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Date of Award

5-2011

Access Type

Campus Access

Document type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Plant and Soil Sciences

First Advisor

Om Parkash (Dhankher)

Second Advisor

Geunhwa Jung

Third Advisor

Elsbeth Walker

Subject Categories

Genetics | Molecular Biology | Plant Biology | Plant Sciences

Abstract

Arsenic is an acute poison and its contamination in soil and water is widespread. Crambe abyssinicaaccumulates significantly higher levels of arsenic as compared to other species of the Brassicaceae. Being a non-food, high biomass crop that is naturally tolerant to heavy metals, crambe has significant potential for phytoremediation of arsenic. In order to identify the pathways involved in arsenic metabolism and detoxification in C. abyssinica , differentially expressed genes in response to arsenic exposure were isolated employing a PCR-Select Suppression Subtraction Hybridization approach. A total of 105 differentially expressed subtracted cDNAs were sequenced which were found to represent 38 genes. Those genes encode proteins functioning as antioxidants, metal transporters, reductases, enzymes involved in the protein degradation pathway, and several novel uncharacterized proteins. The differential expression of transcripts corresponding to the subtracted cDNAs was confirmed by the semi-quantitative RT-PCR.

Arabidopsis homologs of two uncharacterized proteins from this subtracted cDNA library were further characterized for their role in As detoxification in plants. One of these two genes, AtChaC2-1 functions as a gamma-glutamyl cyclotransferase as evident from in vivo studies in yeast as well as in Arabidopsis. It plays a significant role in glutathione homeostasis and participates in gamma-glutamyl cycle to recycle Glu. T-DNA insertion AtChaC2-1 mutant plants were tolerant to arsenic toxicity due to the elevated glutathione contents. AtChaC2-1 over-expression lines were also tolerant to As presumably due to more active gamma-glutamyl cycle and an efficient Glu recycling. Furthermore, AtChaC2-1 overexpression increased the N utilization efficiency as it decreased the de novo synthesis of Glu and thereby N assimilation.

A second gene, AtMATE21, is an efflux protein of MATE family of secondary transporters. Heterologous expression in yeast RM1 mutant strain decreased the As accumulation in yeast presumably by efficient effluxing of As from yeast cells. Arabidopsis plants with T-DNA insertional mutation in the AtMATE21 locus were sensitive to arsenate. The AtMATE21 over-expression lines were more tolerant to arsenate and accumulated a significantly higher amount of arsenic in the aboveground parts. Both AtChaC2-1 and AtMATE21 genes have significant potential to be utilized for developing plant-based strategies for arsenic mitigation in the environment.

DOI

https://doi.org/10.7275/5682958

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