Genome-wide identification and expression analysis of the CHYR gene family in Phaseolus vulgaris under abiotic stress at the seeding stage

ABSTRACT

Really Interesting New Gene (RING) proteins are referred to as CHY zinc and ring finger protein (CHYR) members because they have the CHY domain, a type of protein that responds to abiotic stress. However, there are scant data on CHYR members in Phaseolus vulgaris (common bean). This study identified eight CHYR members (PvCHYRs) in the reference genome. A comprehensive analysis was performed to determine function, location, basic information, evolution, motifs, gene structure, cis-acting elements, collinearity, and expression patterns at the seeding stage. The results demonstrated that PvCHYRs might be involved in response to abiotic stressors, whereas some PvCHYRs were screened and can be used as candidate CHYR genes in further research. This study provides a basis for identifying and analyzing PvCHYR members and provides insights for their screening in future research.

KEYWORDS:

• CHYR members
• Phaseolus vulgaris (common bean)
• identification
• characterization
• expression patterns

Introduction

The Really Interesting New Gene (RING) proteins are important proteins that respond to abiotic stress (Ding et al. 2015), and their ubiquitination may play a significant role in response to environmental stress (including abiotic stresses) (Smalle and Vierstra 2004). These proteins are also called CHY zinc and ring finger protein (CHYR) members because they have a CHY domain (Jia et al. 2021). CHYR members have CHY and RING finger domains, with the N-terminal CHY domain having some conserved cysteine and histidine residues (Cayrol et al. 2007; Finn et al. 2010). Protein sequence alignment depicted that CHYR family members contain CHY zinc-finger, ring finger, and rubredoxin-type fold domains, thus defining them as CHYR family members containing these domains (Matthiadis and Long 2016; Hindt et al. 2017; Rodriguez-Celma et al. 2019a). CHYR members may play a role in binding to zinc ions, physical interactions, protein–protein interactions, and ubiquitination due to the CHY zinc and ring finger domains (Finn et al. 2010; Lee and Kim 2011; Xia et al. 2013), whereas rubredoxin-type fold domain may play a role in regulating iron homeostasis (Hindt et al. 2017; Kobayashi 2019; Rodriguez-Celma et al. 2019).

CHYR members have been reported to perform vital functions in plant growth, development, and various stress responses through ubiquitination and degradation of proteins (Ding et al. 2015; Rodriguez-Celma et al. 2019). The first CHYR member in Arabidopsis was identified in 2013 and named MYB30-Interacting E3 Ligase 1(MIEL1) (Marino et al. 2013), which can stabilize proteins (such as MYB96 and MYB30), facilitate crosstalk between abscisic acid (ABA) and biotic stress (Lee and Seo 2016), and negatively regulate cuticular wax biosynthesis in stems (Lee et al. 2017). More and more results indicated that CHYR members have a role in plant growth, development, and stress response (Jia et al. 2021). Some CHYR members are closely related to reactive oxygen species (ROS), which might play a functional role by regulating ROS under abiotic stresses (He et al. 2018): AtCHYR1 is a CHYR member in Arabidopsis, which stimulates ROS production, closes the stoma to increase ABA content, and responds to drought stress (Ding et al. 2015); PeCHYR1, a CHYR gene in Populus euphratica and a homologous gene of AtCHYR1, exhibited similar phenotypes under drought tolerance, such as stomatal closure and hydrogen peroxide (H₂O₂) production through the regulation of ROS (He et al. 2018); the CHYR gene OsRZF34 has the CHYR domain, which could open stomata and cool leaves under stress (Hsu et al. 2014); MYB30 could interact with MIEL1 and then suppress defense and hypersensitive responses through the regulation of ROS (Lee et al. 2017).

