Transcription factors (TFs), which constitute approximately 8% of the protein-encoding regulators in eukaryotic genomes, are critical transcriptional regulatory factors (Pruneda-Paz et al., 2014). Therefore, functional characterization of transcription factors (TFs) is critical for understanding transcriptional regulatory networks and biological processes (Liu et al., 2014). The basic leucine zipper (bZIP) family is one of the largest and most diverse TF families (Pérez-Rodríguez et al., 2010). The bZIP domain is highly conserved and contains two structural features located on a contiguous α-helix i.e., the leucine zipper composed of several heptad repeats of Leu or other bulky hydrophobic amino acids for dimerization specificity, and the N-x7-R/K-x9 domain for specific binding (Jakoby et al., 2002; Lee et al., 2006; Nijhawan et al., 2008). Apart from the bZIP domain, several domains of the bZIP family have been found to function as transcriptional activators (Liao et al., 2008). To bind DNA, half of the basic region in the N-terminal binds double-stranded DNA, and half of the Leu zipper in the C-terminal undergoes dimerization, leading to the formation of a superimposed coiled structure (Ellenberger et al., 1992).

Members of the bZIP transcription factor family are involved in the regulation of growth and developmental processes such as seed germination, embryogenesis, flower and vascular development, hormonal control, and senescence (Jakoby et al., 2002; Schütze et al., 2008; Toh et al., 2012; Sornaraj et al., 2016). Overexpression of OsbZIP23, a member of bZIP in rice (Oryza sativa), rescued the pre-harvest budding phenotype and the decrease in expression of genes associated with ABA signaling in transgenic plants (Song et al., 2020). CAREB1, an important trans-acting factor of bZIP members, was found to regulate somatic embryogenesis in carrot (Daucus carota) (Guan et al., 2009). Eleven TabZIP genes in wheat (Triticum aestivum) were highly expressed in anthers, suggesting that they were involved in flower development (Li D. et al., 2015). In Arabidopsis, a bZIP transcription factor that control monopteros (MP) output and modulate vascular gene expression (Smit et al., 2020). The bZIP Transcription factor PERIANTHIA interacts with a variety of developmental pathways, including light and plant hormones, both of which participate in meristem formation (Maier et al., 2011). Furthermore, the bZIP members regulate response to abiotic/biotic stresses such as drought, salt, hypoxia, cold, pests, and diseases (Uno et al., 2000; Shimizu et al., 2005; Zander et al., 2012; Alves et al., 2013; E et al., 2014; Amorim et al., 2017). GmbZIP44 and GmbZIP62, the bZIP genes of soybean (Glycine max), conferred tolerance to salt and freezing stress in transgenic Arabidopsis plants (Liao et al., 2008). Overexpression of CabZIP25, a member of bZIP in pepper (Capsicum annuum), enhanced salt tolerance in transgenic Arabidopsis and promoted salt sensitivity by decreasing virus induced gene silencing (VIGS) expression in pepper (Gai et al., 2020). The study by Hsieh et al. (2010) showed that SlAREB, a member of bZIP in tomato (Solanum lycopersicum), regulated stress-responsive genes and improved water logging deficit and salt stress response. Elsewhere, it was reported that AREB1, an Arabidopsis bZIP transcription factor, conferred tolerance to water deficit (including drought and flooding stresses) in modified soybeans overexpressing AREB1 (Fuhrmann-Aoyagi et al., 2021). PPI₁, a bZIP in pepper, regulated expression of genes involved in defense mechanisms (Lee et al., 2002).

