As one of the most widely grown crops in the world, soybean (Glycine max) provides an important source of plant-based protein and edible oil [1]. However, its yield and quality are enormously hindered by germplasm resources and diverse environmental factors, especially water deficiency, high salt, and alkaline [2]. Drought is one of the major natural disasters for the world’s agricultural production. Globally, this extreme weather phenomenon has led to cereal loss of 1820 million Mg during the past four decades ^[3][4][3,4]. Soil saline–alkalization is another worldwide abiotic stress restraining land utilization, grain yield, and local economic development. According to official statistics, more than 6% of the world’s soil resources are affected by saline and alkaline. Furthermore, continuous drought has a great influence on soil salinization and salt accumulation in the root zone [5]. Utilization and management of the saline-alkaline soil are requisite to alleviate the ever-growing population’s demand for food. Consequently, it is meaningful to focus on uncovering the molecular mechanism of plant response to abiotic stress and cultivating crops with stress resistance.

The RING E3 (Really Interesting New Gene) proteins were found to play critical roles in abiotic stress response via protein ubiquitination degradation ^[6][7][6,7]. Previously, a C3H2C3 RING (Really Interesting New Gene) zinc finger domain-containing protein from Arabidopsis was characterized and named as MIEL1 (MYB30-Interacting E3 Ligase1) [8]. According to the conserved RING zinc finger domain, they were also called CHYR (CHY ZINC-FINGER AND RING FINGER PROTEIN) and RZFP (RING ZINC-FINGER PROTEIN) ^[9][10][9,10]. Protein sequence alignment has proved that MIEL1, RZFP, and CHYR were in the same family, with conserved CHY zinc-finger, C3H2C3-type ring finger, and rubredoxin-type fold domain [9]. In addition, when hemerythrin domains appeared in the N-terminus of the CHY zinc finger domain, they were designated BTS/BSTL (BRUTUS/BRUTUS-like) in Arabidopsis, but HRZ (Hemerythrin motif-containing RING-and Zinc-finger protein) in rice ^{[11][12][13][14]}[11,12,13,14]. Above all, we could uniformly define these proteins containing CHY zinc-finger, C3H2C3 -type ring finger, and rubredoxin-type fold domain as the CHYR family.

Increasing evidence has shown the diverse roles of CHYR genes in plant growth, development, and stress responses. MIEL1 was first found to control protein stability of MYB96 and MYB30 in balancing cuticular wax biosynthesis and defense ^[8][15][16][8,15,16]. AtCHYR1 was reported to enhance ABA and drought responses by elevating ROS production and stomatal closure [9]. The homologous gene of Populus euphratica (PeCHYR1) showed similar phenotypes, enhancing drought tolerance, stomatal closure, and H₂O₂ production [17]. However, overexpression of OsRZF34 (AtCHYR1 homologous gene in rice) enhanced stomatal opening, leaf cooling and ABA insensitivity [10]. CHYR proteins with 2–3 additional hemerythrin domains (also known as BTS/BTSL/HRZ) were found to regulate iron response in Arabidopsis and rice ^[11][12][18][11,12,18].

