Cadmium (Cd) is a toxic metal element and the mechanism(s) underlying Cd tolerance in plants are still unclear. Increasingly more studies have been conducted on Cd binding to plant cell walls (CW) but most of them have focused on Cd fixation by CW pectin, and few studies have examined Cd binding to cellulose and hemicellulose. Here we found that Cd binding to CW pectin, cellulose, and hemicellulose was significantly higher in Tor-1, a Cd tolerant A. thaliana ecotype, than in Ph2-23, a sensitive ecotype, as were the concentrations of pectin, cellulose, and hemicellulose. Transcriptome analysis revealed that the genes regulating CW pectin, cellulose, and hemicellulose polysaccharide concentrations in Tor-1 differed significantly from those in Ph2-23. The expressions of most genes such as pectin methyl esterase inhibitors (PMEIs), pectin lyases, xyloglucan endotransglucosylase/hydrolase, expansins (EXPAs), and cellulose hydrolase were higher in Ph2-23, while the expressions of cellulose synthase-like glycosyltransferase 3 (CSLG3) and pectin ethyl esterase 4 (PAE4) were higher in Tor-1. The candidate genes identified here seem to regulate CW Cd fixation by polysaccharides. In conclusion, an increase in pectin demethylation activity, the higher concentration of cellulose and hemicellulose, regulated by related genes, in Tor-1 than in Ph2-23 are likely involved in enhanced Cd CW retention and reduce Cd toxicity.

The xylem sap of vascular plants primarily transports water and mineral nutrients from the roots to the shoots and also transports heavy metals such as cadmium (Cd). Proteomic changes in xylem sap is an important mechanism for detoxifying Cd by plants. However, it is unclear how proteins in xylem sap respond to Cd. Here, we investigated the effects of Cd stress on the xylem sap proteome of Brassica napus using a label-free shotgun proteomic approach to elucidate plant response mechanisms to Cd toxicity. We identified and quantified 672 proteins; 67% were predicted to be secretory, and 11% (73 proteins) were unique to Cd-treated samples. Cd stress caused statistically significant and biologically relevant abundance changes in 28 xylem sap proteins. Among these proteins, the metabolic pathways that were most affected were related to cell wall modifications, stress/oxidoreductases, and lipid and protein metabolism. We functionally validated a plant defensin-like protein, BnPDFL, which belongs to the stress/oxidoreductase category, that was unique to the Cd-treated samples and played a positive role in Cd tolerance. Subcellular localization analysis revealed that BnPDFL is cell wall-localized. In vitro Cd-binding assays revealed that BnPDFL has Cd-chelating activity. BnPDFL heterologous overexpression significantly enhanced Cd tolerance in E. coli and Arabidopsis. Functional disruption of Arabidopsis plant defensin genes AtPDF2.3 and AtPDF2.2, which are mainly expressed in root vascular bundles, significantly decreased Cd tolerance. Several xylem sap proteins in Brassica napus are differentially induced in response to Cd treatment, and plant defensin plays a positive role in Cd tolerance.

The vacuole is an important organelle for nitrate storage, and reuse of vacuolar nitrate under nitrate starvation helps plants adapt to low-nitrate environments. CHLORIDE CHANNEL-b (CLC-b) in the vacuolar membrane is a nitrate transporter; however, its regulation and effects on nitrate efflux have not been established. Here, we evaluated CLC-b expression and its effects on physiological parameters under low nitrate conditions. CLC-b expression increased significantly in the roots of wild-type Arabidopsis (Arabidopsis thaliana) Col-0 under nitrate starvation. Under low nitrate, clcb mutants showed reductions in chlorophyll content and xylem sap nitrate concentration, shoot/root nitrate ratios, shoot/root total N ratios, and biomass. CLC-b overexpression yielded opposite phenotypes and increased nitrogen use efficiency. CLC-b mutants showed elevated chlorate tolerance and increased proportion of vacuolar nitrate relative to the total protoplast nitrate content as compared to the wild type. Yeast one-hybrid, EMSA, and ChIP experiments showed that HRS1 HOMOLOG2 (HHO2), the expression of which is downregulated under low nitrate, binds directly to the promoter of CLC-b. clcb/hho2 double mutants and HHO2-overexpressing clcb plants had similar phenotypes under low nitrate to those of clcb single mutants. Thus, CLC-b mediates vacuolar nitrate efflux and is negatively regulated by HHO2, providing a theoretical basis for improving plant adaptability to low nitrate.

Environmental acclimation ability plays a key role in plant growth, although the mechanism remains unclear. Here, we determined the involvement of Arabidopsis thaliana PLANT DEFENSIN 1 gene AtPDF1.5 in the adaptation to low nitrogen (LN) levels and cadmium (Cd) stress. Histochemical analysis revealed that AtPDF1.5 was mainly expressed in the nodes and carpopodium and was significantly induced in plants exposed to LN conditions and Cd stress. Subcellular localization analysis revealed that AtPDF1.5 was cell wall- and cytoplasm-localized. AtPDF1.5 overexpression significantly enhanced adaptation to LN and Cd stress and enhanced the distribution of metallic elements. The functional disruption of AtPDF1.5 reduced adaptations to LN and Cd stress and impaired metal distribution. Under LN conditions, the nitrate transporter AtNRT1.5 expression was upregulated. Nitrate transporter AtNRT1.8 expression was downregulated when AtPDF1.5 was overexpressed, resulting in enhanced transport of NO₃⁻ to shoots. In response to Cd treatment, AtPDF1.5 regulated the expression of metal transporter genes AtHMP07, AtNRAMP4, AtNRAMP1, and AtHIPP3, resulting in higher Cd accumulation in the shoots. We conclude that AtPDF1.5 is involved in the processing or transmission of signal substances and plays an important role in the remediation of Cd pollution and LN adaptation.

Background: In plants, ammonium metabolism is particularly important for converting absorbed nitrogen into amino acids. However, the molecular mechanism underlying this conversion remains largely unknown. Results: Using wild type Arabidopsis thaliana (Col-0) and AtPDF2.1 mutants (pdf2.1–1 and pdf2.1–2), we found that the small cysteine-rich peptide AtPDF2.1, a plant defensin, is involved in regulating ammonium metabolism in the shoot. Ammonium significantly induced the expression of AtPDF2.1 in the shoot and root, particularly in root xylem vascular bundles, as demonstrated by histochemical analysis. Subcellular localization analysis revealed that AtPDF2.1 was localized to the cell wall. Ammonium concentration was higher in the shoot of mutants than in the shoot of Col-0, but no differences were found for total nitrogen content, root ammonium concentration, and the expression of the ammonium transporter gene AtAMT2.1. The activity of glutamine synthetase was significantly decreased in mutants, and the glutamine synthetase family genes GLN1.3 and GLN1.5 were significantly downregulated in mutants compared to Col-0. The activity of nitrate reductase showed no difference between mutants and Col-0. Conclusions: Overall, these data suggest that AtPDF2.1 affects ammonium metabolism by regulating the expression of GLN1.3 and GLN1.5 through a yet unidentified mechanism.