Root architecture and morphometric analysis of Arabidopsis thaliana grown in metal-gradient agar dishes

This study aims at elucidating root reactions to the toxic heavy metals cadmium, copper and zinc at sub-toxic concentrations (10 μM CdSO4, 5 μM CuSO4, and 150 μM ZnSO4). For this purpose, we devised a new screening strategy using Petri dishes (12 x 12 cm) with a gradient of distances between germinating seeds and a metal-contaminated medium concentrations in order to study alterations in root architecture and morphology of Arabidopsis thaliana (L.) Heinh. (Columbia ecotype; Col-0) when treated with each metal alone (Cd, Cu, Zn) or in triple combination (Cd/Cu/Zn). Each dish was filled with 100 mL melted agarized medium (bacteriological agar supplemented with 0.5% sucrose and 1/4 strength Murashige and Skoog liquid medium without micronutrient and vitamins) + metal . After the medium solidified, the gel was cut diagonally under sterile conditions and the upper half discharged. Then, a metal-free melted medium was poured into the dish in order to fill the empty space. After the second solidification step, top agar (1.5 cm from the upper border) was removed to allow shoot development. Control dishes without metals were kept as controls. Agar final thickness was 0.4 cm. Seeds were sterilized in sodium hypochlorite and let to germinate (eight seeds per plate) at the top of the dishes. To simulate the dark conditions of the soil, the top agar surface (0.3 mm) was covered with activated carbon and the rest of the dish surface covered with a dark foil. Dishes were kept in a growth chamber under controlled conditions. After two weeks, all agar dishes were scanned at high resolution by an image analysis system and the whole root systems analysed. For each plant, the following morphological measurements were carried out: total length of the root system, length of primary root and lateral roots, number of root tips, average root diameter, root density, root growth angle and slope. Metal concentration in the dishes was determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES) on digested agar samples collected along the gradient, and a diffusion coefficient for each metal was calculated. In the presence of all metals, and of Cd in particular, primary root length significantly decreased compared to controls whereas root system total length increased due to the higher root branching. This was confirmed by the significantly higher number of root tips in metal-treated seedlings. The seedlings nearer to the areas with agar + metals showed a marked curvature and a higher root branching. This behavior, together with an observed increase in root diameter in metal-treated seedlings may be interpreted as compensatory growth, and a thicker roots could act as a barrier to protect root from the metals. We therefore propose that the remodelling of the root architecture in response to metals could be a pollution “escaping” strategy aimed at seeking metal-free patches.