CHYR members have been identified in certain plant species, such as soybean (Glycine max) (Jia et al. 2021) and bread wheat (Triticum aestivum) (Liu et al. 2022); however, few studies have been conducted on common bean (Phaseolus vulgaris). Common bean, a legume crop cultivated on approximately 30 million hectares worldwide (Buruchara et al. 2011), is rich in starch, protein, fiber, vitamins, and folate (Mukankusi et al. 2019). It is sensitive to abiotic stress, especially at the seedling stage (Zhang et al. 2021; Zhang et al. 2022); Consequently, the improvement of common bean tolerance to abiotic stresses is of prime interest. Some gene families, including CCCH (Zhang et al. 2020), bZIP (Zhang et al. 2022), PRX (Boubakri et al. 2022a), SWEET (Du et al. 2022a), and LBD (Du et al. 2022b) had been identified in common bean, which had the connection with abiotic stresses. In the present study, the CHYR members were identified in common beans. A systematic analysis (including location, evolution, motif, gene structure, cis-acting elements, and collinearity) of the CHYR members was performed in common bean beside their expression patterns in response to abiotic stressors to filter out potential gene candidates for future functional analyses.

Materials and methods

Identification and analysis of CHYR members

The reference genome of the common bean (Phaseolus vulgaris) was obtained from the Ensembl Plants website (https://plants.ensembl.org/index.html), and PhaVulg1_0 version was used to identify CHYR members. Deoxyribonucleic acid (DNA), protein (pep), coding sequence (CDS), complementary DNA (cDNA), and general feature format (gff) information were obtained from the Ensembl Plants website (Kitts et al. 2016). The domain was provided by the Pfam 35.0 database (Mistry et al. 2021), while hmmsearch and hmmbuild were used to develop models with multiple alignment sequences. The location of CHYR members in the common bean was analyzed using TBtools (Chen et al. 2020) and named accordingly. The evolution was constructed by MEGA X (Kumar et al. 2018), in which LG+G was set as the optimal computing model predicted by MEGA using maximum likelihood. The motifs were analyzed using the MEME Suite (Bailey et al. 2015), and the value of each motif was less than 1e⁻¹⁰. Gene structure and cis-acting elements were analyzed using Gene Structure Display Server (GSDS) and PlantCARE databases, respectively (Lescot et al. 2002; Guo et al. 2007). The multiple collinearity scan toolkit (MCScanX) and Circos were used to analyze the collinearity of CHYR members in the common bean (Krzywinski et al. 2009; Wang et al. 2012). The expression pattern data of CHYR members were obtained from the Phytozome website (https://phytozome-next.jgi.doe.gov/) (Goodstein et al. 2012) and visualized using TBtools (Chen et al. 2020).

Plant materials, treatment and qRT-PCR analysis

Longyun No.5, which was bred by the Heilongjiang Academy of Agricultural Sciences (Harbin, Heilongjiang, China), was used as the plant material, and a local variety was grown in Northeast China and seemed to tolerate stress. The seeds were provided by the Crop Resources Research Institute of Heilongjiang Academy of Agricultural Sciences (Harbin, Heilongjiang, China). The seeds were placed in a wet kit for five days in a 25°C incubator without light to facilitate germination after 10 min of sodium hypochlorite (NaClO) treatment. The sprouts with the same growth trend on the fifth day were chosen for different treatments, including control (CK), salt (70 mmol/L NaCl) (Zhang et al. 2022), and cold (4°C) (Hayford et al. 2017) for 12 h stresses. The cotyledons, hypocotyls, and radicles were included in the control treatment on the fifth day, while radicles in salt and cold treatments were set as samples. Total RNA was extracted using the MolPure® Plant Plus RNA Kit (19292, Yeasen, Shanghai) according to manufacturer’s instructions and tested using NanoDrop (Thermo OneC, United States) and 2% agarose gel electrophoresis. The qualifying RNA was then reverse-transcribed into cDNA using 5X Evo M-MLV RT Master Mix (AG11706, Accurate Biology, Hunan, China). The primers of CHYR members in common bean were designed using Primerpremier 5.0 (Singh et al. 1998), while Pvactin served as the reference gene (Table S1). The qRT-PCR system provided by the Roche platform (480II, Roche, Basel, Switzerland) and Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (11184, Yeasen, Shanghai, China) were used according to the manufacturer’s instructions. The qRT-PCR program was 94°C for 30 s; followed by 40 cycles: 5 s at 94°C, 15 s at 60°C, and 15 s at 72°C, and a final elongation step for 5 min at 72°C. The 2^-ΔCt method (Livak and Schmittgen 2001) was used to calculate the relative expression of common bean CHYR genes (two biological and two technical replicates were performed).

