Distribution of cadmium and induced Cd-binding proteins in roots, stems and leaves of Phaseolus vulgaris

Abstract

Roots, stems and leaves of Phaseolus vulgaris L. (cv. Rubino PF1H) grown in Hoagland's solution supplemented with 1, 2 and 2.5 mM Cd(NO 3) 2 were analyzed. In comparison with control plants grown in a nutrient solution containing equivalent amounts of NO 3 − added as KNO 3, plants grown in the presence of 1 mM Cd(NO 3) 2 showed a significant decrease in fresh weight and per cent dry matter in roots, whereas stems were slightly affected and leaves not at all. At 2 and 2.5 mM Cd concentrations the fresh weight of roots was unaffected, but their dry matter content was strongly reduced; stems showed significant, even limited decrease of both fresh weight and % dry matter. In comparison, the fresh weight and foliar area of leaves were strongly reduced, the dry matter content being unaffected. The distribution of Cd in plant tissues showed that total Cd in roots exceeded by about one and two orders of magnitude total Cd of stems and leaves, respectively. Intercellular spaces of plant tissues were washed successively with water, 5 mM CaCl 2 and 5 mM EDTA. Results showed that most of Cd in the apoplast of root, stem and leaf tissues was extractable by complexing it with EDTA. Water extractable Cd in intercellular spaces was present in ionic form, as Cd 2+. Gel filtration of tissue extracts showed that 83.4% of total Cd was present as free metal ion in extract of leaves, whereas 56.6% and 48.7% was found in stem and roots extracts, respectively. The remaining part of the total Cd was associated with protein fractions. One type of Cd-protein fraction of about 10 kDa molecular weight ( K av 0.54) was present in roots, stems and leaves, binding 24.1%, 43.4% and 16.6% of total Cd, respectively. A second protein fraction with apparent mol. wt. > 30 kDa was present only in roots, binding 27.2% of total root Cd. This result was confirmed by SDS-PAGE electrophoresis, showing a Cd-induced protein band common to leaves, stems and roots with an apparent mol. wt. 9.2 kDa, which can be interpreted as phytochelatins, and an intensively stained Cd-induced band, present only in root extracts, of about 42 kDa apparent mol. wt.