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 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,procona buckets 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 AGAMOUS like 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, 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, 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 sub-cellular protein localization and gene regulation analyses. In this study,procona florida container 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.Abiotic stresses, especially those which affect the water relations of the plant such as low temperatures, may decrease plant growth and yield. The majority of plants will suffer damage when exposed to freezing temperatures , but plants of tropical or sub-tropical origin also suffer damage when exposed to chilling temperatures . Exposure of roots to chilling temperatures decreases root hydraulic conductance , and can result in water stress and chilling injury within a few hours of exposure . The susceptibility to water stress induced by root chilling in species of tropical and sub-tropical origin is a concern for agricultural production in Mediterranean climates such as California, where exposure to cold soils in the spring can affect seedling establishment because soil temperatures under an open canopy may be colder than air temperatures . Cultivated tomato is a classic example of a chilling-sensitive crop . It was domesticated from the wild cherry tomato, which is native to mesic, tropical environments . A related wild tomato species, S. habrochaites, grows in the Peruvian Andes at altitudes up to 3300 m and thrives in xeric habitats and at chilling temperatures detrimental to S. lycopersicum . Upon exposure to root chilling conditions, the root hydraulic conductance of both tomato species decreases, but S. habrochaites closes its stomata rapidly in response to chilling stress, thereby maintaining water potential and shoot turgor, whereas the stomata of S. lycopersicum remains open and the shoots wilt . Other agronomically important crops of tropical or sub-tropical origin such as maize and rice respond to root chilling in a manner consistent with that of cultivated tomato . An improved understanding of the underlying mechanisms of root chilling tolerance in wild S. habrochaites would contribute to a better general understanding of chilling sensitivity in crops of tropical and sub-tropical origins.A review by Venema et al. focused on physiological effects of chilling and noted that wild tomato species were promising sources of genetic tolerance to chilling.
Oyanedel et al. evaluated a back cross inbred line population derived from S. habrochaites acc. LA1777 for growth traits under chilling temperatures and reported QTL for higher biomass accumulation on chromosomes 2, 3, and 9. Elizondo and Oyanedel evaluated tomato introgression lines containing S. habrochaites acc. LA1777 introgressions on chromosomes 2 and 3 in the field under low temperatures . The ILs had higher growth rates but lower fruit set than the parental lines in response to an increase in the number of hours of chilling temperatures. To investigate the genetic basis of shoot turgor maintenance under root chilling, Truco et al. used an interspecific BC1 population derived from chilling-susceptible S. lycopersicum cv. T5 and chilling-tolerant wild S. habrochaites acc. LA1778 to map QTL for this trait. Three QTL for shoot turgor maintenance under root chilling were identified on chromosomes 5, 6, and 9. The largest effect QTL located on chromosome 9 accounted for 33 % of the trait phenotypic variance . We designated this QTL stm9 for shoot turgor maintenance, chromosome 9. Subsequently, QTL stm9 was fine-mapped to a 2.7-cM region on the short arm of chromosome 9 between markers T1670 and T1673 . Easlon et al. determined that tomato ILs containing the short arm of chromosome 9 from chilling-tolerant S. lycopersicoides and S. habrochaites maintained shoot turgor under root chilling. Here we high-resolution mapped QTL stm9 using recombinant sub-near-isogenic lines and compared high resolution mapped QTL stm9 to the S. lycopersicum reference genome for initial identification of potential candidate genes and regulatory sequences . Our longer term goal is to identify and functionally test candidate genes and regulatory sequences from S. habrochaites and determine the causal gene or polymorphisms for QTL stm9.A population of near-isogenic lines containing the chromosome 9 region from S. habrochaites acc. LA1778 in an otherwise completely S. lycopersicum cv. T5 background was marker-selected and used for fine-mapping, as described in Goodstal et al. . For high-resolution mapping of stm9, we created and marker selected recombinant sub-near-isogenic lines as follows.