Statistical analysis

The data of qRT-PCR analysis were analyzed using SPSS 19.0 software (Preacher and Hayes 2004), and significant differences were determined by ANOVA tests.

Results

Identification of CHYR members

After hmmbuild and hmmsearch, 14 members containing the CHY domain were identified. After removing duplicate values, eight members remained that might be identified as CHYR members from the common bean genome (Phaseolus vulgaris). All CHYR members were named PvCHYR01-08 based on their location in the genome (Figure 1). Chrs 1, 2, 3, 6, 9, and 11 had PvCHYR members, with Chr 3 having the most number of PvCHYR members (3). Detailed information is presented in Table 1. The protein length ranged from 221 (PvCHYR08) to 1256 (PvCHYR05), while the isoelectric point of PvCHYR members was between 5.78 (PvCHYR02) and 7.19 (PvCHYR07). The PvCHYR with the largest molecular weight was PvCHYR05 (145479.33), while the lowest molecular weight was 25411.16 (PvCHYR08). The variation ranges of the instability and aliphatic index were 38.34–60.08 and 61.18–65.57, respectively. This detailed information helps to realize the importance of PvCHYRs.

Figure 1. Location of the PvCHYRs. The ruler on the left represents its location on chromosomes on the reference genome. The tightness of the line represents the density of genes. All this information was provided by the Ensembl Plants database.

Table 1. The detailed information of PvCHYRs.

Gene name	Gene Bank	Gene accession	Gene_id	Chr	Location	Protein length	CDS length	Isoelectric point	Molecular weight	Instability index	Aliphatic index
PvCHYR01	ESW33271.1	CM002288.1	Phvul.001G056800	1	6662038–6667903	307	924	6.18	35574.43	58.01	51.07
PvCHYR02	ESW32384.1	CM002289.1	Phvul.002G317800	2	47797468–47807211	1236	3711	5.78	139569.56	53.72	74.88
PvCHYR03	ESW26137.1	CM002290.1	Phvul.003G094000	3	19455085–19461272	628	1887	6.65	70542.93	60.08	65.57
PvCHYR04	ESW26227.1	CM002290.1	Phvul.003G101500	3	24803372–24807416	267	804	6.3	31110.37	58.28	57.94
PvCHYR05	ESW27352.1	CM002290.1	Phvul.003G194400	3	40691734–40702409	1256	3771	6.41	145479.33	53.09	53.09
PvCHYR06	ESW19866.1	CM002293.1	Phvul.006G162000	6	27326977–27330404	267	804	6.58	31141.53	56.66	59.06
PvCHYR07	ESW09085.1	CM002296.1	Phvul.009G099000	9	15095014–15100759	305	918	7.19	34923.05	50.61	62.26
PvCHYR08	ESW04328.1	CM002298.1	Phvul.011G086000	11	8292810–8299636	221	666	7.13	25411.16	38.34	61.18

Evolution of PvCHYRs

MEGA X was used to analyze the evolutionary relationship of PvCHYRs using the maximum likelihood method with 1,000 bootstraps predicted by MEGA based on the protein sequence of PvCHYRs. The Dayhoff+G model is used in this study. The results revealed that the members were divided into three subgroups, with three PvCHYR members in subgroups I and III and only two in subgroup II (PvCHYR04 and 06).

