Instantaneous measurement of fluorescence

Aricia Evlard subjected 41 willow clones from the cloning park of the CRA-W located at Grand-Manil (Gembloux), to a substrate contaminated with heavy metals. Instead of being satisfied with mere empirical observation (phenotypical) of their growth, she investigated the tolerance of the young trees to these pollutants (1).  For two years she used physiological methods which consist in measuring chlorophyll fluorescence, to measure the sugars and pigments present in the leaves and to identify the expression of proteins (known as a “proteomic” approach). “The more the plant becomes stressed by the metals the more it emits fluorescence. This fluorescence can be measured by a fluorometer, a technique which has the advantage of being non-destructive and delivering immediate results. Pigments and sugars also constitute parameters of stress which are interesting to study. During metal stress, the concentration in sugars can increase for example. This is linked to the roles sugars play in the protection of proteins and the maintenance of cellular activities”. Finally, the identification of certain proteins can indicate the presence of reactive oxygen species (ROS), oxygen free radicals that are more toxic than oxygen. “Under normal circumstances, there is always a minimum of ROS produced in the cells. The cell has defense mechanisms that can make them disappear before they can do any damage. These antioxidant defense mechanisms can be enzymatic (again, proteins) or non-enzymatic. However, when the plant is stressed, the ROS are formed more rapidly than they are eliminated. The result of this is that the ROS interact with the macromolecules (DNA, lipids and proteins) to the point of deactivating the antioxidant defense mechanisms. Indirectly, the metals cause various disorders of the cellular metabolism (loss of membrane integrity, altered photosynthesis, accelerated senescence, etc.)” The vital functions of the plant are therefore reduced or inhibited. Finally the plant in question dies.

After having subjected the 41 clones to these four examinations, it clearly emerged that one of them stands out from the rest. Here it is not important for the purposes of the demonstration which species or sub-species is identified. “The main thing is that this clone was not only part of those which produced the most biomass in the presence of metals, but also stood out from the others by having the highest levels of metals in its twigs. In reality, during a supplementary study, it became clear that it was the least tolerant to metal stress from a physiological and proteomics viewpoint. This study was also able to demonstrate that the good individual “phytoextractor” or the most tolerant individual is not neccessarily that which produces the most biomass”.

Accumulation and good health are not synonymous!

The main lesson learned from this research: a tree that “holds its own” against the metals, and which produces a lot of biomass, is not neccessarily in good health due to its weak tolerance to MTEs. For this reason, it is not necessarily a good phytoextractor in the long term. This fact is even more interesting when we consider that the duration of a short rotation coppice-the type of crop generally used for energy purposes-is at least twenty years.

Another observation made during this thesis: while the clones of willow tested at Gembloux seem less interesting from the point of view of remediating a polluted site within a reasonable timescale, they appear, on the other hand, to be more interesting to use in the context of phytostabilisation. “In fact they seem to be tolerant to the metals but slow down the transfer of these to the upper parts of the plant and concentrate them around the area of the rhizosphere. In other words, when this type of individual is planted, the metals are better “handled” (this has yet to be studied in situ) than in the absence of planting: the metals do not travel (or at least very rarely) towards the water tables.  Also, because the transfer of pollutants to the twigs and leaves is reduced, the contamination of the biomass is also reduced as well as the restitution of the metals due to leaf-fall”.

(1) For this purpose she is the beneficiary of assistance from the Gabriel Lippmann Public Research Centre (Luxembourg).