Response of Arabidopsis thaliana seedlings to lead exposure
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Lead (Pb) is one of the most commonly occurring, highly persistent and widely distributed heavy metal contaminants in the environment. It has a tendency to bioaccumulate in animals and plants, and potentially, it is able to enter the human food chain where it poses a hazard to public health. Generally, conventional remediation technologies applied to decontaminate heavy metals from groundwater and soils are very costly. Hence, phytoremediation has emerged as an ecologically friendly and economically attractive technology that uses green plants to clean up heavy metal contaminated sites. However, a lack of knowledge of the biological processes associated with plant responses to Pb (e.g. Pb uptake, accumulation, translocation, and tolerance) has been a bottleneck for the application of Pb phytoremediation in the field. A model genetic system of higher plants, Arabidopsis thaliana, was selected to further examine the physiological, biochemical and molecular events occuring in plants under Pb stress.
The overall aim of this project was to obtain a better understanding of plant responses to Pb contaminants in the early developmental stages of A. thaliana seedlings. This research encompassed the physiological responses of A. thaliana seedlings to Pb exposure, monitoring their antioxidative defence systems, and investigating the participation of annexin 1 in the response to Pb-mediated oxidative stress. This research also assessed the protective effect of nitric oxide on Pb-induced toxicity of A. thaliana seedlings and it isolated a putative Pb tolerant mutant from an EMS-mutagenized M2 population. A multiexperimental approach was adopted to achieve these objectives.
A. thaliana seedlings were grown on modified Huang & Cunningham (1996) nutrient solution containing 0.8% (w/v) agar, with and without Pb(NO3)2, under controlled conditions. A. thaliana seedlings were insensitive to Pb during seed germination. In treatments with up to 200 μM Pb(NO₃)₂, morphological changes and inhibition of root growth were observed in the 7-d-old seedlings. A tolerance index revealed that Pb(NO₃)₂ concentration of 75 μM and higher brought about more than 50% root growth inhibition. Pb was predominantly retained in the roots. Analysis using a graphite furnace atomic absorption spectroscopy indicated that the level of Pb accumulation in A. thaliana roots was greatly dependent on the Pb(NO₃)₂ concentrations, but only a small fraction of the accumulated Pb was translocated to the shoots (18 - 43%). Transmission electron microscopy analysis showed that Pb was mainly immobilized in the cell walls and intercellular spaces. This was interpreted as a mechanism that minimizes the entry of Pb into cells and interference with cellular functions. Pb that gained entry into the cytoplasm was sequestered into the vacuoles.
The toxicity of Pb in the cytosol of A. thaliana seedlings was studied by measuring the H₂O₂ and lipid hydroperoxide levels using a microplate reader. When the Pb(NO₃)₂ concentration in the growth medium was 100 μM, the 7-d-old seedlings contained 2.2-fold higher H₂O₂ and 9.6-fold higher lipid hydroperoxide than the control without Pb(NO₃)₂. This was followed by an up-regulation of the activity of antioxidative enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPX), and general peroxidase (POD) by 2.1-, 3.2-, 2.3-, 1.8- and 4.6-fold, respectively, compared with the control. Pb toxicity is known to trigger oxidative stress, but A. thaliana seedlings appeared to be capable of activating cell rescue, defending themselves against harmful oxidative stress and also acclimating to Pb. Data from physiological and biochemical analysis indicate that a combination of avoidance and tolerance mechanisms exists in Pb-treated A. thaliana seedlings to maintain the essential cellular metabolism for survival.
Real-time reverse-transcription polymerase chain reaction was used to show the involvement of AnnAt1 in the response of 7-d-old A. thaliana seedlings to a high threshold concentration of Pb. When the seedlings were treated with 100 μM Pb(NO₃)₂, AnnAt1 message levels were up-regulated by 2.12-fold. Pb-mediated oxidative stress may be a component of AnnAt1 gene expression. AnnAt1 potentially could be invoked to reduce the toxic effects of Pb stress by acting as ROS and/or Ca²⁺ signals, as a membrane protector, in detoxification of excessive ROS, or in sequestration of Pb.
Pb stress symptoms were less evident in seedlings pre-treated with 1 mM sodium nitroprusside (SNP), a nitric oxide (NO) donor. The present study found that exogenous NO did not alter Pb transport into the plants or efflux pumping of Pb at the plasma membrane. However, NO conferred protection to 7-d-old A. thaliana seedlings primarily by acting as an antioxidant or a signal for actions to scavenge excessive ROS level. The application of exogenous NO before subjecting to 100 μM Pb(NO₃)₂ decreased H₂O₂ back to its original level, and reduced 50% lipid hydroperoxide in the Pb-treated seedlings. As a result, the antioxidative enzyme activities in Pb-exposed seedlings pre-treated with SNP were 23 - 45% lower than those without SNP pre-treatment. Less antioxidative enzyme activities were probably needed to counteract the reduced amount of Pb-induced ROS in A. thaliana seedlings.
A post-germination procedure involving prolonged exposure to 150 μM Pb(NO₃)₂ was developed to screen an EMS-mutagenized M2 population of A. thaliana. Potential Pb tolerant mutants were selected based on the ability to grow with their roots penetrating into the medium and maintain purple-green leaves without wilting. A minority of the survivors appeared to go into a resting stage and they seemed to have altered transporters that prevented Pb from entering the cells. Only one putative Pb mutant (M3-1) was recovered from the rescue and set seeds. The M₄ generation of this putative Pb mutant was re-screened for phenotypic confirmation and to determine the regulation of AnnAt1. The 7-d-old putative Pb mutant seemed to display enhanced root and shoot growth in the presence of 150 μM Pb(NO₃)₂ compared to the wild-type seedlings. The transcript level of AnnAt1 in this putative Pb tolerant mutant increased by 2.19-fold when exposed to 150 μM Pb(NO₃)₂.