Motifs and gene structure of PvCHYRs

MEME was used to analyze the motifs of PvCHYRs, where the length of the amino acids of the motifs ranged from 10 to 50, and the e-value was less than 1e⁻²⁰. The motif results of PvCHYRs revealed that all members had motifs 1, 2, 3, and 4, whereas members of each subgroup had motifs of the same kind (Figure 3). Motifs 5 and 9 were present in subgroups I and II, while motifs 6, 8, and 10 were exclusive to subgroup III members. These results suggest that the PvCHYRs in each subgroup may have similar functions. In addition, GSDS was used to analyze the gene structure of PvCHYRs. The sequence of the PvCHYRs was divided into the untranslated region (UTR) (green squares), coding sequence region (CDS) (yellow squares), and intron region (black lines). In addition, the sequence encoding the CHY domain was determined and is depicted as pink squares. The gene structure results of the PvCHYRs revealed that the members of each subgroup had a similar gene structure (Figure 3). Subgroup II members (PvCHYR04 and 06) had a longer CDS region, while the coding CHY domain was located farther forward compared to the other subgroups. These results demonstrated that the PvCHYRs in each subgroup might share a similar structure.

Figure 3. The motifs and gene structure analysis of PvCHYRs. (A) The evolution of PvCHYRs; (B) The motifs of PvCHYRs. Ten squares with different colors represent different motifs; (C) The gene structure of PvCHYRs. The green squares represent the UTR region, the yellow squares represent the CDS region, and the pink squares represent the sequence coding CHY domain.

Comprehensive analysis of CHYR members

The CHYR members of five species (Arabidopsis, rice maize, soybean, and common bean) were analyzed. These members were divided into three subgroups in the evolutionary analysis, with more members in subgroups I (15) and III (20) than in subgroup II (12). This finding was similar to the evolutionary relationship observed in PvCHYRs (Figures 2 and 4). Analysis of CHYR members depicted that CHYR members in each subgroup had similar motifs and gene structures, indicating that CHYRs in each subgroup may have similar functions.

Figure 2. The evolutionary analysis of PvCHYRs. The arc shapes with different colors represent subgroups I, II, and III.

Figure 4. The evolution, motifs, and gene structure analysis of CHYR members in five species, Arabidopsis, rice, maize, soybean, and common bean. Different colored backgrounds represent different subgroups. The outer ring is the motif analysis, while the inner ring is the gene structure analysis of CHYR members.

Cis-acting elements of PvCHYRs

Plant care was used to analyze the cis-acting elements of PvCHYRs and predict their functions (Table S2). The cis-acting elements were divided into three categories: hormone-(red), stress-(blue), and sprout-related (yellow). This analysis revealed that almost all members had hormone-related elements, except PvCHYR08, in which PvCHYRs might respond to hormones. More than half of the members had stress-related elements, indicating that some PvCHYRs may be associated with stress. Some members contained sprout-related elements, indicating that PvCHYRs may have participated in the sprouting stage (Figure 5).

Figure 5. The cis-acting elements analysis of PvCHYRs. (A) The evolution of PvCHYRs. (B) The cis-acting elements of PvCHYRs. The red ones represent the hormone-related elements, the blue ones represent stress-related elements, and the yellow ones represent sprout-related elements.

Collinearity of PvCHYRs

The collinear relationship between PvCHYRs was analyzed in this study. The results are depicted in Figure 6. Only two pairs of PvCHYRs depicted a collinear relationship (PvCHYR02 and 03, PvCHYR04 and 06). PvCHYRs had more collinear pairs with soybean (10) than Arabidopsis (4), suggesting that these collinear genes may have similar functions to PvCHYRs.

Figure 6. The collinearity analysis of PvCHYRs. (A) The collinear relationship of PvCHYRs. Two middle rings represent the gene density of the genome. (B) The collinear relationship of PvCHYRs with soybean and Arabidopsis. Green squares are common bean’s chromosomes; yellow ones are soybean chromosomes, while red ones represent Arabidopsis’s chromosomes. Red lines represent the collinear relationship with soybean, and blue lines represent the collinear relationship with Arabidopsis.

