Potential use of charophytes in the phytoremediation of cadmium-contaminated soils
Clabeaux, Bernadette Louise
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Cadmium (Cd) is a ubiquitous and toxic heavy metal. A cost-effective approach to Cd removal from soil is phytoremediation, the use of plants to remediate soil. My research investigated the potential use of Chara australis (R. Br.) for the phytoremediation of cadmium-contaminated soil. This requires that Chara tolerate Cd in the soil, and accumulate it within easily harvestable tissues. To determine phytotoxicity I evaluated the effects of Cd on Chara growth. I showed that Chara shoots tolerated up to 10 mg added Cd (kg soil) -1 , while rhizoids were tolerant up to 20 mg added Cd (kg soil) -1 . Liquid chromatography/mass spectrometry (LC-MS) was used to measure changes in glutathione (GSH) levels in plant tissues in response to Cd. This tripeptide is known to decrease in response to stress, and increase with the development of plant tolerance to environmental stresses. Chara explants treated with 2 to 35 mg Cd (kg soil) -1 for 97 days had GSH levels ranging from 200 to 350 nmol GSH (g DW) -1 in shoot tissues, significantly less than control levels of 678 nmol GSH (g DW) -1 . Histochemical staining with dithizone showed Cd in both rhizoids and shoots. This indicates that Cd is moved from rhizoids to shoots, an important characteristic of plants used for phytoremediation. Localization of Cd to shoot cell walls suggests apoplastic transport of Cd (outside the plasma membrane), while localization of Cd to the cytoplasm indicates symplastic transport (within the cytoplasm). I quantified Cd accumulation using inductively coupled plasma mass spectrometry (ICP-MS) and graphite furnace atomic absorption spectrometry (GF-AAS), and assessed whether Chara was hyperaccumulating Cd based on three criteria: Cd accumulation exceeding 100 mg Cd (kg DW)-1, and translocation factors (TF=Cd concentration shoots /Cd concentration rhizoids ) and bioconcentration factors (BCF=Cd concentration tissue /Cd concentration soil ) greater than 1.0. I showed a linear trend between the concentration of Cd in the soil and the total Cd accumulated by shoots and rhizoids. However, Chara did not meet all hyperaccumulator criteria, since the maximum concentration did not exceed 100 mg Cd (kg DW) -1 , and TFs did not exceed 1.0. The BCFs were >1.0 in the shoots of plants cultured in 35 mg added Cd (kg soil) -1 and rhizoids cultured in ≥ 25 mg added Cd (kg soil) -1 . I tested whether Cd accumulation could be increased by the addition of the chelator, EDTA (ethylenediamine-tetraacetic acid), to cadmium-contaminated soil by increasing the solubility of Cd. Moderate levels of EDTA (1 and 2 g EDTA (kg soil) -1 ) increased Cd accumulation in shoots and rhizoids, but it did not exceed 100 mg Cd (kg DW) -1 . Cd BCFs of shoots also increased in plants cultured in soil containing Cd and low EDTA (1 g (kg soil) -1 ) and exceeded 1.0. TF did not exceed 1.0 for any treatment. To test the hypothesis that Zn transporters may be a point of entry for Cd in Chara, I tested whether Zn competed with Cd for uptake. I found no significant interactions between Cd and Zn on metal accumulation by Chara shoots or rhizoids (p>0.05), failing to support our hypothesis. Overall, Chara 's high tolerance for Cd and ability to phytoextract Cd from soil make it a good candidate for the phytoremediation of cadmium-contaminated soil. I also investigated the role of auxin in the control of apical dominance in Chara, a phenomenon found in higher plants, in which lateral branch outgrowth is inhibited by auxin stemming from the apex of the main branch. I hypothesized that the apex of Chara shoots similarly produces auxin which is transported to the axillary branches and inhibits their development. I tested this by removing the apical shoot and found, as predicted, that the number of axillary branches produced by decapitated C. australis explants was significantly greater than in intact controls. However, I was unable to reverse the effect by exposing the plants to auxin in solution, and so could not confirm its role in the process. To determine whether preventing basipetal transport altered the number of basal branches, Chara internodes were ligated with silk thread. My results showed that the ligated explants produced significantly more rhizoids and axillary branches below the ligation than controls, indicating that the prevention of transfer of some substance to the lower node stimulates the production of new lower branches and rhizoids. This is the first experimental evidence for apical dominance in Chara.