A transcriptomic study of the early responses of Medicago sativa to mercury

SUMMARY One of the greatest environmental concerns of the XXI century is soil contamination by toxic metals, due to various anthropogenic activities such as the chemical industry or mining. Toxic metals such as cadmium (Cd), mercury (Hg), aluminum (Al) or metalloids like arsenic (As), can cause toxicity problems in plants but also in higher levels of the trophic chain where large amounts of the toxic could be accumulated throughout the ingestion. Mercury is a very toxic metal for most living organisms. Its persistence in the environment and its accumulation throughout the food chain has led to episodes of severe diseases in humans. In Spain, large accumulations of Hg have been found in soils used for agricultural activity, causing a great risk to human health in the widely known mining area of Almaden. Thus, assessments of toxicity in autochthonous flora, in local and alternative crops such as alfalfa (Medicago sativa), constitute a vital requirement to avoid the deterioration of ecosystems and the human health. In this work, we have characterized the early response of alfalfa seedlings to the stress caused by Hg in order to understand its molecular basis. We analyzed the transcriptional profile of alfalfa roots by cDNA microarrays in parallel to several physiological parameters during the first 3, 6 and 24 hours of exposure to HgCl2. Mercury accumulation in plants subjected to doses above 10 ? M Hg for 3 hours caused a dramatic reduction in the root growth and increased the lipid peroxidation and hydrogen peroxide accumulation, enhancing the oxidative stress. During the first 3 to 6 hours of exposure to a low Hg dose (3 ? M), alfalfa seedlings suffered the most pronounced transcriptional response. Among the up-regulated genes were those involved in the secondary metabolism, related to cell wall and sulphur assimilation, heat-shock activated genes, biotic stress-related, or those related to metabolism and perception of phytohormones such as ethylene. Functional assays with Arabidopsis thaliana were useful to confirm the involvement of the ethylene-signaling pathway in Hg sensitivity by reducing the production of reactive oxygen species. The transcriptional profile of M. sativa under Hg stress was also compared to that of Medicago truncatula and Hordeum vulgare exposed to Hg. A common transcriptional response was found where stress-related genes coding glutathione-S-transferases, heat shock proteins, pathogenesis related proteins and several lignin biosynthesis related enzymes were induced. Moreover, a group of genes related to phytohormones signaling events, particularly ethylene, where among those up-regulated. This suggests that they may play a role in Hg perception and homeostasis common in all the three plant species. The Hg-responses of alfalfa seedling were compared with those produced by As, a widespread toxic metalloid. Similar to Hg, alfalfa roots treated with As (V) showed a reduction of the growth proportional to the toxic dose. Interestingly, the growth reduction caused by As seemed to be independent to the oxidative stress. The comparative analysis of both transcriptional patterns indicated the existence of a common gene expression response, as 20% of the genes differentially expressed as compared to untreated plants, were co-regulated. Furthermore, a differential response caused by each metal was also detected: Arsenic treated plants increased the expression of genes related to photosynthesis, protein degradation, and RNA processing. Meanwhile, in Hg-exposed plants, genes related to cell wall, protein synthesis, and DNA associated histones were induced. Finally, the role of the phytohormone ethylene in the signaling pathways leading to the observed plant physiological responses to Hg was assessed. The growth responses of the Arabidopsis ethylene-insensitive mutants, ein2-5, demonstrated a close relationship between ethylene and oxygen reactive species to negatively regulate root growth. Moreover, the ethylene-auxins interrelation at moderate Hg doses seemed to positively control the formation of new lateral roots. We also analyzed in detail the expression of genes involved in the heat shock response. A general over-expression of genes encoding several small heat sock proteins (shsp) was observed in response to Hg, in both alfalfa roots and epidermal cells roots of transgenic Arabidopsis carrying the Hahsp17.7G4::GUS construction. New tools for gene expression analysis greatly facilitate the study of plant responses to toxic metals. This study yield a broader understanding of the physiological, molecular and transcriptional responses of plants subjected to soil contamination, what will probably lead to find alternatives to control the pollution throughout phytoremediation or phyto-restauration techniques. Furthermore, we conclude that ethylene signaling attenuation could be useful in future phytotechnological applications to ameliorate stress symptoms in Hg-polluted plants improving crop production.