Progress 10/01/00 to 09/30/06
Outputs
Plants, yeast and algae synthesize phytochelatins enzymatically to detoxify metals such as cadmium (Cd), lead and arsenic. Several genes that code for the phytochelatin synthase enzyme have been identified and a few have been characterized enzymatically. All of the sequences reported share a conserved N-terminal(from plants, yeast and one animal) and the sequences from plants are very similar over the length of the entire gene. Upon exposure to Cd, most marine algae produce phytochelatins in proportion to intracellular Cd. Two species, however, have been found to synthesize a great excess of phytochelatin over intracellular Cd (Thalassiosira weissflogii and Thalassiosira pseudonana). This is particularly surprising in light of recent findings regarding how the metal-dependent reaction occurs in higher plants. Other researchers have used a purified recombinant phytochelatin synthase from Arabidopsis thaliana to show that both the Cd-bis-glutathione complex and free
glutathione are required substrates for the enzyme. The lack of a stoichiometric relationship between phytochelatin and Cd in these two coastal diatoms suggests a deviation from this mechanism found in higher plants. Though we were able to clone a gene with significant homology to phytochelatin synthase from T. weissflogii, we were unable to conclusively measure phytochelatin synthase activity in E. coli clones expressing the gene. We then turned our attention to the three separate genes with homology to PCS found in T. pseudonana genome. We found that all three of these genes are constitutively transcribed and that there were slight increases in mRNA copy numbers when the algal cultures were subjected to Cd stress. We designed primers and obtained a clone of E. coli containing one of the genes and had difficultly obtaining clones for the remaining two. We observed some evidence (in vitro and in vivo) that the enzyme is active though its activity seems different from the native
enzyme. We had no trouble getting activity with a clone containing the gene from Arabidopsis. Following numerous attempts to get better activity with our E. coli clone, we decided that we may need to use a eucaryotic expression system to get functional protein. Tiffany Gupton, a MS student, has been working with a graduate student in the laboratory of Carl Batt using the yeast Pichia pastoris to express all three proteins using vectors that will generate product inside and outside the yeast cell. We are currently in the process of testing the activity of enzyme generated with the vector containing TpPCS1 that yields extracellular product. We also did experiments during the last year comparing the activity of phytochelatin synthase in extracts of different algae species. The activity of phytochelatin synthase was quite similar in extracts of T. pseudonana and T. weissflogii but were significantly different from those measured in extracts of Dunaliella tertiolecta and Emiliania
huxleyi.
Impacts
Physiological evidence suggests that marine diatoms may contain a novel form of phytochelatin synthase, an enzyme that is responsible for the production of metal detoxification compounds. Once we obtain a functional enzyme in sufficient amounts we will be able determine whether it has useful engineering applications such as transformation into higher plants that could be used for phytoremediation.
Publications
- Wei, L. 2003. Phytoplankton-metal interactions: Phytochelatin production, exudation and degradation in natural seawater, and molecular studies of phytochelatin synthesis in marine microalgae. Cornell University PhD Thesis.
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Progress 01/01/02 to 12/31/02
Outputs
Phytochelatins are synthesized enzymatically by plants, yeast and algae to detoxify metals such as Cd and Cu. Several genes that code for the phytochelatin synthase enzyme have been identified and a few have been characterized enzymatically. All of the sequences reported share a conserved N-terminal (from plants, yeast and one animal) and the sequences from plants are very similar over the length of the entire gene. Upon exposure to Cd, most marine algae produce phytochelatins in proportion to intracellular Cd. Two species, however, have been found to synthesize a great excess of phytochelatin over intracellular Cd (Thalassiosira weissflogii and Thalassiosira pseudonana). This is particularly surprising in light of recent findings regarding how the metal-dependent reaction occurs in higher plants. Other researchers have used a purified recombinant phytochelatin synthase from Arabidopsis thaliana to show that both the Cd-bis-glutathione complex and free glutathione are
required substrates for the enzyme. The lack of a stoichiometric relationship between phytochelatin and Cd in these two coastal diatoms suggests a deviation from this mechanism. Using degenerate PCR primers we obtained a fraction of the sequence from Cd-stressed T. weissflogii that had 49 percent homology (46 out of 93 amino acids matching) to that of phytochelatin synthase in Typha latifolia (a marine cattail) and 39 percent homology (39 out of 93) to phytochelatin synthase in Arabidopsis thaliana (on the basis of a BLAST analysis). We then used 3'and 5' RACE to obtain a full sequence of similar size and that has significant homology with other PSC genes reported in GenBank. There are problems with the sequence that could not be resolved. For example there is a stop codon following the initiation codon. Sequencing based on a PCR product employing the genomic DNA revealed an identical sequence to that obtained from the cDNA. We were unable to find PCS enzymatic activity in E. coli
clones expressing the gene product. The complete genome for T. psuedonana has recently been completed and three separate genes with homology to PCS were reported. We designed primers and obtained clones containing two of the three genes. We have observed some evidence (in vitro and in vivo) that the enzyme is active though its activity seems different from the native enzyme. We are in the process of trying to purify the protein from one of the clones. In addition we have investigated the use of comparative RT-PCR to examine and potentially quantify genetic regulation of the phytochelatin synthase enzyme under different environmental conditions. In higher plants, the enzyme has been found to be constitutive, but this has yet to be demonstrated in marine algae. We have preliminary evidence that transcription is induced in the presence of Cd though we did not see quantitative differences with increasing Cd.
