Similar to the findings for BrpHMA2, our results suggest that these TF genes may respond to Cd. The coding sequences of the three NAC TFs and three AREB TFs listed above were cloned and submitted to the NCBI database. The last three or four numbers of each gene’s full name was used as the gene name. MEGA5 was used to create a phylogenetic tree of these NAC TF or AREB TF genes and Arabidopsis NAC or AREB genes using the neighbor-joining method. The results revealed that the BrpNAC4584 and BrpNAC895 sequences were closer to those of Arabidopsis ANAC046 and ANAC087, respectively ; in addition, the BrpABI227 and BrpABI678 sequences were closer to that of AtABF4, and the BrpABI449 sequence was more comparable to that of AtABF3 .Our results reveal that BrpHMA2 could be activated by Cd2+ , which is similar to the results found for HMA2 in Arabidopsis. Results suggest that BrpHMA2 is involved in the Cd response of plants. BrpHMA2 was also found to be expressed explicitly in the vascular tissues of roots, stems, leaves, flowers, siliques, and carpopodia, and its protein was localized in the plasma membrane . These results are consistent with previous findings for HMA2 in Arabidopsis, OsHMA2 in rice, and TaHMA2 in wheat. The protein plasma membrane localization and the vascular-specific expression pattern of the genes revealed that HMA2 might function as a membrane transporter in long-distance transport in plants. In recent years,best indoor vertical garden system some studies have investigated the function of HMA2. Most of these studies demonstrated that HMA2 is involved in Zn2+ and Cd2+ transmembrane transport and influences root-to-shoot Zn/Cd translocation.

For example, HMA2 in Arabidopsis is thought to be involved in the outward transport of Zn2+ and Cd2+ from the cell cytoplasm, and HMA2 mutants are more sensitive to Cd stress and exhibit higher Zn or Cd accumulation than wild-type plants in the presence of high levels of Zn2+ or Cd2+ 14,15. The over expression of OsHMA2 in wheat, rice, and Arabidopsis improves root-to-shoot Zn/Cd translocation. In addition, the transformation of TaHMA2 in yeast enhances the resistance of cells to Zn/Cd. In rice, the suppression of OsHMA2 decreases the Zn and Cd concentrations in leaves, increases the retention of Zn in roots and reduces the translocation of Cd and Zn from roots to shoots compared with the results obtained with wild type plants. According to the literature, HMA2 is responsible for Zn2+/Cd2+ efflux from cells, plays roles in Zn and Cd loading to the xylem, and participates in the root-to-shoot translocation of Zn/Cd. However, Yamaji et al. found that OsHMA2 is localized at the pericycle of the roots and in the phloem of enlarged and diffuse vascular bundles in the nodes. Their insertion lines of rice showed decreased concentrations of Zn and Cd in the upper nodes and reproductive organs. The study revealed that the heterologous expression of OsHMA2 in yeast is associated with the influx transport of Zn and Cd. These researchers suggested that OsHMA2in the nodes plays an important role in the preferential distribution of Zn and Cd through the phloem to the developing tissues. Our results also revealed that, in the presence of Cd2+, transgenic Arabidopsis seedlings and yeast over expressing BrpHMA2 showed higher concentrations of Cd and enhanced Cd2+ sensitivity compared with the controls . Thus, we propose that BrpHMA2 functions in Cd2+ transport in the phloem tissue of vascular systems through influx into cells, and the efflux from phloem cells during long-distance transport may be performed by other transporters. The differential function of HMA2 from various plants might come from the tiny difference in amino acids in their function domains; this puzzle requires further investigation.

