Phytoextraction of cadmium by species of the Brassicaceae

© 2015 Kerryn Michelle PopaThis thesis contributes to the knowledge of metal-tolerant and metal-accumulating plant species for use in the field of phytoremediation. A broader range of species of Brassicaceae has been assessed for cadmium tolerance and uptake than has previously been tested. Cadmium (Cd) was chosen as the target metal for phytoremediation due to its high toxicity at low contaminating concentrations. The Brassicaceae family is an important potential source of species for phytoremediation as it is host to the largest number of metal hyperaccumulating species and also includes metal-tolerant species capable of accumulating various metals, including Cd. The capability of untested members of the family Brassicaceae to accumulate and tolerate Cd, the effect of Cd exposure and concentration on shoot nutrient homeostasis and the effect of Cd exposure and concentration on the expression of metal transporter HMA4 was investigated in hydroponic experiments. The feasibility of candidate species to phytoextract Cd from contaminated soils and biosolids was assessed in both glasshouse and field conditions. In addition, the localisation of Cd in aerial tissue was investigated. Of 19 previously untested species encompassing four genera of the Brassicaceae, five species, B. oleracea gongylodes (kohl rabi), B. oleracea capitata (cabbage, variety Golden Acre), B. oleracea (collards), B. oleracea acephala (kale, Half Scotch Tall) and Erysimum scoparium (wall flower), were identified as Cd tolerant from hydroponic studies. The concentration of Cd in the aerial tissue in the species tested varied from 60 to 370 µg Cd g–1 DW. Cadmium tolerance was not correlated with aerial Cd concentration (µg g-1 DW), however, total aerial Cd (µg plant–1 DW) was proportional to total aerial biomass (mg plant–1 DW) (r=0.92), demonstrating the importance of biomass in the phytoextraction of Cd. Exposure to Cd resulted in decreased Fe and Zn concentrations and increased Ca, Na and Mg concentrations in aerial tissues. Orthologues of HMA4, a gene encoding a metal transport protein capable of root-to-shoot transport of Cd, were detected in kohl rabi, pak choi, collards, wild mustard, senposai, kale, turnip and radish. Putative paralogues were observed in half of these species. The presence of identical sequences in Brassica species originating from different genomic backgrounds as well as more distantly related species of Brassicaceae, suggests an ancestral duplication event of HMA4 occurred before the evolution of these species. An expression analysis using reverse-transcriptase PCR of turnip, senposai, kohl rabi and kale detected HMA4 in the root tissue only, with no expression detected in kale. When plants were exposed to increasing Cd concentrations, HMA4 expression increased in both turnip and senposai, corresponding to medium to high levels of Cd accumulation respectively. Expression of this gene did not change in kohl rabi, which is consistent with low levels of Cd accumulation in this species. This finding supports the role of HMA4 in root to shoot transport of Cd. In the glasshouse pot trial, the candidate species (radish, tsoi sim, kale and senposai) demonstrated the capability to grow in biosolids provided as 100% substrate and survived in soil and biosolids contaminated with 8 µg Cd g-1 and 25 µg Cd g-1, respectively. In contrast to the high values observed in the hydroponics trial, the Cd concentrations in the aerial tissues of the candidate species ranged from 2 to 8 μg g-1 when grown in contaminated soil, and from 15 to 40 μg g-1 when grown in contaminated biosolids. There was no measurable reduction of Cd content in the contaminated soil or biosolids in which the plants had been grown. Biomass production of plants grown in contaminated biosolids was more substantial in the field trial. This was reflected in the greater amount of Cd extracted per plant than in the glasshouse pot trial, despite lower aerial tissue concentrations (5 to 9 µg g-1 DW). Kale and senposai extracted 100 and 50 µg Cd per plant respectively, however, this is too low to be economically viable. The attempt to identify the major sequestration sites within the aerial tissue of senposai and radish plants grown in the biosolids field using micro- Proton-Induced X-ray Emission/ Energy-Dispersive X-ray (PIXE/EDX) was not successful. The localisation of Cd in aerial tissue of these species remains elusive. Naturally occurring, high biomass, Cd accumulating plant species suitable for the phytoextraction of Cd from contaminated substrates are yet to be identified. Two major approaches to develop phytoextraction technologies are through breeding and transgenics. Of all the species examined, kale and senposai have the greatest potential for the phytoextraction of Cd. They tolerate and accumulate Cd, produce an extensive multi-branched root mass, are fast growing and easily harvested and they extracted more Cd per plant than the other species tested. Kale and senposai should be considered as suitable candidates for improving phytoextraction through genetic transformation with key hyperaccumulating genes such as MTP1 and HMA3 & -4