Impact of long-term heavy metals exposure in Salix viminalis roots

Abstract

A few years ago, it was observed that Salix viminalis grown in a soil polluted by a mixture of heavy metals were more winter-tolerant than plants grown in unpolluted soil. Salix aboveground organs being extremely frost-hardy, it was hypothesized that the difference in winter-tolerance was the consequence of an increased frost-tolerance at the root level. While this observation is intriguing, it is known that plant response to a combination of stress is unique and cannot be deduced a priori from the response to each of the stresses applied individually. The aim of this thesis was to investigate this metal-induced frost-tolerance to identify markers of frost-tolerance in the roots of woody perennials. The amount of publicly available molecular data on S. viminalis being scarce at the beginning of the thesis, the first objective was to identify reference genes stably expressed in the root of S. viminalis. Then, these genes were used to select the plants that were studied from a transcriptome and proteome point of view. Since regulation of the cellular redox homeostasis is an important component in plant stress responses and a potential candidate for the observed increase of root frost-hardiness, a particular emphasis was put on it. Contrary to our initial hypothesis, the simultaneous exposure to cold and metals did not seem to induce the antioxidative machinery. These results were confirmed by an analysis of different antioxidant enzymes and metabolites. However, proteins and transcripts related to the synthesis of lignans and sugars (especially raffinose) and proteins involved in oxidative damage repair mechanisms were found to be significantly more abundant in roots exposed to cold and metals compared to roots only exposed to one stress. Since these observations were made on plants grown under controlled conditions, a follow up study was done by growing S. viminalis cuttings outside during a full growth season and analysing their roots at several time points. The simultaneous exposure to cold and metals did not increase the root phenolic content or the antioxidative capacity. In addition, the simultaneous exposure to metals did not impede sucrose accumulation, a known metabolic response to cold, in the cold-acclimated roots. During the work presented here it was also observed that while at the individual gene level exposure to multiple stresses gave results that cannot be predicted from studies using single stress exposure, this is much less the case at the gene-ontology level. While needing confirmational studies with different stress combinations, such approach has the potential to accelerate molecular studies on the effects of multiple stress exposure. Based on the different experimental setups a small number of genes was identified that show a synergetic effect in plants exposed to both stresses. These are potential candidates for breeding efforts to produce plants with an increased resistance to cold at the root level. Further study is however needed to confirm this.