Gomphrena claussenii, a Zn and Cd hyperbioindicator species

<h4>Gomphrena claussenii, a Zn and Cd hyperbioindicator species</h4> A small group of plant species called metallophytes have evolved the ability to grow in highly metal-enriched soils that are toxic to other plants. Some of these metal-tolerant species have evolved the ability to accumulate high levels of metals or metalloids, such as nickel (Ni), zinc (Zn), arsenic (As), cadmium (Cd) and lead (Pb). Important progress towards understanding the physiological and molecular basis of metal and metalloid homeostasis in plants has been made by studying these metal hyperaccumulator species, which may also be useful for as the basis of phytoremediation technologies in which plants are used to stabilize or extract metals from soil. Gomphrena claussenii Moq. (Amaranthaceae), is a previously uncharacterized plant species which grows in the metal‑rich soils of a Zn mining area in the state of Minas Gerais, Brazil. This thesis describes the investigation of G. claussenii to determine the molecular basis of its ability to tolerate and accumulate Zn and Cd. <strong>Chapter 2</strong> presents a detailed comparative investigation of the physiological impact of Zn and Cd exposure on G. claussenii and the closely-related non-tolerant species G. elegans Mart. growing in soil or in hydroponic conditions. The impact of Zn/Cd in each species was determined by measuring growth characteristics such as biomass and root elongation. It was found that G. claussenii plants growing in the field in the Zn mining area accumulated up to 10434 &micro;g Zn and 96 &micro;g Cd per gram of shoot dry weight. Under hydroponic conditions, G. claussenii tolerated up to 3000 &micro;M Zn and up to 100 &micro;M Cd, showing only slight metal toxicity symptoms at the highest concentrations and no significant decrease in biomass or root length. In contrast, G. elegans showed significant toxicity symptoms at 100 &micro;M Zn and 5 &micro;M Cd. It was also found that both species accumulated more Zn and Cd in roots than shoots and that metal accumulation in G. claussenii showed a bioindicator-like response. Finally, the concentrations of other minerals such as Fe and Mn were not affected by Zn/Cd in G. claussenii shoots but declined dramatically in G. elegans in the presence of Zn/Cd. Taken together, these results indicated that G. claussenii is extremely tolerant to Zn and Cd and accumulates high levels of these metals in shoots, making it potentially valuable for phytoremediation applications. <strong>Chapter 3</strong> addresses the distribution of Zn/Cd in G. claussenii stem and leaf tissues, and metabolic profiles were used to investigate the involvement of metabolites in the sequestration of Zn/Cd. G. claussenii plants were exposed to high concentrations of Zn/Cd and analysed by scanning electron microscopy using energy dispersive X-ray (SEM-EDX) and micro-proton-induced X‑ray emission (micro-PIXE) technologies. We also investigated the dynamic profiles of primary metabolites in roots and shoots exposed to high levels of Zn/Cd to identify potential ligands for these metals. We observed the presence of abundant calcium oxalate (CaOx) crystals in the stem and leaf tissues of G. claussenii plants exposed to control and high levels of Cd, but intriguingly the number of crystals declined in the presence of Zn. Cd was shown to co‑localize with calcium (Ca) in the CaOx crystals, indicating that Cd sequestration in vacuolar CaOx crystals in G. claussenii is part of a tolerance mechanism to deal with excess Cd accumulation. Furthermore, citrate, malate and oxalate levels all increased in the shoots of G. claussenii exposed to Zn/Cd suggesting these organic acids are involved in metal chelation and contribute to metal tolerance. <strong>Chapter 4 </strong>focuses on the molecular genetic aspects of hypertolerance in G. claussenii. The comparative transcriptomics analysis of G. claussenii and G. elegans was used to identify genes potentially responsible for the adaptation of G. claussenii to high Zn/Cd exposure. The transcriptional response of both species to high Zn/Cd concentrations was investigated by RNA-Seq analysis. Transcript sequences were annotated, and differential expression induced by Zn/Cd exposure was analysed in G. claussenii and G. elegans roots and shoots. Orthologous transcript pairs were identified between both species, allowing the direct comparison of gene expression profiles. G. elegans showed a stronger transcriptional response to metal exposure than G. claussenii, featuring the significant modulation of 10&ndash;20 times as many genes. Many of these transcripts encode proteins involved in metal homeostasis or stress responses. Metal hypertolerance in G. claussenii therefore appears to be a constitutive expression trait, based on adaptations in the metal homeostasis and general stress response. <strong>Chapter 5</strong> summarizes and evaluates the knowledge gained by the investigation set out in this thesis, focusing on the relevance of the information obtained from G. claussenii and its contribution to our current understanding of metal hypertolerance. It is also discussed the benefits of G. claussenii for phytoremediation applications, as well as its potential for future research activities. &nbsp