Impacts
Physiological evidence suggests that the marine diatom Thalassiosira weissflogi may have a novel form of the enzyme phytochelatin synthase. Once we obtain the DNA sequence of phytochelatin synthase and are able to generate sufficient amounts of the enzyme, we will be able determine whether it has useful engineering applications.
Publications
- Xie, Chaoyun. 2002. Identification of the putative gene sequence for phytochelatin synthesis in Thalassiosira weissflogii, a coastal diatom. Cornell University MS Thesis. August 2002.
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Progress 01/01/01 to 12/31/01
Outputs
Phytochelatins are synthesized enzymatically by plants, yeast and algae to detoxify metals such as Cd and Cu. Recently several genes that code for the phytochelatin synthase enzyme have been identified. All of the sequences reported share a conserved N-terminal (from plants, yeast and one animal) and the sequences from plants are very similar over the length of the entire gene. Upon exposure to Cd, most marine algae produce phytochelatins in proportion to intracellular Cd. Two species, however, have been found to synthesize a great excess of phytochelatin over intracellular Cd (Thalassiosira weissflogii and Thalassiosira pseudonana). This is particularly surprising in light of recent findings regarding how the metal-dependent reaction occurs in higher plants. Other researchers have used a purified recombinant phytochelatin synthase from Arabidopsis thaliana to show that both the Cd-bis-glutathione complex and free glutathione are required substrates for the enzyme.
The lack of a stoichiometric relationship between phytochelatin and Cd in these two coastal diatoms suggests a deviation from this mechanism. We have used four conserved regions of the published sequences and design two sets of degenerate PCR primers. Nested PCR was performed on cDNA prepared from Cd-stressed T. weissflogii. Nested PCR is a method used to improve the specificity of the PCR reaction. The amplified product from the first round of PCR (using the outside set of primers) is used as a template for the second round of PCR (using the inside set of primers). A single PCR product was obtained and sequenced. We obtained one sequence (279 base pairs in length) that has significant homology to the higher plant sequences. BLAST searches performed in GenBank revealed that our sequence has 49% homology (46 out of 93 amino acids matching) to that of phytochelatin synthase in Typha latifolia (a marine cattail) and 39% homology (39 out of 93) to phytochelatin synthase in Arabidopsis
thaliana. Using the partial sequence we now have for T. weissflogii, we will use 3', 5' RACE to obtain the complete sequence. Once we have obtained the completed sequence we will work with S. Clements' laboratory to insert the sequence into a strain of Schizosaccharomyces pombe that lacks phytochelatin synthase to test for functionality of our T. weissflogii enzyme (TwPCS1). We will also work with the Clemens' laboratory to express TwPCS1 in order to produce enough protein to develop antibodies as well as to examine the metal binding affinity of the newly obtained enzyme. Clemens' group is currently working on phytochelatin synthases obtained from higher plants and yeast. In addition we will generate radiolabeled probes to be used in Northern blots to examine and potentially quantify genetic regulation of the phytochelatin synthase enzyme under different environmental conditions. In higher plants, the enzyme has been found to be constitutive, but this has yet to be demonstrated in
marine algae. If the transcription of the gene is under some Cd regulation this could yield some potentially useful promoter sequences.
Impacts
Physiological evidence suggests that the marine diatom Thalassiosira weissflogi may have a novel form of the enzyme phytochelatin synthase. Once we obtain the DNA sequence of phytochelatin synthase and are able to generate sufficient amounts of the enzyme, we will be able determine whether it has useful engineering applications.
Publications
- No publications reported this period
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