Improving phytoextraction efficiency with Salix caprea

Heavy metal contaminated soils pose an increasing worldwide problem to agriculture and to human and animal health. Although heavy metals are generally disadvantageous for plants, some species are adapted to contaminated areas and accumulate heavy metals at high concentration in above ground organs. A promising, low cost and sustainable technology that uses metallophytes to clean up polluted sites is phytoextraction. However phytoextraction is slow and thus fast growing perennial plants are needed that tolerate and accumulate heavy metals in harvestable organs such as leaves and stems. In particular, energy crops of the Salicaceae (willow) family naturally colonize metallicolous sites are easy to propagate, develop an extensive root system and are able to transfer heavy metals such as Cd and Zn to the foliage. In a preceding project consortium we have characterized Salix caprea plants from metallicolous and non- metallicolous areas that fulfill the criteria of good Cd/Zn accumulators. The aim of this multidisciplinary project is to better understand the mechanisms that are responsible for this increased hyperaccumulation efficiency and tolerance. Using a range of different techniques we focus on following questions: i) where and how much of the heavy metals are stored in leaves and roots and ii) if the subcellular Cd/Zn allocation, iii) the root architecture, and iv) growth behaviour differ between isolates with opposing heavy metal accumulation efficiencies, tolerance and biomass production. Based on our previous results, we will further functionally characterize two genes that are specifically induced upon Cd/Zn exposure in a good accumulator isolate. Whereas for a homolog of one of these genes an involvement in mineral responses has been reported, nearly nothing is known for the other. To determine structure/function relationships we will establish growth/response assays that will further serve as tool to study its activity on other plant species. This multidisciplinary project aims to clarify why S. caprea is able to tolerate and accumulate Cd/Zn, how the root system is responding to Cd/Zn contaminations and where these metals are deposited in leaves. Furthermore, the functional characterization, usability and transferability of promising novel Cd/Zn responsive genes will provide the basis for further developments to improved phytoextraction technologies.

The overall aim of the project was to improve our understanding of the structural and molecular basis that influences heavy metal accumulation of the goat willow Salix caprea Thus genotypes previously characterized to exhibit contrasting Zn and Cd accumulation capacities were used to determine root architecture, the localization of metals in leaves and roots and the identification of genes induced by Zn and/or Cd. The distribution of heavy metals was determined using a X-ray microanalysis for which a reliable fixation method was established. The results uncovered different strategies against toxic elements in genotypes isolated from polluted and unpolluted sites. In roots, Zn, Ca, Mg, Na and Si were enriched in the peripheral bark, K and S in the phloem and Cd in both vascular tissues. Si levels were lower in the superior Cd translocator genotype. In leaves, higher concentrations were found in the lamina and the midrib than in hairs. In stems, the primary xylem contained more heavy metals than the secondary xylem. Increased concentrations of heavy metals were also found in the pith, the cortex and the epidermis. X-ray dotmapping identified separted Ca and Si rich crystals in leaves and stem sections. Furthermore the development of apoplastic barriers and tissue organization in roots might reflect an adaptive predisposition of genotypes from different natural origins. Furthermore S caprea did not show preferential root growth towards heavy metal polluted patches and thus no avoidance of polluted patches could be observed suggesting that the high Cd and Zn uptake efficiency may be related to physiological rather than root foraging processes.Expression analyses in leaves revealed 213 genes that were in the good accumulator genotype. Among them were components of the cell wall and apoplast. Three of these genes were cloned and constitutively overexpressed in the model plant Arabidopsis thaliana. Detailed phenotypic analyses revealed for two of them the predicted plasma membrane/cell wall associated localization. Furthermore, Arabidopsis seedlings expressing these genes exhibited growth phenotypes upon heavy metal exposure supporting their role in heavy metal responses. One of the genes was recombinantely expressed and biochemically characterized. The purified protein exhibited binding abilities to different heavy metals. Since this gene/protein is novel and no close homologs were identified in most other sequenced plant genomes, the BOKU-University of Natural Resources and Life Sciences considered a patent application. Most of these data have been published now.A continuation project has been submitted in June 2013 to the FWF for evaluation. This project would allow a detailed functional and biochemical characterization of the previously detected genes/proteins. Furthermore the expression analysis will be extended to the full genome level with state of the art next generation sequences techniques. With this approaches molecular pathways should be revealed that are necessary to support the high tolerance and accumulation capability of S. caprea upon long term exposure to heavy metals a condition relevant for phytoextraction strategies.