Cd transporters are considered to play central roles in various physiological activities

Excess Cd uptake in plants normally induces the accumulation of reactive oxygen species in plants and has severe consequences, such as chromosome aberrations, protein inactivation, membrane damage, and and further leading to leaf chlorosis and root growth inhibition. Furthermore, accumulation of Cd in crops enhances the risk of Cd poisoning in humans and animals. Brassica species have been identified as Cd hyperaccumulators. Brassica parachinensis L.H. Bailey is a leafy vegetable widely consumed in China, Europe, and other regions of the world. Thus, elucidating the molecular mechanisms of Cd accumulation in this plant is essential for developing effective strategies to control Cd accumulation in the plant’s edible parts. Cd accumulation in plant tissues generally involves a three-step process: absorption and accumulation of Cd in roots from the soil, translocation of Cd to the shoot via vascular tissue, and Cd storage in leaves. The HMA , ZIP , and Nramp families are among the transporter families that have been identified as being involved in these processes. Our previous transcriptome analyses of B. parachinensis also showed that differentially expressed genes enriched in the gene ontogeny terms ‘transmembrane transport’ and ‘metal ion transport’ may be involved in response to Cd, including genes encoding members of some transporter families, such as the subfamily C of ATP-binding cassette proteins and HMAs. HMAs,growing strawberries vertically which belong to the P1B subfamily of the P-type ATPase super family, have been extensively investigated in the model plant Arabidopsis as well as in some crop plants, and the main focus of these studies has been on their functions.

For example, eight members of HMAs have been identified in Arabidopsis thaliana, and among these, AtHMA1–AtHMA4 are thought to specifically transport divalent cations, such as Zn2+, Cd2+, Co2+, and Pb2+ [10]. AtHMA2 is generally regarded as a Zn2+- ATPase. It contains a conserved short metal binding domain in the N-terminus and a long metal binding domain in the C-terminal end; Zn2+-binding affinity was detected in both domains, and Cd2+- and Cu+-binding affinity was detected in the Nterminal domain. Some studies showed that AtHMA2 functioned as an efflux to drive the outward transport of metals from the cell cytoplasm and responsible for cytoplasmic Zn2+ homeostasis and Cd detoxification. Some researchers proposed that AtHMA2 together with AtHMA4 played key roles in the long-distance root to-shoot transport of Zn2+ and Cd2+ by loading these ions into the xylem. Similar results were also reported in wheat TaHMA2. However, it seems that OsHMA2 in rice has a different role. The enhanced sensitivity to Cd and tolerance to zinc deprivation afforded by heterologous expression of OsHMA2 in yeast cells suggest that OsHMA2 functions as a Cd influx transporter. These studies showed that HMA2 and its subfamily members in different plants may function differently. There is a lack of thorough knowledge of the role of BrpHMA2 in Cd hyper accumulation in the leafy vegetable B. parachinensis. The function of BrpHMA2 and the mechanisms that regulate its expression must be elucidated. Previous studies have indicated that plants employ a universal and conserved approach to regulate the transcription of heavy metal uptake and tolerance genes. For example, in a bean , PvMTF-1 , which could be induced by PvERF15 , may regulate the expression of the stress-related gene PvSR2 and confer Cd tolerance to the plant.

