OM PARKASH (DHANKHER)'s LAB

The current focus in the lab is to understand the molecular mechanism of tolerance and detoxification of arsenic in plants. My research approach involves a multidisciplinary application of methodologies from molecular biology, genomics (cloning of genes conferring detoxification or tolerance, ion transport and sequestration), physiology and biochemistry to decipher the underlying molecular mechanism of metal tolerance. The underlying molecular mechanism of arsenic uptake, tolerance, translocation and detoxification in plants has not been explored yet. These studies will help to develop efficient strategies for phytoremediation of arsenic in the environment.

Rotation students will learn many molecular techniques Primer design, PCR amplification, gene cloning, Northern, and western blotting, protein purification and physiological and biochemical assays. Also, the successful rotation students will the author/co-author in the subsequent research publications. Following the projects available in my lab.

1. Amplification and cloning of two putative Arsenite Transporter genes in Arabidopsis
A preliminary study in my lab has shown that Arabidopsis plants uptake not only arsenate (AsV) but arsenite (AsIII) as well. It is known that plant uptake arsenate along with phosphate uptake system using high affinity phosphate transporters. However, the mechanism of AsIII transport has not been reported before. From the Arabidopsis thaliana genome, I have found two putative AsIII transporters homologues to Arsenite -binding proteins from human. In this project we will PCR amplify and clone two putative arsenite transporters from Arabidopsis thaliana. First, these two genes will be PCR amplified using synthetic oligos, cloned into pBlueScript plasmid and will be sequenced. The function of these genes will be tested by functional complementation in E. coli mutant strain ( arsAB) deficient in arsenite transporters arsAB. If these genes are able to complement the arsenite transporter function in E. coli strains, then this project will be pursued further to overexpress these transporters into Arabidopsis thaliana. The resultant plants will be analyzed for arsenite tolerance and hyperaccumulation. Also, we will purify these two proteins by His-tag affinity chromatography to raise poly or monoclonal antibodies and also to carry out biochemical assays for functional analysis of these two transporters.

2. Knockdown of two putative Arsenite transporters in Arabidopsis by RNAi
The function of these two putative arsenite transporter genes in Arabidopsis will be tested using the reverse genetic approach. We will knock down the expression of these genes by RNA interference (RNAi) technique. In order to make a gene-specific RNAi construct, we will amplify the 3' untranslated region (3' UTR) of these genes and these RNAi construct will be introduced into Arabidopsis plants. The reduction in the level of mRNA and corresponding protein expression of these genes will be analyzed in RNAi knock down plants by RT-PCR and western analysis, respectively. These RNAi plants will be analyzed on various concentrations of arsenic for phenotypic difference and accumulation.

3. Isolation and cloning of Arsenic-induced genes by subtractive PCR suppression hybridization
In plants the mechanism of arsernic tolerance and detoxification is unknown. My lab focus is to develop technologies for phytoremediation of arsenic and decipher the underlying molecular mechanism of arsenic tolerance and detoxification. For efficient phytoremediation of toxic metals and metalloids, it is important to engineer fast growing high biomass plants with less agronomic requirements. It is also equally important that engineered crop plants should not be food crops and easily distinguishable even by layman in order to avoid accidental human exposure. We have selected Crambe species (a member of Brassica family) an ideal crop for subsurface arsenic phytoremediaon. My lab is using Crambe as a model system because it is highly tolerant to and accumulates various heavy metals and metalloids. We are interested in studying the molecular mechanism of arsenic tolerance and isolating the potential genes from Crambe. In this project we will make subtractive cDNA libraries from arsenic-induced mRNAs isolated from Crambe grown on arsenic media. The representative cDNAs will be cloned and screened for arsenic tolerance and hyperaccumulation by functional complementation in E. Coli mutant strains. This project will provide some insights of arsenic tolerance mechanism in plants, which will ultimately lead to development of efficient strategies of arsenic phytoremediation.

4. Overexpression of Bacterial arsenate reductase (ArsC), and gamma-glutamylecysteine synthetase ( -ECS) genes into Crambe sp.
Previously, I have co-expressed these two bacterial genes into Arabidopsis. The double transgenic plants were highly tolerant and accumulated 3-fold more arsenic in aboveground tissues (Dhankher et al., 2002, Nature Biotech. 20: 1140-45). Also, suppression of an endogenous arsenate reductase (AtACR2) in Arabidopsis by RNAi enhanced the translocation of arsenic from root to shoot. The RNAi knock down plants accumulated 10- to - 15-fold more arsenic in shoot tissues (Dhankher et al., 2005 Submitted). Our objective here in this project is to transfer this genetics-based phytoremediation strategy for arsenic removal in fast growing high biomass accumulating non-food plants for field trials. Crambe is an ideal plants for this purpose as it is a natural hyperaccumulator and highly tolerant to arsenic. Our ultimate goal is to combine the overexpression of several genes that involve uptake, detoxification, sequestration and hyperaccumulation of arsenic in aboveground tissues. This project will be a first step in this direction. The overexpression of these proteins will be detected by immunoblotting using the Anti-ArsC and Anti- ECS monoclonal antibodies available in my lab. These plants will be analyzed for arsenic tolerance and hyperaccumulation.

5. Genetic transformation of Crambe plants
As Crambe has not been genetically transformed, we will develop an efficient genetic transformation protocol using Agrobacterium-mediated transformation methods. In this project we will vacuum-infiltrate Crambe flowers and also co-cultivate leaf discs with Agrobacterium harboring GUS reporter construct. We have a construct called SRS1pt::GUS containing GUS reporter gene under a soybean leaf-specific promoter/terminator cassette SRS1pt. The resultant plants will be analyzed for GUS activity in shoot tissues. This project will be used to evaluate the transformation efficiency of Crambe by flower dip and leaf-disc methods.

There are many other projects currently on-going in my lab. If you are willing to join, please come and see me for further information (202 French hall, parkash@psis.umass.edu, tel: 545-0062, http://www.bio.umass.edu/plantbio/faculty/parkash.html).