Expression patterns of PvCHYRs in silico and qRT-PCR analysis at seeding stage

The expression patterns of PvCHYRs in different tissues were extracted from the Phytozome database. The results demonstrated that PvCHYRs had tissue-specific expression patterns. Some PvCHYRs (such as PvCHYR05) had a higher expression in roots, whereas PvCHYR01 had a higher expression in mature green pods, and the expression pattern of PvCHYRs at the seeding stage was analyzed by qRT-PCR. The results depicted that almost all PvCHYRs (such as PvCHYR01, 02, 03, and 07) had tissue-specific expression patterns, whereas others (such as PvCHYR04) did not. A greater number of PvCHYRs were highly expressed in radicles, which might be used to target tissues during the seeding stage (Figure 7).

Figure 7. The expression pattern analysis of PvCHYRs in different tissues at the seeding stage. (A) The expression pattern analysis of PvCHYRs in silico. Different colored backgrounds represent different subgroups. The color of the squares, from blue to red, represent the expression level from low to high, respectively. (B-I) The expression pattern analysis of PvCHYRs by qRT-PCR analysis. The columns of three colors represent cotyledons, hypocotyls, and radicles, respectively. The different letters on the column represent the significance (P < 0.05).

Expression pattern of PvCHYRs under abiotic stress

This study determined the expression levels of PvCHYRs at the seedling stage under abiotic stress using qRT-PCR. The results displayed that almost all members had significantly different expression levels under abiotic stress conditions (P < 0.05). Some PvCHYRs (such as PvCHYR01, 05, 07, and 08) were upregulated under stress, whereas others (such as PvCHYR03 and 04) were downregulated. All these results demonstrated that PvCHYRs have closely related to abiotic stress, and they may be used as candidate members in further research (Figure 8).

Figure 8. The expression pattern analysis of PvCHYRs in radicle at the seeding stage under abiotic stress. The columns of three colors represent control, salt, and cold, respectively. The different letters on the column represent the significance (P < 0.05).

Discussion

CHYR members have been identified in some plants such as Arabidopsis, rice, maize, soybean, and wheat (Xia et al. 2012; Hsu et al. 2014; Rodriguez-Celma et al. 2019; Jia et al. 2021; Liu et al. 2022). However, the number of CHYR members in different plant species varies substantially; 18 TaCHYR members have been identified in the bread wheat (Triticum aestivum) genome (Liu et al. 2022), while 16 GmCHYR members have been identified in the soybean (Glycine max) genome (Jia et al. 2021). In this study, only eight CHYR members were identified in the reference genome of the common bean (Phaseolus vulgaris), which is lower than the number of TaCHYRs and GmCHYRs. This phenomenon could be due to the small size of the common bean genome compared to that of wheat and soybean (Zhang et al. 2022) beside the differential evolutionary history of each plant species (Feller et al. 2011). The CHYR members in plants emerged during evolution, resulting in the appearance of three subgroups (Ding et al. 2015; Liu et al. 2022); in which subgroups I and II CHYR members appear after angiosperm differentiation (Liu et al. 2022). TaCHYR and GmCHYR members were also divided into three subgroups based on evolutionary analysis (Jia et al. 2021; Liu et al. 2022). Additionally, the evolution of PvCHYRs demonstrated that their members were divided into three subgroups, with subgroup II including fewer members than subgroups I and III. The evolution of CHYR members in the five species (containing monocots and dicots) depicted three subgroups, which may be characteristic of PvCHYRs.