In Arabidopsis, two basic helix–loop–helix transcription factors , FIT and PYE , modulate iron deficiency responses by regulating the expression of IRT1 and FRO2, whereas the bHLH TFs IAA-leucine resistant 3 and bHLH104 can form heterodimers and bind to specific elements in the promoter of PYE to regulate PYE. NAC TFs are members of the most prominent TF families in plants. These TFs play essential roles in diverse biological processes, such as growth, development, senescence, and morphogenesis, and are widely involved in various signaling pathways in response to different phytohormones and multiple abiotic and biotic stresses. For example, NAC019, NAC055, and NAC072 negatively regulate drought stress-responsive signaling. NAC096 is associated with drought stress. It could exert its function via a mechanism like that of basic leucine zipper protein -type TFs to bind specifically to abscisic acid – responsive elements in the promoters of several drought stress-responsive genes. This finding implies that NAC096 and bZIP-type TFs can sometimes regulate the same target genes. Studies have also shown that the core DNA-binding sequences of NACRE and ABRE are PyCACG and PyACGTGG/TC , respectively. In a previous study, we identified a few NAC and AREB TFs triggered by Cd stress in B. parachinensis. However, their functions remain unclear. To clarify the molecular mechanisms of Cd accumulation in B. parachinensis, the function of a Cd-responsive metal ion transporter gene BrpHMA2 and the coregulation of BrpHMA2 transcription by two TFs were examined in this study. The findings reveal a precise regulatory mechanism in B. parachinensis in response to Cd stress.We previously analyzed the Cd-induced mRNA transcriptome of B. parachinensis and found that several HMA homologs were substantially expressed under Cd stress. We cloned one of the HMA2 homologs and constructed the phylogenetic tree of this HMA2 homolog with other HMAs in A. thaliana, Oryza sativa, Zea mays, and Alfred stone crop by the neighbor-joining method using MEGA5. The results revealed that the sequence of this HMA2 homolog is closer to that of the AtHMA2 gene , and thus it was named BrpHMA2.

The transcript level of BrpHMA2 in seedlings grown hydroponically was examined using reverse transcription–quantitative PCR to investigate the expression pattern of BrpHMA2 in B. parachinensis. According to the results, BrpHMA2 was expressed at higher levels in leaves than in roots. Cd stress may increase BrpHMA2 expression in leaves and roots, although BrpHMA2 expression in leaves fluctuates owing to developmental regulation . The GUS gene was transformed and expressed in Arabidopsis using the promoter of BrpHMA2 to corroborate the expression pattern, and histochemical assays were performed. Instant β-glucuronidase staining for 0.5 hours showed that the GUS signal was visible in the vascular bundles of the leaves and roots of the plants treated with 50 μM Cd 2 for 2 days, but not in vascular bundles ofseedlings that were not treated with Cd . Results from an examination of transcripts of the GUS gene in the reporter line were also consistent with these findings . This showed that BrpHMA2 could be induced by Cd stress. However, when the pBrpHMA2::GUS transgenic seedlings were subjected to GUS staining for 3 hours, a strong GUS signal could be observed in the vascular bundles of the cotyledons, true leaves, stems, petals, filaments, and the carpopodium of the seeds in young siliques. The blue GUS signal was particularly strong in the tissue junction regions where the vascular bundles were clustered . These results indicate that BrpHMA2 may function primarily in transport in vascular tissues. The fluorescent signal of BrpHMA-GFP was detected at the plasma membrane by transient expression analysis in protoplasts of B. parachinensis leaf cells , indicating that BrpHMA2 is localized at the plasma membrane.To further analyze the function of BrpHMA2, BrpHMA2 fused with the galactose-inducible promoter was transformed into a Cd-hypersensitive yeast mutant, ycf. In the presence of the transcriptional inducer galactose, Cd2+ considerably inhibited the growth of yeast cells with heterologous expression of BrpHMA2 compared with that of cells transformed with the empty vector . However, when gene expression was suppressed by the presence of glucose, no growth differences were detected between the cells transformed with BrpHMA2 and those transformed with the empty vector. The Cd content in the heterologous transgenic