In this study, we identified the NAC TF gene BrpNAC895, a homolog of Arabidopsis ANAC087 , which could be induced by Cd2+ stress . We confirmed that BrpNAC895 has a role in the response of B. parachinensis to Cd2+ stress by upregulating BrpHMA2 expression through direct binding to the BrpHMA2 promoter using EMSA, ChIP–qPCR, and the transient transformation method with B. parachinensis protoplasts . Previous studies have demonstrated that Arabidopsis ANAC087 is associated with plant programmed cell death . It functions along with the TF ANAC046 to show partial redundancy in coregulating the expression of some PCD genes in the root columella, including ZEN1, BFN1, and RNS3. Whether ANAC087 could participate in regulating Cd transporters in plants has not been reported. Our findings on BrpNAC895 show that this NAC TF has a novel role in upregulating BrpHMA2 expression in response to Cd2+ stress. We also identified the Cd-responsive AREB TF BrpABI 449 , which is a homolog of Arabidopsis ABF3 and can bind to the promoter of BrpHMA2 . ABF3 modulates the response to drought, salt, and other osmotic stresses as a master component in ABA signaling. This TF can also regulate the expression of multiple genes, such as the AGAMOUSlike MADS-box TF family gene SOC1, which is a floralintegrator regulating flowering in response to drought, and the AREB TF ABI5, which is a core component in the ABA signaling pathway in the regulation of seed germination and early seedling growth during exposure to ABA and abiotic stresses. In general, ABF3 can form protein complexes with other TFs. For example, ABF3 forms homodimers or heterodimers with AREB1/AREB2 and acts cooperatively to regulate ABRE dependent gene expression. ABF3 forms a complex with NF-YC3 to promote the expression of the SOC1 gene and thus accelerate flowering and drought-escape responses; ABF3 interacts with NAC072 to regulate RD29A and RD29B expression in response to ABA. Thus, complex formation might be the important functional mechanism by which ABF3 regulates gene transcription.

Using EMSAs and ChIP–qPCR assays, we found that BrpABI449 could directly bind to regions of the BrpHMA2 promoter . The interaction of BrpABI449 and BrpNAC895 was further confirmed by pull-down and BiFC assays . The inhibition of BrpABI449 on the transcriptional regulatory role of BrpNAC895 was detected in the B. parachinensis protoplast transient system . The inhibition by BrpABI449 of the transcriptional regulatory role of BrpNAC895 complex, likely interferes with BrpNAC895’s activity in the transcriptional activation of BrpHMA2 in response to Cd stress. It has also been reported that Cd stress can induce a stress response via ABA signaling. Our results showing that BrpNAC895 and BrpABI449 are upregulated by Cd stress also support this point. The uptake or homeostatic regulation of heavy metals needs proper modulation to ensure plant health. Previous studies have shown that Cd stress induces the MYB TF gene MYB49 in Arabidopsis. This TF may further positively regulate the downstream TF gene bHLH38 and bHLH101 by directly binding to their promoters, and activate iron-regulated transporter 1 to enhance Cduptake. In contrast,growing strawberries vertically Cd stress upregulates the expression of ABI5. ABI5 interacts with MYB49, prevents its binding to the promoters of downstream genes, and functions as a negative regulator to control Cd uptake and accumulation. Our present results also demonstrate a mechanism for controlling the expression of the heavy metal transporter gene BrpHMA2 under Cd stress. We propose that Cd2+ induces the expression of BrpNAC895 and BrpABI449, which might be mediated by ABA signaling. The activation of BrpHMA2 enhances Cd2+ uptake and may induce cell damage. Negative regulation of BrpHMA2 is then achieved by the upregulation of another AREB TF, BrpABI449, which interacts with BrpNAC895 and forms BrpNAC895-BrpABI449 protein complexes to inhibit the BrpHMA2 transcription activated by BrpNAC895 . BrpABI449 could also bind to the promoter of BrpHMA2 directly to compete with BrpNAC895 in binding to the BrpHMA2 promoter. This negative regulation may play a supplementary role in the uptake and transport of Cd.Many plant species of Brassicaceae, including Arabidopsis, turnip, and oil seed rape, can be genetically modified, but the creation of transgenic B. parachinensis remains difficult. Therefore, we over expressed BrpHMA2 in Arabidopsis to investigate the function of BrpHMA2 and established a transient transformation system in B. parachinensis protoplasts to perform gene regulatory network analysis. Protoplasts have been widely used for subcellular protein localization and gene regulation analyses. In this study, the transient transformation of B. parachinensis protoplasts was demonstrated to be a powerful system for ChIP–qPCR analysis. Previous studies have applied a similar approach to Populus trichocarpa and Brassica napus. Although the transient transformation system of B. parachinensis protoplasts was successfully used in this study of molecular mechanisms, the system cannot be easily used for phenotype and physiological analyses. The lack of BrpNAC895 and BrpABI449 transgenic B. parachinensis is a problem that severely limits research on this plant.

