A major latex-like protein is a key factor in crop contamination by persistent organic pollutants.

This is the first report, to our knowledge, to reveal important factors by which members of the Cucurbitaceae family, such as cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo), pumpkin (Cucurbita pepo), squash (C. pepo), and zucchini (C. pepo), are selectively polluted with highly toxic hydrophobic contaminants, including organochlorine insecticides and dioxins. Xylem sap of C. pepo ssp. pepo, which is a high accumulator of hydrophobic compounds, solubilized the hydrophobic compound pyrene into the aqueous phase via some protein(s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of xylem sap of two C. pepo subspecies revealed that the amount of 17-kD proteins in C. pepo ssp. pepo was larger than that in C. pepo ssp. ovifera, a low accumulator, suggesting that these proteins may be related to the translocation of hydrophobic compounds. The protein bands at 17 kD contained major latex-like proteins (MLPs), and the corresponding genes MLP-PG1, MLP-GR1, and MLP-GR3 were cloned from the C. pepo cultivars Patty Green and Gold Rush. Expression of the MLP-GR3 gene in C. pepo cultivars was positively correlated with the band intensity of 17-kD proteins and bioconcentration factors toward dioxins and dioxin-like compounds. Recombinant MLP-GR3 bound polychlorinated biphenyls immobilized on magnetic beads, whereas recombinant MLP-PG1 and MLP-GR1 did not. These results indicate that the high expression of MLP-GR3 in C. pepo ssp. pepo plants and the existence of MLP-GR3 in their xylem sap are related to the efficient translocation of hydrophobic contaminants. These findings should be useful for decreasing the contamination of fruit of the Cucurbitaceae family as well as the phytoremediation of hydrophobic contaminants.

A20/AN1 zinc-finger proteins positively regulate major latex-like proteins, transporting factors toward dioxin-like compounds in Cucurbita pepo.

The Cucurbitaceae family accumulates dioxin-like compounds in its fruits. We previously showed that A20/AN1 zinc finger protein (ZFP) genes were highly expressed in the zucchini (Cucurbita pepo) subspecies pepo, which accumulates dioxin-like compounds at high concentrations. Transgenic tobacco (Nicotiana tabacum) plants overexpressing A20/AN1 ZFP genes show accumulation of dioxin-like compounds in their upper parts. However, the mechanisms underlying the accumulation of dioxin-like compounds regulated by the A20/AN1 ZFPs remain unclear. Here, we show that A20/AN1 ZFPs positively regulate the expression of the major latex-like protein (MLP) and its homolog genes in N. tabacum and C. pepo. MLPs are involved in the transport of dioxin-like compounds from the roots to the upper parts of C. pepo. Overexpression of A20/AN1 ZFP genes in N. tabacum leads to the upregulation of pathogenesis-related protein class-10 genes with the binding ability toward dioxin-like compounds. Our results demonstrated that A20/AN1 ZFPs upregulate MLP and its homolog genes in N. tabacum and C. pepo, resulting in the accumulation of dioxin-like compounds.

High temperatures promote the uptake of hydrophobic pollutants by Cucurbita pepo via altered gene expression levels of major latex-like proteins.

Cucurbitaceae family members are accumulators of hydrophobic pollutants. Such pollutants have been detected in cucurbits at levels above the maximum residue limit. Since major latex-like proteins (MLPs) are involved in hydrophobic pollutant uptake, changes in MLP expression can increase or decrease contamination. MLP expression levels were altered in the roots of Cucurbita pepo 'Magda,' and MLP-PG1 was detected in the xylem sap of Magda when cultivated at a high temperature (35°C). Day length also influenced MLP expression levels but only induced minor changes in the amount of MLPs. The concentration of pyrene, a hydrophobic pollutant, significantly increased with increasing MLP levels in the xylem sap of Magda when cultivated at 35°C. Thus, high temperatures promote the pollution of cucurbits by hydrophobic pollutants. These results can be used to develop novel techniques to reduce crop contamination and establish efficient phytoremediation.

Factors regulating the differential uptake of persistent organic pollutants in cucurbits and non-cucurbits.

Contamination with persistent organic pollutants (POPs) has become a worldwide concern owing to their the toxicity to humans and wildlife. Pumpkin, cucumber, and squash (Cucurbitaceae) accumulate POPs in their shoots in concentrations higher than those in non-cucurbits; to elucidate the underlying molecular mechanisms of this accumulation, POP transporters were analyzed in the xylem sap of cucurbits and non-cucurbits. The 17-kDa xylem sap proteins detected in all cucurbits but not in non-cucurbits readily bound polychlorinated biphenyl (PCB) in all tested cucurbits, except in cucumber and loofah, and to dieldrin in all tested cucurbits. Ten genes encoding major latex-like proteins (MLPs) responsible for the accumulation of PCBs in zucchini plants were cloned from cucurbits. Phylogenetic analysis using MLP sequences identified two separate clades, one containing Cucurbitaceae MLPs and the other containing those of non-cucurbit members. Recombinant MLPs bound PCB and dieldrin. Western blotting with anti-MLP antibodies identified translocatable and non-translocatable MLPs between root and stem xylem vessels. Translocation of MLPs from the root to stem xylem vessels and POP-binding ability of MLPs are important for selective accumulation of MLPs in cucurbits. This study provides basic knowledge about phytoremediation through overexpression of MLP genes and for breeding cucurbits that accumulate less contaminants.

Zinc finger protein genes from Cucurbita pepo are promising tools for conferring non-Cucurbitaceae plants with ability to accumulate persistent organic pollutants.

Some cultivars of cucumbers, melons, pumpkins, and zucchini, which are members of the Cucurbitaceae family, are uniquely subject to contamination by hydrophobic pollutants such as the organohalogen insecticides DDT. However, the molecular mechanisms for the accumulation of these pollutants in cucurbits have not been determined. Here, cDNA subtraction analysis of Cucurbita pepo cultivars that are low and high accumulators of hydrophobic contaminants revealed that a gene for zinc finger proteins (ZFPs) are preferentially expressed in high accumulators. The cloned CpZFP genes were classified into 2 types: (1) the PBG type, which were expressed in C. pepo cultivars Patty Green, Black Beauty, and Gold Rush, and (2) the BG type, which were expressed in Black Beauty and Gold Rush. Expression of these CpZFP genes in transgenic tobacco plants carrying an aryl hydrocarbon receptor-based inducible gene expression system significantly induced ß-glucuronidase activity when the plants were treated with a polychlorinated biphenyl (PCB) compound, indicating that highly hydrophobic PCBs accumulated in the plants. In transgenic tobacco plants carrying CpZFPs, accumulation of dioxins and dioxin-like compounds increased in their aerial parts when they were cultivated in the dioxin-contaminated soil. In summary, we propose that addition of CpZFP genes is a promising tool for conferring noncucurbits with the ability to accumulate hydrophobic contaminants.