The application of genome sequencing has led to identification of bZIP family members (Jakoby et al., 2002), in Arabidopsis (Jakoby et al., 2002), rice (E et al., 2014), Carthamus tinctorius (Li H. et al., 2020), Chinese jujube (Ziziphus jujuba) (Zhang Q. et al., 2020), Olive (Olea europaea) (Rong et al., 2020), common bean (Phaseolus vulgaris) (Zhang et al., 2021), and potato (Solanum tuberosum) (Herath and Verchot, 2020). However, few studies have investigated bZIP family members in adzuki bean (Vigna angularis). Adzuki bean (Vigna angularis) is an important dietary legume crop that was first cultivated in China (Han et al., 2005). Its grains have high protein content, a low-fat content, and high iron content. They contain several bioactive substances such as triterpenoids, flavonoids, and saponins. It was traditionally used as an iron supplement, to remove damp and swelling (Amarowicz et al., 2008; Yang et al., 2015). Being a sensitive species, adzuki beans are particularly vulnerable to environmental stressors such as cold, drought, salt, and heavy metals (Srivastava et al., 2018; Li W.-Y. et al., 2020). In this study, bZIP members in the adzuki bean were identified, and characterized in terms of phylogeny and evolutionary expansion in different tissues under different stress conditions such as drought (D), cold (C), salt (NaCl) and heavy metal (CdCl₂). The findings will provide new insights about bZIP members which can be applied in resistance breeding.

The bZIP members are present in various species, and the number of members vary from one species to another. There are 78 members in Arabidopsis (Dröge-Laser et al., 2018), 80 members in potato (Solanum tuberosum) (Herath and Verchot, 2020), 160 members in soybean (Glycine max) (Zhang et al., 2018), 89 members in rice (Oryza sativa L.) (Nijhawan et al., 2008) and 69 members in tomato (Solanum lycopersicum) (Li X. et al., 2015). In this study, VabZIP members were identified from the reference genome of the adzuki bean, which contained 72 members. The number of VabZIP members was found to exceed the number of bZIP members in tomato, while the number of bZIP members was less than in Arabidopsis, potato, soybean, and rice. These findings suggest that the number of bZIP members may be related to the size of the reference genome and that after differentiation from their early ancestors, the adzuki bean may have experienced fewer genomic replication events, when compared to other species.

Evolutionary analysis revealed that VabZIP members could be divided into 14 subfamilies in the unrooted Maximum Likelihoodphy (ML) tree, and the result of evolutionary combined with bZIP members in Arabidopsis and adzuki bean was also revealed that bZIP members had 14 sub-families, which was similar to the results in wheat (Triticum aestivum) (Li D. et al., 2015) and Chinese jujube (Ziziphus jujuba Mill.) (Zhang Q. et al., 2020). With regards to motif and gene structures of VabZIP members, motif constitutions differed in different sub-families. Within the same sub-family, the motifs were similar and the motif of the coded bZIP (motif-1) was highly conserved (Zhou et al., 2017). Motif-3 and motif-5 were only found in sub-family X while motif 9 was only found in subfamily XII, which was similarly found in tartaty buckwheat (Fagopyrum tataricum) (Liu et al., 2019). VabZIP members from the same subfamily exhibited a similar gene structure, whereas VabZIPs from sub-families I, II and, III had no more than two introns, suggesting a relationship between the low number of introns and stress responses in the three sub-families (Zhao et al., 2016). The combined results of the motif and gene structure for two species of bZIP members revealed similar results.

Cis-elements in promoter regions of VabZIP members regulate the functions of VabZIPs that contain related cis-elements (Lescot et al., 2002). Similarly, bZIP members have cis-elements that are comparable across species: ABRE, TATC-box, TCA-element, and P-box are hormone-related cis-elements in VabZIP and bZIP members in common bean and potato (Wang et al., 2021; Zhang et al., 2021), implying that VabZIPs may regulate hormones involved in plant growth. Stress-related cis-elements, such as MBS and LTR, were found in VabZIPs and sesame bZIP transcription factor members (Wang et al., 2018), leading to the hypothesis that VabZIPs are involved in abiotic stress responses. Moreover, since they contain the RY element, which is similar to that found in the common bean, VabZIP members may have had an effect at the bud stage (Zhang et al., 2021).

