT Premix for qPCR (AG11706, Accurate Biology, Hunan, China).
Subsequently, qRT-PCR analysis was performed on a Light Cycler system (Roche 480II, Roche, Switzerland) using TransStart® Top Green qPCR SuperMix (AQ131-04, TransGen Biotech, Beijing). The common bean ACTIN-11 gene was used as the reference gene for signal normalization [42]. The relative expression levels of PvSULTRs were analyzed using the 2 −△△t method. Three biological and technical replicates were performed. The primers used for the qRT-PCR analysis are listed in Supplemental Table S1.

Statistical analysis
Microsoft Office 2013 (Redlands, CA, USA) and SPSS version 17.0 (SPSS Inc., Chicago, IL, USA) were used for data analyses [43]. Data are presented as the mean values with standard deviation (±SD) of three independent experiments.

Identification and characterization of the SULTR genes in common bean
HMM profile, InterPro and SMART analysis identified 15 PvSULTR genes in common beans ( Table 1). The 15 PvSULTR genes were named PvSULR1 -PvSULTR15 based on their chromosomal position ( Figure 1). The number of amino acid residues of 15 predicted PvSULTR proteins ranged from 628 (PvSULTR4) to 709 (PvSULTR10), and their predicted molecular weight varied from 68,123.47 Da (PvSULTR4) to 77,639.56 Da (PvSULTR10) ( Table 1). The pI values ranged from 8.49 (PvSULTR6) to 9.66 (PvSULTR7), and all members exhibited pI values >8.
The 15 PvSULTR genes were distributed on 10 of 11 chromosomes ( Figure 1). Chromosome 1 contained the largest number of PvSULTRs (3) genes, whereas no PvSWEET genes were found on chromosome 11. In addition, PvSULTRs were mostly distributed at both ends of the chromosomes.

Phylogenetic analysis of the predicted PvSULTR proteins
To describe the phylogenetic history of the predicted SULTR in common bean and classify it, we constructed a phylogenetic tree based on the alignment of amino acid sequences within the conserved SULTR domain. The analysis included 12, 6, 11, and 25 SULTR domain genes of Arabidopsis, maize, rice, and soybean, respectively ( Figure 2). According to the phylogenetic analysis, different SULTRs were classified into four major clades (I-IV). Among the PvSULTR proteins, clade I contained five members, clade II and III contained three members each, and clade IV contained four members ( Figure 2 and Supplemental Figure S1). Different colors represent a different clade: yellow, clade I; blue, clade II; red, clade III; and green, clade IV. The red stars represent common bean SULTR gene family members, while the dark blue, wathet blue, green, and yellow circles represent rice, maize, A. thaliana and soybean SULTR gene family members, respectively.

Conserved motifs and gene structure analysis of PvSULTRs
The conservative motifs of 15 predicted PvSULTRs were analyzed (Figure 3b). Ten motifs were visualized in different colors and designated as motifs 1 to 10. The predicted PvSULTR members had similar motifs in the same subfamily. Notably, PvSULTR13 in subfamily I contained the fewest motifs (7), and nearly 70% of PvSULTRs contained 10 motifs. Moreover, motifs 1 to 6 were detected in all the PvSULTRs.
The exon-intron structures of the PvSULTR members are shown in Figure 3c. The number of exons varied from 2 to 7 in PvSULTRs. Also, the gene structures of PvSULTRs in the same subfamily were similar.

Analysis of cis-regulatory elements in PvSULTRs
Nine types of cis-regulatory elements were identified in the 1.5-kb upstream sequences of SULTR genes, including hormone and resistance-related elements (Figure 4, Supplemental Table S2). Regarding the hormone-related elements, multiple copies of ABRe were identified in 10 PvSULTR genes, whereas  AuxRR-core was only found in PvSULTR3. Concerning the resistance-related elements, multiple copies of ARe were found in 12 PvSULTR genes, whereas MBS was only identified in PvSULTR13.

Collinearity analysis of PvSULTR genes
Collinearity analysis of PvSULTR genes identified two PvSULTR genes (PvSULTR12vsPvSULTR14 and PvSULTR11vsPvSULTR13) as duplicate gene pairs in common bean (Figure 5a). Meanwhile, collinearity analysis of PvSULTRs in Arabidopsis identified 12 gene pairs. A total of 21 gene pairs were identified in soybean, whereas one gene pair was found in rice, which was collinear with PvSULTRs in common bean (Figure 5b, Supplemental Table S3).

