Effects of root applied glutathione (GSH) on gene expression profile in the root of Brassica napus

Glutathione (GSH) is a tripeptide involved in controlling heavy metal movement in plants. Our previous study demonstrated that GSH, applied to plant roots site-specifically, inhibited Cd translocation from roots to shoots in oilseed rape plants (Brassica napus) cultured hydroponically. One of the factors of this inhibitory effect was due to activation of Cd efflux from root cells. To investigate the molecular mechanism triggered by root applied GSH in more detail, the Cd movement was monitored non-invasively using a positron-emitting tracer imaging system (PETIS). The Cd absorption and efflux process in roots were visualized successfully. The effects of GSH on Cd efflux from root cells were estimated by analyzing obtained imaging data. Another image analysis suggested that Cd return was activated by GSH, applied to roots, at the shoot base. Cutting the shoot base of oilseed rape plants significantly inhibited Cd efflux from root cells. These experimental results demonstrated the shoot base is playing important roles in distributing Cd in the plant bodies. Furthermore, DNA microarray analysis revealed that over 300 genes in the roots of oilseed rape plants responded to root applied GSH. Among them, transporter proteins, related to heavy metal movement in plants, and proteins related to changing the structure of cell walls were involved. Seeds of oilseed rape (Brassica napus L. cv. Nourin No.16, Kaneko Seed Co. Ltd., Gumma, Japan) were germinated in submerged vermiculite. The solution used for the bottom flooding is a nutrient solution for hydroponics. This solution is containing 0.5 mM NH4H2PO4, 3.0 mM KNO3, 2.0 mM Ca(NO3)2, 1.0 mM MgSO4, 90 µM Na2EDTA (ethylenediamine-N, N, N’, N’-tetra acetic acid disodium), 90 µM FeSO4, 22.5 µM H3BO3, 10 µM MnSO4, 0.1 µM (NH4)6Mo7O24, 0.35 µM ZnCl2, 0.20 µM CuCl2 (Nakamura et al., 2008). The pH of this solution was adjusted to 5.5 using KOH solution. After one week, plants were transferred to hydroponic culture condition in 3 L plastic containers (6 plants per one container) with 2 L of Hoagland solution and aeration. The Nutrient solution was freshly replaced once a week. 2 – 3 week old seedlings were used for each experiment. The plants were cultivated in 10 µM CdCl2-containing hydroponics solution with or without 100 mM GSH (glutathione, reduced form) for 2 days.