Calamine wastes highly contaminated with trace metals (TMs) are spontaneously inhabited by a legume plant Anthyllis vulneraria L. This study determined an adaptation strategy of metallicolous (M) A. vulneraria and compared it with that of the non-metallicolous (NM) ecotype. We hypothesized that TMs may lead to (i) leaf apoplast modifications and (ii) changes in the antioxidant machinery efficiency that facilitate plant growth under severe contamination. To verify our hypothesis, we implemented immunolabelling, transmission electron microscopy and biochemical measurements. NM leaves were larger and thicker compared to the M ecotype. Microscopic analysis of M leaves showed a lack of dysfunctions in mesophyll cells exposed to TMs. However, changes in apoplast composition and thickening of the mesophyll and epidermal cell walls in these plants were observed. Thick walls were abundant in xyloglucan, pectins, arabinan, arabinogalactan protein and extensin. The tested ecotypes differed also in their physiological responses. The metallicolous ecotype featured greater accumulation of photosynthetic pigments, enhanced activity of superoxide dismutase and increased content of specific phenol groups in comparison with the NM one. Despite this, radical scavenging activity at the level of 20% was similar in M and NM ecotypes, which may implicate effective reduction of oxidative stress in M plants. In summary, our results confirmed hypotheses and suggest that TMs induced cell wall modifications of leaves, which may play a role in metal stress avoidance in Anthyllis species. However, when TMs reach the protoplast, activation of antioxidant machinery may significantly strengthen the status of plants naturally growing in TM-polluted environment.
Keywords: antioxidants; apoplast; calamine; cell wall; kidney vetch; trace metals.