New techniques, such as the transient reprogramming of plant traits via the transfection of RNA based viral vectors using Agrobacterium and gene editing combined with fast-treated Agrobacterium coculture, may be useful approaches for comprehending gene function concerning physiology and for the further application of modifications of gene function to effectively control the accumulation of Cd in B. parachinensis.Reduced pod shattering is an important breeding target in many crops, including common bean . In the wild, many legumes benefit from seed dispersal mediated by explosive pod dehiscence, known as pod shattering. During the domestication process, the trait has been strongly reduced across most legume taxa . Despite this, some market classes of common bean have persistently high levels of pod shattering, leading to reduced yields and a constrained harvest window. This issue is particularly problematic in semiarid environments, which cause pods to become brittle and fracture more easily . Common bean is a vital source of protein and micro-nutrition for hundreds of millions of people globally . The crop was independently domesticated in Middle America and the Andes , leading to the species’ two major domesticated gene pools. These are additionally subdivided into several ecogeographic races, each with a long history of adaptation to specific environmental conditions . In particular, members of the Middle American ecogeographic race Durango are adapted to the semiarid highland environments of northern Mexico and the southwestern USA, whereas the Middle American raceMesoamerica inhabits humid lowland regions of Mexico, Central America and lowland South America. Useful alleles from any major gene pool can readily be moved into others, and crosses between races have major untapped potential for breeders . Seven independent domestication events occurred in the Phaseolus genus, including close relatives of common bean such as Lima bean , runner bean year bean and tepary bean . An improved genetic understanding of pod shattering in common bean will be useful for improvement of numerous other domesticated legumes that suffer from pod shattering . Several genes are known to influence resistance to pod shattering in common bean , and the genes involved vary by gene pool. In the Middle American domesticated beans, the locus Phaseolus vulgaris Pod dehiscence 1 on chromosome Pv03 is associated with a major reduction in pod shattering . The shattering resistance allele is found at high frequency in race Durango, but is nearly absent in market classes belonging to race Mesoamerica or the Andean gene pool . This is a major target for improvement in these classes. Orthologs of this Pv03 gene may also regulate pod shattering in other species, such as cowpea , chickpea and soybean , where the orthologous locus plays a role in adaptation to arid climates by modifying the extent of twisting in pod valves . A possible second locus on chromosome Pv08 in Middle American beans has been proposed to reduce pod shattering , but a relatively small sample size of these individuals has hindered the study of this allele. The Pv08 QTL is also believed to have a major effect in Andean beans, so a deeper investigation of this QTL could provide insight on whether it has evolved in parallel between domestication events. A recently discovered QTL on Pv05, in immediate vicinity of PvMYB26, is associated with a loss of dehiscence in the Andean gene pool . This locus was mapped in detail in a biparental recombinant inbred population , which also found significant QTLs on Pv04 and Pv09 in the same population. The role of the Pv05 and Pv09 loci were identified in parallel in a diversity panel of Andean beans , which also identified significant loci on Pv03 and Pv08. PvMYB26 was subsequently found to be differentially expressed between dehiscent and non-dehiscent individuals, leading to major differences in development of cell walls in the suture . Other loci, including St and To , control strong fiber development in pod sutures and pod walls , respectively, and the mutant variants are found only in snap beans grown as a vegetable. St has been mapped to Pv02, and To has been mapped to Pv04 .