Zinc And Cadmium Tolerance In Salix: A Search For The Role Of Polyphenols

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The enhanced metal accumulation and tolerance properties found in the Salix species have generated interest in using these plants in phytoremediation applications. However, little is known about the mechanisms behind these traits. Many aspects await elucidation, such as the molecular basis of metal uptake, tolerance and accumulation. In this investigation an attempt is made to shed light on a possible ligand involved in metal sequestration in willow plants. Polyphenols, abundant in willows, are examined as potential candidates. Already known for their roles in plant growth, development and defense, these compounds might be a part of the metal tolerance mechanism. In the first phase of the investigation, the potential role of polyphenols as ligands is explored by confirming their ability to chelate metals, namely Zn and Cd. Modeling exercises with representative phenolic moieties indicated that molecules with vicinal oxygens are more suitable chelators due to their ability to complex metal cations in a bidentate manner. A combination of acid titrations and infrared spectral analysis confirmed the ability of a representative polyphenol, tannic acid, to bind Zn and Cd over a physiologically relevant pH range. In the second phase of the investigation, the effect of metal exposure on phenolic levels in plants is explored; the hypothesis being that if phenols are a part of the mechanism that confers enhanced metal tolerance in Salix, metal exposure will induce an upregulation of these compounds as part of a defensive response. A total of 20 plants from three willow species, S. alba, S. viminalis and S-301, were subjected to one of two Zn and Cd treatments in a hydroponic setting for a period of two weeks. Leaf tissue was then harvested for analysis. Metal levels were determined via a methanol extraction of fresh leaf tissue and analysis with atomic absorption spectroscopy (AAS). Colorimetric methods were performed on dried leaf tissue to ascertain gross phenolics as well as levels of condensed tannins and leucoanthocyanins. Results showed that metal levels increased in plants as exposure concentrations increased but metal exposure appeared not to have a significant effect on the phenolic status of plants. However, these results do not necessarily disprove the above hypothesis. Small sample sizes as well as the considerable variations known to occur both between and within species could have obscured metal-induced changes in phenolic levels. Therefore, further investigation is warranted. More plants should be exposed to metals for longer periods of time with metal and phenol status assessed along the way. Moreover, assays should be employed to assess the levels of phenolics such as salicylates and hydrolyzable tannins. Pursuing additional avenues of investigation aside from phenolic quantification would also be sensible. A combination of size exclusion chromatography techniques coupled with UV-VIS or other detectors and X-ray absorption spectroscopy could go a long way to solving the mystery of which plant constituents are responsible for enhanced metal tolerance. In summary, while this examination did not yield conclusive results it provides a solid foundation for further investigation. The next steps briefly outlined above will determine if polyphenols involved in metal tolerance. If they are ruled out, the focus can shift to other compounds, such as organic acids. However, if it is shown that polyphenols do figure into the process then science will be one step closer to sorting out the metal tolerance mechanism in Salix.
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