The motifs and gene structure of PvCHYRs demonstrated that each subgroup member had similar motifs and gene structures similar to those of TaCHYRs and GmCHYRs (Liu et al. 2022). The conserved motifs, including CHY and ring finger domains, have been displayed to have an important function in response to adverse stresses, especially abiotic stress (He et al. 2018). These results suggest that PvCHYRs may be related to abiotic stress. According to the analysis of the cis-acting elements, ARE, MBS, and LTR were identified as three cis-acting elements related to abiotic stress in the initiation region of PvCHYRs. These cis-acting elements also appeared in the CHYR members in other plants or other gene families related to abiotic stress tolerance, such as SULTR and Grxs (Boubakri et al. 2022b). PvCHYRs also were found to have a collinear relationship with AT1G18910, AT1G74770, and AT5G18650, AT1G18910 and AT1G74770 that regulated ion homeostasis and responded to ion import into the cell (Colangelo and Guerinot 2004; Rodriguez-Celma et al. 2019), whereas AT5G18650 was found to be involved in stress response (Marino et al. 2013). These findings suggest that PvCHYRs might have a role in abiotic stress response in common bean.

Previous studies have reported that CHYR members exhibit tissue-specific expression (Liu et al. 2022). In this study, PvCHYRs also revealed tissue-specific expression patterns. In addition, CHYR genes have been reported to participate in various biological processes and stress responses (Li and Lan 2015; Leng and Hall 2019). Some GmCHYRs exhibit a potential role in the stress response, as their expression levels are upregulated or downregulated in response to abiotic stresses (Jia et al. 2021). ATCHYR1 is a CHYR gene that improves tolerance by promoting stomatal closure and ROS production (Matthiadis and Long 2016). CaASRF1, a CHYR member, positively modulates abiotic stress via the ABA pathway (Moore and Purugganan 2003). Moreover, certain H-type thioredoxins (Trxs) were previously reported to be induced by abiotic stresses in Phaseolus vulgaris and the authors have suggested that these members might be used to engineer new salt-tolerant lines (Boubakri et al. 2021). Similarly, in our present study, specific PvCHYR members (PvCHYR01, 05, and 08) were found to be upregulated under abiotic stresses suggesting that these genes may also be good candidates for further functional analyses.

Conclusions

This study identified eight CHYR members through the common bean reference genome and named PvCHYR01–08 based on their location information. A comprehensive analysis including evolution, motifs, gene structure, and cis-acting elements revealed that the PvCHYRs in each subgroup were similar. Analysis of cis-acting elements, collinearity, and expression patterns under abiotic stresses demonstrated that some PvCHYRs may be connected to abiotic stresses and might be used as candidate CHYR genes in future functional studies.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All data generated or analyzed during this study are included in this manuscript.

Additional information

Funding

This research was funded by “The Research Funds of the Scientific Research Institution of Heilongjiang Provincial (CZKYF-2-C004)” and “The China Agriculture Research System of MOF and MARA (CARS-08)”.

Notes on contributors

Xianxin Meng

Meng Xianxin, Associate professor, Institute of Crop Resources, Heilongjiang Academy of Agricultural Sciences, is engaged in genetic breeding of edible beans for stress resistance (especially environmental interaction).

Zhengong Yin

Yin Zhengong, Associate professor, Istitute of Crop Resources, Heilongjiang Academy of Agricultural Sciences.

Guangdong Yang

Yang Guangdong, Professor, Istitute of Crop Resources, Heilongjiang Academy of Agricultural Sciences.

Shuhong Wei

Wei Shuhong, Professor, Istitute of Crop Resources, Heilongjiang Academy of Agricultural Sciences.

Yifan Guo

Guo Yifan, Associate professor, Istitute of Crop Resources, Heilongjiang Academy of Agricultural Sciences.

Wei Zhang

Wei Zhang, Associate professor, Istitute of Crop Resources, Heilongjiang Academy of Agricultural Sciences.

Qiang Wang

Wang Qiang, Professor, Institute of Crop Resources, Heilongjiang Academy of Agricultural Sciences, is engaged in genetic breeding and resource innovation of edible beans.