CADMIUM EXCLUSION FROM BARLEY ( Hordeum Vulgare L.): DEVELOPMENT OF PHYSIOLOGICAL AND MOLECULAR MARKERS.

The entry into the food chain of excessive amounts of heavy metals, due to the consumption of foodstuffs from crops grown on contaminated soils, is of increasing concern for public health. Among heavy metals, Cd results particularly dangerous since it is easily taken up by roots and translocated to vegetative and reproductive organs of plants without obvious symptoms of phytotoxicity. In particular, Cd can accumulate in grains of cereals such as rice, wheat and barley. The Codex Alimentarius Commission of the Food and Agricultural Organization/World Health Organization set the maximum permissible concentration of Cd for human consumption at 0.1 µg g-1 for cereal grains, excluding rice (0.4 µg g-1). Among the strategies to limit the risk of introducing Cd into the human food chain, the identification and/or constitution of plant genotypes able to exclude the metal from the shoot or from the edible parts seems to be the most promising line of enquiry for the future. Among cereals, barley ranks fourth in terms of both yearly-produced amounts and cultivated area in the world. In recent years, a correlation between presence of barley in the diet and reduced risk of coronary heart diseases has been suggested, inducing a progressive increase in the demand for the cereal in countries where its consumption was traditionally limited. Although some evaluations of genotypic differences in Cd accumulation in barley grain have been described, very little information is available about the physiological basis of the observed variability. Specific aims of the research were: a) to analyze six barley cultivars among the most cultivated in Tunisia for their tolerance to relatively high Cd concentrations and ability to limit the accumulation of the metal in shoot and grain; b) to identify the molecular and physiological basis of the behavior of the two most divergent cultivars, i.e. the highest and the lowest Cd accumulator, in order to develop markers useful in the selection of low-Cd grain cultivars. Among the six Tunisian barley cultivars, a large variability in their sensitiveness to Cd exists. The concentrations of the metal in the roots of plants grown in hydroponic solution in the presence of Cd did not significantly differ among the six cultivars, whereas wide differences were apparent in the shoots, where Lemsi and Manel showed the highest and the lowest values, respectively. Despite similar transpiration fluxes, the six barley cultivars loaded into the xylem and translocated to the shoots different amounts of Cd. A close linear correlation between the concentrations of the metal in the xylem sap and those measured in the shoots was observed. The measurements of concentration-dependent influx of Cd in the roots revealed marked differences between Lemsi and Manel. Lemsi showed a clearer saturable component in the low Cd concentration range; the maximum influx (Vmax) for Cd was about threefold higher in Lemsi. Although the Cd concentrations were not different in the roots of the two cultivars, the amounts of phytochelatins and the ability to retain Cd were lower in Lemsi than in Manel. The different Cd retention in roots between the two cultivars cannot be ascribed to a differential expression of the HvHMA3 gene encoding a tonoplast-localized transporter mediating the vacuolar sequestration of the metal. In detail, the Cd-treatments decreased the steady state levels of HvHMA3 mRNA in the two cultivars at the same extent. Exhaustive extraction combined with a fractionation procedure showed that in the roots of Lemsi the percentage of free non-chelated Cd ions (Cd2+), i.e. the form potentially available for xylem loading , was twice that present in Manel. Since the expression levels of the gene HvHMA2, encoding a protein actively extruding Cd2+ from the parenchyma cell of the root stele towards the xylem vessels, did not differ between Lemsi and Manel, it is reasonable to conclude that the larger amount of metal loaded in the xylem in the former cultivar is due to the higher amount of substrate (Cd2+) available for the HvHMA2 protein. When plants were grown on Cd-contaminated soil, the levels of the metal in the grain, as well as in flag leaves and husk, were higher in Lemsi than in Manel. This suggests that the reallocation of Cd from the leaves to the spike during grain filling does not involve mechanisms able to override the differences imposed by the differential Cd root uptake and root-to-shoot translocation described for the two cultivars. In conclusion, the activity of mechanisms mediating the uptake of Cd into the root, and, particularly, the efficiency of the phytochelatin-dependent system chelating and sequestering Cd in the root, emerge as critical points in controlling low the concentration of Cd in barley.