Full-length BrpNAC895 and BrpABI449 were fused to the C-terminal and N-terminal halves of enhanced yellow fluorescent protein , respectively.The results demonstrated that BrpABI449 could be detected in the precipitates of MBP-BrpNAC895 and indicate that BrpNAC895 can interact with BrpABI449. The cotransfection of BrpABI449 with BrpNAC895 reduced the BrpNAC895-activated transcription of BrpHMA2 ; moreover, BrpABI449 could interact with BrpNAC895, which suggests that the interaction of BrpABI449 and BrpNAC895 inhibits the ability of BrpNAC895 to bind with the promoter of BrpHMA2. To confirm this speculation, we used an EMSA to analyze the binding ability of BrpNAC895 with the pF1 locus of the BrpHMA2 promoter in the presence of the BrpABI449 protein. The results demonstrated that the binding of BrpNAC895 on the promoter fragment pF1 of BrpHMA2 was reduced by the BrpABI449 protein . However, BrpABI449 could not bind to pF1 directly, as shown in Fig. 4c. These results demonstrate that the interaction between BrpABI449 and BrpNAC895 interferes with the binding of BrpNAC895 to the BrpHMA2 promoter.B. parachinensis is a popular leafy crop; however, it may collect significant levels of heavy metals, particularly Cd, when grown on Cd-polluted substrate soil. Transcriptomes of B. parachinensis under Cd stress were previously generated to elucidate the mechanisms underlying Cd accumulation.We reveal that BrpHMA2,how to set up a vertical farm which is differently expressed in plants, is involved in Cd uptake and accumulation .
Furthermore, BrpHMA2 expression is controlled by BrpNAC895 and BrpABI449, which operate as activators and inhibitors, respectively .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, 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 overexpression 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.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, 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. BrpNAC895 then promotes the transcription of BrpHMA2 by binding directly to its promoter . 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,what is vertical growing 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 RNAbased 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 .Copper and its compounds have been known to have the ability to inhibit fungi since ancient times and have been used widely in agriculture as fungicides, algaecides,pesticides,and herbicides.There are at least 209 pesticide products registered in California that use copper oxide as an active ingredient.In addition, due to steady increase of drug resistance of bacteria, synthesis and application of novel antibacterial/anti anti-fungal Cu nanoparticles has increased.Besides antibacterial applications, Cu NPs also have application as additives of livestock and poultry feed.There is increasing concern about the potential for bio-accumulation and toxicity of Cu NPs after their release to the environment. It has been shown in several studies that nano-Cu triggers reactive oxygen species generation and induces oxidative stress in cells, bacteria, and zebra fish.
However, very few studies have focused on the toxicity of Cu NPs on terrestrial plants, especially crop plants. Lee et al.,documented that Cu NPs are toxic to mung bean and wheat at concentrations of 335 and 570 mg/L, respectively. Hong et al.reported that even at the level of 5−20 mg/L, Cu NPs significantly reduced the root length of alfalfa and lettuce and altered their nutrient uptake. Cucumber plants are generally more sensitive to contaminants and their bio-accumulation is higher than many other plants due to their high transpiration rate. Our previous study showed both CeO2 and ZnO NPs triggered more physiological changes in cucumber plants compared to corn plants, which have lower transpiration rates.Here we hypothesized that nano-Cu would induce physiological responses in cucumber plants. To evaluate this, we selected metabolomics studies as a novel approach to understand plant−nanoparticle interactions. We selected a 1 H nuclear magnetic resonance -based environmental metabolomics platform to detect the induced alteration, because NMR can simultaneously detect a variety of metabolites with simple sample preparation.In addition, compared with other “omics”, metabolomics reveals effects downstream of DNA and simultaneously provides a nonspecific assessment of the end result of multiple biological responses.Therefore, 1 H NMR has been employed to evaluate the toxicity of a large variety of environmental contaminants on different organisms.The toxicity and toxicity mechanism of titanium dioxide NPs to earthworms and rats was evaluated via an environmental metabolomics platform using 1 H NMR. Furthermore, most previous studies of the effect of various NPs to plants have concentrated on toxicity. Potential detoxification mechanisms have been less reported. It is well known that approximately 30−40% of all photosynthetically fixed carbon will be transferred to the rhizosphere as root exudates, including organic acids, amino acids, sugars, proteins, phenolic compounds, and CO2. These compounds play an important role in plant stress tolerance and external exclusion of pollutants.