Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsSeveral xylem sap proteins in Brassica napus are differentially induced in response to Cd treatment, and plant defensin plays a positive role in Cd tolerance.
Highlights
The xylem sap of vascular plants primarily transports water and mineral nutrients from the roots to the shoots and transports heavy metals such as cadmium (Cd)
Functional disruption of plant defensin decreased cd tolerance in Arabidopsis To further show that plant defensin plays a positive role in Cd tolerance, we generated two homozygous knockout mutants of plant defensin AtPDF2.2 and AtPDF2.3 using clustered regularly interspaced short palindromic repeats/ associated protein 9 (CRISPR/Cas9) technology (Fig. 7a)
The proteomic approach used in this study identified and quantified 672 proteins in the xylem sap of Brassica napus, which is considerably higher than the 69 proteins previously identified in 2-DE [6], though a recent study found 643 proteins in tomato xylem sap using a similar shotgun method [25], indicating that the shotgun labelfree analysis used in this study is very sensitive
Summary
The xylem sap of vascular plants primarily transports water and mineral nutrients from the roots to the shoots and transports heavy metals such as cadmium (Cd). Proteomic changes in xylem sap is an important mechanism for detoxifying Cd by plants. The accumulation of Cd in a plant shoot is mainly determined by the plant’s capacity to transport xylem sap long distances. Xylem sap proteomic research can be used to illustrate the impact of long-distance transport mechanisms in plants in response to Cd stress. The long-distance transport of xylem sap is mainly driven by transpiration and root pressure [13]. Proteomic studies of xylem sap in many plant species have been reported over the past three decades [14,15,16,17,18,19,20,21,22,23,24,25,26]. With regards to stress responses, in one study, the xylem sap proteome changed in response to iron and manganese deficiencies in tomato (Solanum lycopersicum) [25], and in another, nitrogen under- and over-supply induced distinct protein responses in the xylem sap of maize [26]
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