Plant materials, growth conditions, and agrochemical treatments
The promoter regions of MLP genes were cloned, and MLP gene pro (MLP-PG1, MLP-GR3, MLP-GR3-1, or MLP-GR3-2)::β-glucuronidase (GUS) fusion was each transformed into tobacco (Nicotiana tabacum) plants, as described previously (Fujita et al. 2020a, 2022). The sterile seeds were sown on Murashige and Skoog medium and incubated overnight at 4 °C in the dark, and the seedlings were cultivated under a 16/8 h light/dark cycle at 24 °C.
The seed coat of C. pepo subspecies pepo cultivars ‘Black Tosca’ (BT) (Sakata Seed Corp., Ltd., Kanagawa, Japan) and ‘Raven’ (RA) (Johnny's Selected Seeds, Albion, ME, USA) was peeled off. The seeds were incubated overnight in tap water at 4 °C and sown in a glass jar supplemented with 60 or 180 g of sterile soil (Hyponex Japan Corp., Ltd., Osaka, Japan) containing 0.125 mmol/kg or 1.25 mmol/kg pyrene.
The agrochemicals (Getter® [Nippon Soda Co., Ltd., Tokyo, Japan], Theek® [Mitsui Chemicals Crop & Life Solutions, Inc., Tokyo, Japan], Betofighter® [Nippon Soda], Masterpiece® [Nisso Green Co., Ltd., Tokyo, Japan], Trigard® [Syngenta Crop Protection, Tokyo, Japan], Diana SC® [Sumitomo Chemical Co., Ltd., Tokyo, Japan], Movent® [Bayer CropScience, Tokyo, Japan], Chess® [Syngenta Crop Protection], Basilex® [SDS Biotech K.K., Tokyo, Japan], Tomato-tone® [Sumitomo Chemical Garden Products Inc., Tokyo, Japan], and Jasmomate® [Mitsui Chemicals Crop & Life Solutions, Inc.]) were treated at the recommended timing at the recommended dose. The agrochemical Oryzemate® (Mitsui Chemicals Crop & Life Solutions, Inc.) was treated at the recommended number of times with doses (one-three hundredths the recommended dose, one-two hundredths the recommended dose, one-hundredth the recommended dose, one-fiftieth the recommended dose, one-eight the recommended dose, one-fourth the recommended dose, one-second the recommended dose, or the recommended dose).
Collection of the xylem sap and roots
The xylem sap of three-week-old C. pepo ssp. pepo cultivars BT and RA was collected as described in our previous report (Inui et al. 2013). The stem was cut below the cotyledons, a glass tube was inserted into the stem, and it was left overnight. Xylem sap was stored at 4 °C. The roots were washed with tap water to remove the soil particles and stored at −80 °C.
GUS assay
The 13-day-old transgenic tobacco seedlings with MLP-GR3 pro::GUS were transferred to tap water and incubated at 24 °C overnight. The seedlings were transferred to tap water containing 5 µg/mL, 50 µg/mL, and 500 µg/mL agrochemical active ingredients (probenazole, imidacloprid, fipronil, dieldrin, and CuCl2) in a 1% dimethyl sulfoxide (DMSO) solution and incubated under a 16/8 h light/dark cycle at 24 °C. The 14-day-old transgenic tobacco seedlings with MLP-PG1, MLP-GR3, MLP-GR3-1, or MLP-GR3-2 pro::GUS were transferred to deionized water and incubated at 24 °C overnight. The seedlings were transferred to deionized water containing 25, 50, and 75 µg/mL probenazole in 1% DMSO solution and incubated under a 16/8 h light/dark cycle at 24 °C. After 48 hours, the roots were collected and stored at −80 °C until the time of use. Root proteins were extracted by homogenization in protein extraction buffer (50 mM sodium phosphate buffer [pH 7.0], 10 mM EDTA [pH 8.0], 0.1% [v/v] Triton X-100, 0.5% [w/v] N-lauroylsarcosine sodium salt, and 0.072% [v/v] 2-mercaptoethanol). After centrifugation at 20,700 ×g for 5 min at 4 °C, the supernatants were collected and centrifugated at 20,700 ×g for 5 min at 4 °C again. The concentrations of root proteins in the supernatant were quantified using the Bradford method (Bradford 1976). The supernatant was subjected to a GUS assay as described previously (Fujita et al. 2020a).
Western blot analysis
Western blotting was performed as previously described (Goto et al. 2019). Protein extraction buffer was added to homogenize the roots of C. pepo BT and RA plants. After the centrifugation at 20,700 ×g for 5 min at 4 °C, the supernatant was collected and centrifugated at 20,700 ×g for 5 min at 4 °C again. The concentration of root proteins in the supernatant was quantified using the Bradford method. After adding the sample buffer solution with a reducing reagent for SDS-PAGE (Nacalai Tesque, Inc., Kyoto, Japan) and heat treatment, the samples were subjected to SDS-PAGE on a 15% acrylamide gel. Separated proteins were transferred onto polyvinylidene difluoride membranes. These were incubated in TTBS buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% [v/v] Tween 20) supplemented with 1% BSA and incubated with anti-MLP-PG1 and anti-MLP-GR3 antibodies (Medical & Biological Laboratories Co., Ltd., Aichi, Japan) in TTBS buffer. After washing thrice with TTBS, the membranes were incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (Proteintech, Rosemont, IL, USA) in TTBS. After washing with TTBS buffer thrice, the bands were detected by the reaction with 75 μg/mL nitroblue tetrazolium and 50 μg/mL 5-bromo-4-chloro-3'-indolyl phosphate p-toluidine salt in AP9.5 buffer (10 mM Tris−HCl [pH 9.5], 100 mM NaCl, 5 mM MgCl2). Band intensities were quantified using the ImageJ software version 1.53n (Schneider et al. 2012).
Quantification of pyrene
The pyrene concentration in the xylem sap was quantified as previously described (Fujita et al. 2020a). The 50 µL of xylem sap from C. pepo ssp. pepo cultivars BT and RA plants was mixed with 50 µL of DMSO in a 96-well black microtiter plate. Pyrene fluorescence was measured using a microplate reader (Microplate Reader SH-9000; Corona Electric Co., Ltd., Ibaraki, Japan) at excitation and emission wavelengths of 330 and 390 nm, respectively.
Competitive binding assay
Recombinant MLP-PG1 and MLP-GR3 were purified as described in our previous study (Fujita et al. 2020b) and diluted with sodium phosphate buffer (50 mM sodium phosphate buffer [pH 7.0], 200 mM NaCl, and 150 mM imidazole) to a concentration of 4.8 µM. Probenazole (1, 5, and 10 mM) dissolved in DMSO and pyrene (1 mM) were dissolved in DMSO and mixed in equivalent quantities, and 1 µL of these mixtures was added to the 99 µL of MLP solutions. The concentrations of MLP, probenazole, and pyrene in the reaction solutions are 4.8 µM, 5, 10 or 20 µM, and 5 µM, respectively. These solutions were incubated at 25 °C for 60 min with continuous mixing, and pyrene fluorescence was measured as described above.
Statistical analysis
Dunnett’s multiple comparison test or Welch’s two-sample t-test were used to determine significant differences among treatments using RStudio software version 2022.12.0+353 (http://www.rstudio.com).