Tissue-specific expression profiles of PvSULTR genes
The tissue-specific expression patterns of PvSULTR genes were determined according to the Phytozome database ( Figure 6). A total of 15 PvSULTR genes showed different expression patterns in various tissues. Specifically, PvSULTR8 was up-regulated in roots, PvSULTR2 was up-regulated in stems, PvSULTR1 and PvSULTR11 were up-regulated in leaves, PvSULTR4, PvSULTR7, and PvSULTR9 were up-regulated in flower . mega X was used to construct a maximum-likelihood phylogenetic tree with the Jtt + g model. buds or flowers, PvSULTR10, PvSULTR12, and PvSULTR13 were up-regulated in young pods or mature green pods, and PvSULTR3, PvSULTR6 and PvSULTR14 were up-regulated in root nodules.qRT-PCR was used to analyze the expression of PvSULTR genes in different parts of common bean seeds after 5 days of germination. The results showed that the 15 PvSULTR genes were up-regulated in hypocotyls, radicles, and cotyledons, except a few PvSULTR4/9/11 transcripts detected in hypocotyls (Figure 7).

Stress-induced expression of PvSULTR genes
The expression levels of 15 PvSULTR genes were examined in the radicles of the germinating seeds of common bean under NaCl, CdCl 2 and HgCl 2 stresses to      ) radicles under 70 mmol/l nacl (na), 0.5 mg/l cdcl 2 (cd) and 60 mg/l hgcl 2 (hg) treatments. gene expression levels in common been radicles under distilled water treatment (cK) were set to 1 as the normalization for qRt-pcR analysis using the operational formula 2 −△△ct . the common bean ACTIN-11 gene was used as the reference gene for signal normalization. Data are expressed as means ± SD of three independent experiments (each with three technical repeats). For the same treatment, different letters indicate statistically significant differences (p < 0.05) among different treatment durations. For the same treatment duration, * indicates statistically significant differences (p < 0.05) between cK and treatment, nS denotes no significant difference between cK and treatment conditions. manner; however, the expressions of these genes were unaffected under CdCl 2 condition.