2. Identification and Phylogenetic Analysis of CHYR Genes from Soybean and Arabidopsis

To identify soybean CHYR genes, protein sequences of published Arabidopsis CHYRs ^{[8][9][11][12][19]}[8,9,11,12,19] were used to construct a Hidden Markov Model (HMM) [20]. Whole soybean and Arabidopsis protein sequences were downloaded from Phytozome to carry out the local search. Finally, 16 soybean and 7 Arabidopsis CHYR genes were identified. The 23 proteins were proven to contain at least three conserved domains, including CHY zinc-finger (PF05495), C3H2C3-type ring finger (PF13639), and zinc ribbon domain (PF14599) according to Pfam and SMART analysis. For convenience’s sake, soybean CHYR genes were renamed GmCHYR1 to GmCHYR16 based on their order on the chromosomes, and genes from Arabidopsis were relabeled as AtCHYR1 to AtCHYR7. Their involved information (including sequence length, hydropathicity, predicted protein location, classification, alternative name, and functions) were listed in Table S1. As we could see from Table S1, amino acid numbers of GmCHYRs and AtCHYRs ranged from 234 to 1262. Their grand average of hydropathicity was all negative, indicating that GmCHYRs and AtCHYRs are hydrophilic proteins. Furthermore, these CHYR proteins were predicted to localize in the cytoplasm, or nucleus, or chloroplast. The cytoplasm and nucleus distribution of AtCHYR6/MIEL1 in Arabidopsis cells could support this result [8]. To further investigate the phylogenetic relationship of GmCHYRs, their protein sequences were aligned with 7 AtCHYRs. All 23 CHYR proteins contained conserved CHY zinc-finger (PF05495), C3H2C3-type ring finger (PF13639), and zinc ribbon 6 domain (PF14599) (Figure S1). Then, a phylogenetic tree was generated based on this multiple alignment by using MEGA 7.0 with the Maximum-Likelihood (ML) method with 1000 bootstrap replications. As shown in Figure 1A, soybean and Arabidopsis CHYRs could be classified into three groups according to their topological analysis and bootstrap values. In detail, both Group I and Group II consisted of 5 GmCHYRs and 2 AtCHYRs. The rest, 6 GmCHYRs and 3 AtCHYRs were allocated to Group III. Furthermore, their conserved domains and motifs were analyzed. As expected, all 16 GmCHYRs and 7 AtCHYRs contained CHY zinc-finger, C3H2C3-type ring finger, and zinc ribbon (Figure 1B). Besides, there were 2-3 hemerythrin domains in the N terminus of Group III members. Group III members were also called BTS/BTSL in Arabidopsis, and HRZ in rice ^[12][18][12,18]. This is consistent with former reported results that there were 2 BTSL (AtCHYR2/3) and 1 BTS (AtCHYR4) in Arabidopsis [12]. All of them have been reported to regulate iron homeostasis [11]. Meanwhile, we employed the MEME program to predict conserved motifs (Figure 1B). In accordance with conserved domains, GmCHYRs within each group displayed similar motif distribution. Among the detected 15 motifs, motif 1, 5, 9, 12 in the N terminus made up CHY-zinc finger. Motif 3 and 4 formed the Ring finger domain. Motif 2 served as Zinc_ribbon domain. Additionally, the hemerythrin domain of Group III members constitutes motif 7, 10, 11, 14, 15. Additionally, a conserved motif 6 and 8, which was close to the hemerythrin domain, could be found in Group III members. However, their function still needs further investigation.

3. Identification and Classification of CHYR Members in Green Plants

The above results showed that only Group III members contained 2–3 additional hemerythrin domains in the N terminus, which are of great importance in regulating iron homeostasis. We wondered whether Group III CHYR proteins gained these hemerythrin domains during evolution, or Group I and II lost these domains. Therefore, the local proteome sequences of 21 representative plant species, including Dicots, Monocots, Basal Angiosperms, Pteridophyta, Bryophyta, Chlorophyta, and Gymnosperm were searched to identify potential CHYR genes by using the former Arabidopsis HMM. At last, a total of 107 nonredundant sequences were obtained from 21 detected plant species (Table 1 and Table S2). Pfam and SMART were further used to detect the three conserved domains for CHYR proteins, including the CHY zinc-finger domain, C3H2C3-type ring finger domain, and zinc ribbon domain. To explore their evolutionary relationship, 107 CHYR members were aligned using ML (Maximum-likelihood), NJ (Neighbor-joining), and ME (Minimum-evolution) methods to construct unrooted phylogenetic trees based on their protein sequences (Figure 2, Figure S2, and Figure S3). As the three phylogenetic trees depicted, three methods presented a similar topology. According to their evolutionary relationship, 107 CHYR members could be further divided into three groups (Group I, II, III) as well. Though Group I and Group II were clustered together, CHYR members from Bryophyta, Pteridophyta, and Gymnosperms could be only found in Group I, implying the possibility of gene acquisition during evolution. From this result, we speculated that Group II might appear after Group I. Group III did coexist with the other two groups, but was far away from the others in topology, which indicated that they might come from different ancestors. Interestingly, there were only 4 CHYR members in Chlorophyta, two of them were from Chlamydomonas reinhardtii, the others were from Volvox carteri. While CreCHYR2 and VocarCHYR1 were clustered with Group I and Group II, CreCHYR1 and VocarCHYR2 were grouped Group III, indicating the existence of CHYR members throughout green plants evolution. A previous study has reported the up-regulation of CreCHYR1 under iron deficiency [21], suggesting the conserved role of Group III members in iron regulating. The above findings implied the early emergence of CHYR members and their persistence in the evolution of green plants.