Figure 7 summarizes the postulated events leading to the inhibition of tomato pedicel abscission in the THyPRP-silenced plants. This sequence of events is based on regulatory genes whose expression was altered in the FAZ of the silenced plants at zero time and early after flower removal, compared to their expression in the WT FAZ. The role of these regulatory genes, which are active at different regulatory levels in the abscission process, was described and discussed above. The TAPG4 promoter was found to be a very good candidate for controlling gene silencing in the FAZ, since THyPRP expression in the silenced plants already decreased at zero time. Consequently, alternation in the expression of regulatory genes, including epigenetic modifiers, TFs, post-translational regulators, and transporters, already occurred before flower removal . We detected genes that were specifically upregulated in the WT FAZ at 4 h after flower removal, and their expression continued to increase later on for different periods . These upregulated genes, including genes related to ethylene biosynthesis, GA perception, TFs, post-translational regulation, and exocytosis, were inhibited in the FAZ of the THyPRP-silenced plants. Down regulation of the ethylene biosynthesis genes in the THyPRP-silenced plants probably leads to reduced ethylene production in the FAZ of these plants at 4 h, and to down regulation of other genes listed in Fig. 7b, which delay and inhibit the acquisition of the competence of the FAZ cells to respond to ethylene signaling. This inhibition could have resulted from altered expression of regulatory genes at zero time , or from the low expression of THyPRP during the 4-h period after flower removal . These two effects,vertical plant tower resulting from the THyPRP silencing, lead in turn to down regulation of the genes involved in abscission execution , finally resulting in the inhibition of the abscission phenotype .

Our data suggest that the effect of THyPRP silencing on pedicel abscission was not mediated by its effect on auxin balance, but by decreased ethylene biosynthesis and response. Additionally, THyPRP silencing revealed new players, which were demonstrated for the first time to be involved in regulating pedicel abscission processes. These include: GA-perception; Ca2+/CaM signaling; SERPINS and SUMO proteins involved in post-translational modifications; Synthaxin and SNARE-like proteins, which participate in exocytosis, necessary for cell separation. Taken together, our results suggest that THyPRP is a master regulator of pedicel abscission in tomato, predominantly by playing a role in the regulation of the FAZ competence to respond to ethylene signals.Cadmium is one of the major environmental pollutants and a potential hazard to worldwide agriculture. 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, which belong to the P1B subfamily of the P-type ATPase superfamily, 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+ . 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,indoor vertical farming 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 stonecrop 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 BrpHMA2to 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 of seedlings 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 investigate the physiological role of BrpHMA2 in plants, transgenic A. thaliana lines expressing BrpHMA2were generated. The Cd distribution and accumulation in seedlings of Col and BrpHMA2– over expressing lines were investigated. Dithizone staining showed that Cd was mainly located in the epidermal hairs of the leaves in both Col and OEBrpHMA2 seedlings, but more Cd-dithizone precipitates were found in the OE-BrpHMA2 lines .

Although Cd stress inhibited the growth of both Col and transgenic plants, the extent of growth inhibition in the OE-BrpHMA2 plants was stronger than in the Col plants after 6 days of Cd exposure . Moreover, Cd content assay revealed that the roots and shoots of the OE-BrpHMA2 plants had considerably more Cd than those of the Col plants .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, 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 .