Discussion
Sulfate transporters facilitate the transport and absorption of sulfate and participate in plant growth and abiotic stress responses [12,15,24,25,44]. However, functional identification of SULTR genes in common bean has not been reported. In this study, we identified 15 PvSULTR genes in common bean and divided them into four subgroups: subgroup I (5, 33.3%), subgroup II (3, 20%), subgroup III (3, 20%), and subgroup IV (4, 26.7%). Phylogenetic analysis showed that the PvSULTR classification in common bean is consistent with previous reports [11,14,15]. Gene structure and intron-exon analysis showed that PvSULTR genes vary among subfamilies but share a conserved structure within the same subfamily. This may be because members of different subfamilies have undergone splicing or insertion of gene fragments during evolution [45,46]. The shared conserved sequences and gene structures within the PvSULTR subfamily imply that these genes possibly have similar biological functions, further suggesting that the motifs of the PvSULTR gene family were conserved during evolution. The PvSULTR gene family is larger than most of the reported SULTR families in other crops. For example, there are 8 in maize [43], 11 in rice [12], 10 in wheat [2] and 8 in tea [15]. This variation can be attributed to the expansion of PvSULTR genes. Gene replication during evolution can increase genome content and promote gene functional differentiation, which is crucial for plant environmental adaptation and speciation [47,48]. Comparative analysis of the genomes of common bean, Arabidopsis, rice and soybean can provide a valuable reference for further study of the biological functions of PvSULTRs. Collinearity analysis showed that some PvSULTR genes might have been produced by gene replication. GmSULTR1;2b, a soybean sulfur transporter homologous to PvSULTR, was confirmed to play a crucial role in plant sulfur absorption and sulfur deficiency tolerance [14]. The PvSULTR homolog AtSULTR3;4 in Arabidopsis was co-expressed with AtSULTR3;1 in response to drought stress [49]. A previous study showed that plastidal SULTR3;1 gene in Arabidopsis and Medicago was significantly up-regulated under drought and salt stress [50]. Therefore, the collinear PvSULTRs of common bean identified in this study potentially participate in the plant response to abiotic stress.
The tissue-specific expression of genes is related to their function [51]. In A. thaliana, subgroup I of SULTR genes belongs to the high-affinity sulfate transporters, among which, AtSULTR1;1 and AtSULTR1;2 are expressed in root epidermis and cortex and participate in sulfate transport and absorption, whereas AtSULTR1;3 is expressed in phloem and participates in sulfate transport from source to sink [11,18,19]. In this study, phylogenetic analysis showed that five sulfate transporters (PvSULTR2/3/10/11/13) were clustered with Arabidopsis high-affinity sulfate transporters, indicating their key roles in sulfate absorption and transport.
There is a symbiotic relationship between common bean and rhizobia [52]. Common beans provide a conducive environment and necessary carbon source for the rhizobium bacteria, which convert nitrogen from the air into nitrogen-containing material that plants can absorb. Notably, we found that PvSULTR3 in subgroup I, PvSULTR14 in subgroup II, and PvSULTR6 in subgroup III were highly expressed in common bean root nodules, suggesting that they may be involved in nitrogen fixation and transport. In Arabidopsis, sulfate transport from root to shoot is mediated by two low-affinity sulfate transporters in subgroup II [20]. In this study, three sulfate transporters (PvSULTR 8/12 /14) were clustered in subgroup II of common bean and expressed in different tissues, indicating that they are widely involved in sulfate transport in plants. Arabidopsis AtSULTR4;1 and AtSULTR4;2, clustered in subgroup IV, localized with vacuolar membranes, and were involved in the transfer of sulfate from pericycle and xylem parenchyma cells in roots to xylem [53]. Zuber et al. [54] confirmed that AtSULTR4;1 is associated with sulfur content in Arabidopsis seeds. In common bean, PvSULTR7 and PvSULTR4 clustered in subgroup IV, and were highly expressed in flowers and flower buds, suggesting that they might facilitate sulfate transport in flower organs. In this study, qRT-PCR analysis of PvSULTR gene expression showed that most PvSULTRs were highly expressed in hypocotyl, radicle and cotyledon, indicating that PvSULTR genes are involved in the development of common bean at the sprouting stage. effective seed germination and seedling establishment are critical for crop yield in adverse soil conditions such as salt and heavy metal stress [55]. In this study, many cis-regulatory elements were identified in the promoter region of PvSULTRs, including ARe, MBS and LTR, suggesting that PvSULTRs might be involved in abiotic stress response. The expression patterns of 15 PvSULTR genes in the radicle of common bean at the sprouting stage were determined by qRT-PCR under different stress conditions, including salt, cadmium, and mercury stress. The results showed that 40% of PvSULTRs were down-regulated under the three abiotic stresses, while nearly 30% were up-regulated. Furthermore, the expression levels of these genes increased with the duration of treatment. These results imply that PvSULTR responds to multiple abiotic stresses. Adhikari et al. [56] showed that sulfate could regulate maize sulfur metabolism and oxidative defense system to reduce cadmium accumulation and improve maize tolerance to cadmium. In this study, PvSULTR1/2/7 was down-regulated in CdCl 2 and HgCl 2 stress conditions compared to the control, while their expressions were unaffected by NaCl treatment, suggesting that these genes may be expressed under specific stress conditions. In addition, PvSULTR11/12 were up-regulated under NaCl and HgCl 2 stress conditions compared with the control, while their expression levels were unaffected under CdCl 2 stress, indicating that PvSULTR11/12 respond to specific abiotic stresses. The variations in the expression patterns of PvSULTRs under different stress treatments indicate that various PvSULTR genes operate via special regulatory mechanisms in response to different stress treatments.

Conclusions
In this study, 15 PvSULTR genes were identified according to the published reference genome of common bean. Phylogenetic analysis indicated that SULTRs from common bean, Arabidopsis, maize, rice, and soybean were clustered into four groups. expression analysis of PvSULTRs showed that PvSULTRs are expressed differentially in different tissues. Most PvSULTR gene family members responded to metal stresses, including NaCl, CdCl 2 , and HgCl 2 stresses. This study provides a reference for further research to understand PvSULTR family members in common bean and reveal their potential roles in the growth, development, and resistance of plants to metal stress.