Copper microlocalisation, ultrastructural alterations and antioxidant responses in the nodules of white lupin and soybean plants grown under conditions of copper excess

Abstract

Copper (Cu) is a heavy metal which, at high concentrations, is toxic to organisms. Some plants, however, possess systems for dealing with excess Cu, such as its cell localisation, and have antioxidant enzymes that help to reduce the oxidative stress. The present work examines the microlocalisation of Cu and the antioxidant enzyme activity present in the nodules of white lupin and soybean plants grown hydroponically for 35 days in the presence of 1.6μM (control) or 192μM (excess) Cu. The effect of these conditions on nodule (ultra)structure was also examined. Energy-dispersive X-ray microanalysis showed the cell walls to be the main area of Cu binding in the inner and outer cortex and infected zone of white lupin nodules grown under the excess Cu conditions, while in soybean a high Cu signal was detected inside cells (cytoplasm or vacuoles) both in the inner cortex and infected zone. At the tissue level, an increasing Cu gradient was seen from the outer towards the inner nodule cortex in white lupin nodules, while the opposite was seen in soybean. Cu excess also induced oxidative stress and promoted damage to the ultrastructure of nodules. In the white lupin infected cells, a breakdown of the peribacteroidal membrane was seen, along with an increased number of vesicles in the cytosol of these cells. In the infected cells of the soybean nodules, the bacteroidal membrane became degraded and precipitation was seen within the vacuoles of the infected and uninfected cells. Finally, the white lupin nodules seemed to be more sensitive to Cu excess than those of soybean, with the nodulation process, N2 fixation, and the ultrastructure of bacteroids more strongly affected. A less effective antioxidative stress response against Cu was also seen in white lupin than in soybean nodules: the excess copper treatment induced a smaller increase in the total thiol content and ascorbate peroxidase activity in white lupin nodules than in soybean nodules, and promoted a greater reduction in catalase activity.