Collinear analysis allows the transfer of functional information from a well-studied taxon to a less-studied taxon (Ghiurcuta and Moret, 2014). In this study, 25 VabZIPs pairs exhibited collinearity with an Arabidopsis member, which was found to be involved in plant growth regulation, abiotic stress responses, responses to hormones, and germination in plants. AT4G37790, AT1G45249, and AT2G36270 were the collinearity genes for VABZIP26, VaBZIP48, and VaBZIP18 respectively, which play a function in salt stress responses (Lopez-Molina et al., 2001; Nakashima et al., 2006; Liu et al., 2016). Collinearity members for Vigna angularis such as VaBZIP23, VaBZIP46, VaBZIP18, and VaBZIP47 in Arabidopsis, have a role in the bud stage of the plant: AT1G75390, the collinearity member for VabZIP23 and VabZIP46, positively regulates plant seed germination rate. Its knock-out was associated with significantly slower germination rate (Iglesias-Fernández et al., 2013). AT2G36270 had collinearity with VabZIP18, which increases proteins for preventing seed germination (Piskurewicz et al., 2008); AT4G38900, the collinearity gene for VabZIP47 was expressed in meristematic tissues and negatively modulates Arabidopsis growth (Lozano-Sotomayor et al., 2016). In Arabidopsis, VaBZIPs member collinearity genes, such as AT1G22070 (VaBZIP21) were shown to be involved in the salicylic acid (SA) signaling pathway (Zhou et al., 2000), whereas AT1G45249 (VaBZIP48) and AT2G36270 (VaBZIP18) were found to be involved in abscisic acid (ABA) responses (Fujita et al., 2005). The collinear analysis results indicate that VaBZIP members are involved in responses to hormones, coping with environmental pressures, and regulating the bud stage.

The bZIP members have previously been reported to exhibit tissue-specific expressions, including in Olive (Olea europaea) (Rong et al., 2020), radish (Raphanus sativus) (Fan et al., 2019), and poplar (Zhao et al., 2021). Expressions of VabZIPs at the bud stage revealed tissue-specificity, with the radicle having higher expressions than other tissues, indicating that the radicle could be used as a target tissue for VabZIPs’ research. Gene expression changes under abiotic stress conditions might lead to abiotic stress responses in plants, and differentially expressed genes under abiotic stress can be used as candidate genes for further research on abiotic stress responses (Qi et al., 2021). In this study, expressions of selected VabZIPs under abiotic stress indicated that VabZIPs are involved in abiotic stress responses. Expressions of VabZIP06, VabZIP11, VabZIP21, VabZIP47 and VabZIP51 were markedly up-regulates under drought, cold, salt and heavy metal stress, implying that these bZIP members are involved in abiotic stress responses. Moreover, some bZIP members are involved in abiotic stress responses in other plants, such as StbZIP25 in potato (Wang et al., 2021), TabZIP96 in wheat (Liang et al., 2022) and CabZIP25 in pepper (Gai et al., 2020). Expressions of bZIP members under abiotic stress revealed that some bZIPs can be used in plant breeding for abiotic stress resistance, such as in watermelon (Yang et al., 2019), sesame (Wang et al., 2018), and apple (Zhao et al., 2016). These results indicate that bZIPs might be useful in molecular breeding under abiotic stress and the VabZIPs that were differentially expressed under stress can be used for further research, particularly in stress-resistance breeding.

In this study, 72 VabZIP members were identified and divided into 14 subfamilies. The members of each sub-family had motifs and gene structures that were comparable. These VabZIP members exhibited hormonal responsiveness, environmental stress, and germination cis-elements, indicating that the VabZIPs might be involved in plant hormone and abiotic stress regulation. The VabZIPs, whose expressions were tissue specific, might be involved in abiotic stress responses. And VabZIP17 and VabZIP56 were located on the nuclear in subcellular localization ananlysis. Furthermore, expressions of VabZIPs under stress conditions such as drought, cold, salt, and heavy metal stress at the bud stage revealed that some VabZIPs (such as VabZIP06, VabZIP11, VabZIP21, VabZIP47 and VabZIP51) might regulate abiotic stresses responses in the adzuki bean. This study provides valuable information and insights into the development of VabZIPs and establishes a foundation for the use of related characteristics of VabZIPs in adzuki beans.