Major latex-like proteins show pH dependency in their binding to hydrophobic organic pollutants

Abstract

The Cucurbitaceae family accumulates hydrophobic organic pollutants in its aerial parts at high concentrations. Major latex-like proteins (MLPs) were identified in zucchini (Cucurbita pepo) as a transporting factor for hydrophobic organic pollutants. MLPs bind to hydrophobic organic pollutants in the roots, are secreted to xylem vessels as complexes, and are transported to the aerial parts. However, the suitable conditions for binding MLPs to hydrophobic organic pollutants remain elusive. In the present study, we show that MLPs bind to the hydrophobic organic pollutant pyrene with higher affinity under acidic conditions. Our results demonstrated that pH regulates the binding of MLPs to hydrophobic organic pollutants.

Keywords: Cucurbita pepo; acidity; crop contamination; hydrophobic organic pollutants; major latex-like protein.

Fig. 1. Major latex-like proteins (MLPs) are detected in the xylem and phloem sap of Cucurbita pepo. C. pepo subspecies ovifera cultivar ‘Starship’ (ST) and ssp. pepo cv. ‘Black Tosca’ (BT) were cultivated under a 16/8 hr light/dark cycle at 26°C for three weeks. (A) Xylem and (B) phloem sap were collected and boiled in sample buffer solution, and the mixture was subjected to SDS-PAGE. Bands were detected using Coomassie Brilliant Blue (CBB) staining. Western blot analysis was performed by the reaction with the anti-MLP–PG1 or anti-MLP–GR3 antibodies, and bands were detected. The different lanes showed biological replicates.

Fig. 2. Major latex-like proteins (MLPs) bind to 1-pyrenecarboxylic acid under acidic conditions. (A) Immobilization reaction of 1-pyrenecarboxylic acid to NH₂ magnetic beads. (B) Binding assay of recombinant MLPs with 1-pyrenecarboxylic acid. 1-Pyrenecarboxylic acid immobilized with NH₂ magnetic beads reacted with recombinant MLP–PG1 and MLP–GR3 in the buffer with different pH (5.6, 7.2, and 8.8) at 4°C for 4 hr. After washing eight times by magnetic separation, the supernatant from the eighth wash was collected. The buffer and sample buffer for SDS-PAGE were added to the beads, and the solution was boiled at 98°C for 5 min. After magnetic separation, the supernatant was collected as heat elution. The samples were subjected to SDS-PAGE, and bands were detected by silver staining.

Fig. 3. Fluorescence of pyrene mixed with major latex-like proteins (MLPs) increased under acidic condition. (A) Increase in the fluorescence of pyrene through the binding of MLPs to pyrene. (B) Pyrene fluorescence assay mixed with recombinant MLPs. Recombinant MLP–PG1 and MLP–GR3 (0.2 mg/mL) diluted in the buffer with different pH (5.6, 7.2, and 8.8) was mixed with 0, 10, or 20 µM pyrene dissolved in dimethyl sulfoxide at room temperature, and the pyrene fluorescence was measured.