cells grown in liquid medium was higher than that in the control cells . These results indicate that BrpHMA2 functions as an affluxtype Cd transporter.To determine the TFs responsible for BrpHMA2 expression in B. parachinensis, a cis-element analysis of 2000 bp of the BrpHMA2 promoter was performed. In the promoter region, three ABRE cis elements were identified,vertical farming equipment all of which contain the G-box family core sequence ACGT . The NAC recognition site CGTG is likewise present in these ABREs. In the promoter of BrpHMA2, two additional NAC recognition motifs, CDBS and CACG, were found. Three ABREs , four NACRESs, and four CDBS cis elements were found in the promoter of BrpHMA2 . These findings suggest that certain transcription factors, such as NACs or AREBs, may control BrpHMA2 in B. parachinensis via these cis elements. To confirm this deduction and identify the regulatory pathways involved in the response to Cd stress, the transcriptome of B. parachinensis as mentioned above was used to collect data for the NAC and AREB genes that showed differential expression following Cd stress. Eighteen NAC genes and 11 AREB genes were selected to create a heat map, and three NAC TFs and three AREB TFs were identified as Cd-induced TFs . Their transcription levels were further analyzed by RT–qPCR. The results showed that the NAC TF genes BraA03000895, BraA010004584, and BraA10002796 were upregulated in the roots of the plants exposed to Cd for 1 day . After 4 days of Cd exposure, the AREB TF gene BraA01000449 was induced in roots, and BraA05001227, BraA01000449, and BraA01003678 were induced in leaves . 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 .Electrophoretic mobility shift assays were conducted to investigate whether the BrpNAC895 protein directly binds to the promoter of BrpHMA2. Three probes containing NACRES and CBDS motifs on the BrpHMA2 promoter were designed and used for the EMSA. The results revealed that the BrpNAC895-MBP fusion protein could bind to the three probes in vitro . A chromatin immuno precipitation assay was performed using an anti-GFP antibody to precipitate BrpNAC895-GFP fusion proteins expressed in B. parachinensis protoplasts, and three fragments covering the NACRES and CBDS motifs on the BrpHMA2 promoter were designed and used for PCR. Moreover, there is only one base interval between the last two NACRES cis elements, so they were considered as one fragment . Approximately 1.5- to 2-fold enrichment of fragments pF1, pF2, and pF3 was detected compared with those found in the control . The results demonstrate that BrpNAC895 can promote the expression of BrpHMA2 by binding directly to the NACRES and CBDS motifs of its promoter.To investigate the mechanism of BrpHMA2 coregulation by BrpNAC895 and BrpABI449, a ChIP assay was performed by expressing BrpABI449-GFP in B. parachinensis protoplasts to analyze the binding affinity of BrpABI449 with the promoter of BrpHMA2. A qPCR analysis revealed that the BrpABI449 protein was enriched with fragments containing pF2 and pF3 of the BrpHMA2 promoter . We further performed an EMSA to confirm the binding of BrpABI449 to ABRE motifs in the promoter of BrpHMA2. The results proved that BrpABI449 could bind directly to the probes containing ABRE cis elements in the pF2 and pF3 regions of the BrpHMA2 promoter .The roles of BrpNAC895-binding loci in BrpHAM2 transcriptional regulation were investigated by constructing a series of BrpHMA2 promoter mutants by changing CACG/CGTG in the NACRES or CBDS to AAAA . A dual LUC assay was performed using the effector p35S::BrpNAC895 vector and the reporter vector was cotransformed into B. parachinensis protoplasts. Compared with pBrpHMA2::LUC, the cotransformation of p35S::BrpNAC895 with pMUT1::LUC or pMUT3::LUC resulted in much reduced LUC activity, but the cotransformation of the p35S::BrpNAC895 effector with pMUT2::LUC resulted in considerably higher LUC activity . Among the cotransformations of the promoter of BrpHMA2 with two or more mutations, substantially weaker LUC activity could only be seen in the transformations with promoters mutated at both locus 1 and locus 3 . These findings indicate that the mutation in the first and third NACRES motifs reduced the BrpNAC895-activated transcription of BrpHMA2, and these two binding loci may play central roles in the BrpNAC895-activated transcription of BrpHMA2.To elucidate the relationship between NAC and AREB TFs, a bimolecular fluorescence complementation approach was used.