BLACKSBURG, Va., — Understanding the functions of the genes in the plant Arabidopsis could help with research in the fields of agriculture, medicine, and energy; and Virginia Tech researchers have received a grant from the National Science Foundation’s Arabidopsis 2010 Project Program to help understand those genes.
Asim Esen and Brenda Winkel of biology and David R. Bevan of biochemistry, along with researchers at the University of Iowa, have received a $2-million NSF grant for three years to study the "Functional Genomics of Arabidopsis Beta-glucosidase and Beta-galactosidase Gene Families." Virginia Tech will receive $1.1 million, which includes a $200,000 sub-award to Virginia State University at Petersburg, and The University of Iowa will receive $0.9 million.
A possible future use of this research could be the development of plants that can defend themselves better without pesticides or plants that can be used as a source of sugar for food stocks or for the production of alcohol.
"NSF is excited to begin this important endeavor of understanding the functions of each gene in Arabidopsis," said NSF director Rita Colwell. "While the task is daunting, it is also essential to this growing area of biotechnology research and its many applications. Only by understanding the fundamental processes of each gene can we piece together the puzzle of how DNA determines, for example, the rate of growth, resistance to disease, and many other factors in plants."
The plant Arabidopsis and the 2010 Project to understand its genes are important to biologists, the NSF said. "By studying this humble plant in the mustard family, scientists can better understand how all sorts of living organisms behave genetically, with potentially widespread applications for agriculture, medicine, and energy," the NSF press release said. Arabidopsis is a useful model because its entire genome consists of a relatively small set of genes that dictate when the weed will bud, bloom, sleep, or seed, the NSF said. Also, those genes have counterparts in crop plants with much larger genomes.
The Virginia Tech researchers will be looking at a sequence of 125 million nucleotides, or building blocks that make up a gene. "We predict there will be about 25 thousand genes their encoding protein," Esen said. Protein makes up and perfects the functions of our cells. Most of the proteins are actually enzymes. The sequence of protein is encoded in the sequence of the gene. About 25,000 genes in Arabidopsis encode protein, and the purpose of the 2010 project is to determine the functions of all the 25,000 proteins.
"We know the sequences of all genes," Esen said, "but that does not allow us to know what each gene does. In some cases, we know because of a similar gene in another organism and can extrapolate its function in Arabidopsis. But we know only about 10 percent of these genes’ functions directly. For a large percentage, we can predict the function, but that then has to be proven. For a substantial number, we have no idea what the genes or their protein products do. The goal is to determine that." Information gained from Arabidopsis can be extrapolated to other plants such as wheat, corn, cabbage, and soy beans.
Esen and his colleagues will attempt to determine the function of beta-glucosidase and beta-galactosidase genes. In Arabidopsis, about 44 genes encode the beta-glucosidase enzyme family and about 18 genes encode the beta-galactosidase enzyme family, Esen said. These two enzymes catalyze similar reactions. "Enzymes are known for their specificity, which is the reason we need thousands of enzymes in cells," he said.
Esen and his colleagues at Virginia Tech and the University of Iowa will split the work and try to determine the functions of the individual genes within each family. To learn what a gene does, the researchers must first learn what its protein does, since they cannot use the gene itself directly.
Each gene is divided into coding and non-coding sequences. The plant transcribes the gene into ribonucleic acid (RNA), with sections called introns making up the non-coding sequences and exons making up the coding sections. Then the cells remove the non-coding areas, leaving and splicing the coding genes (exons) to produce mature messenger RNA (mRNA).
The mRNA information is then translated to protein sequences and the protein is synthesized using the mRNA sequence as a guide. The particular sequence of nucleotides in an mRNA specifies the sequence of amino acids in the protein. "Each gene has a unique sequence and encodes a unique protein," Esen said.
Esen and his fellow researchers will take the mRNA part and make DNA out of it through reverse transcribing back into complementary DNA (cDNA), which has no introns, or non-coding sequences, and which contains only the coding information for the protein. The cDNA can then be cloned into an expression vector and introduced into bacterial, yeast, or insect cells that function as a protein synthesis factory. "Basically, we’re cloning and expressing the complementary DNA," Esen said.
The host cells will express the new cDNA or gene and make that protein exactly the way it occurs in Arabidopsis, Esen said. Since these proteins can be produced on demand, the scientists can make them in large quantities, purify them, and study their biochemical characteristics. "That’s how we learn what a gene does, by learning what its protein product does," Esen said.
The enzymes from this procedure may break down some 60 substrates, which are called beta-glucosides, beta-glucosinolates, and beta-galactosides, producing glucose (sugar) and nonsugar (aglycone) substance. The scientists then must identify what substrates correspond to each enzyme and in what plant part the substrates are, as well as when they are produced. "The shoot, leaf, stem will have different enzymes or genes active in them as well as different substrates," Esen said. "Now we will know about the enzyme coded by each gene and what substrate it works on in the plant. That will tell us what function it performs—chemical defense, growth, germination, response to cold or hormones, cellulose degradation, and so forth."
The products from this biomass conversion are reusable, Esen said. The sugar obtained from the breakdown of the cellulose could be used for food and animal feed or in the production of alcohol or other fuels, for example.
One well-known function of enzymes is in plant defense. "Plants have to defend themselves on their own ground," Esen said. "A way to do that is through toxic materials that are activated by some of these enzymes." For example, he said, the taste of mustard or horse radish comes from the products of these enzymes. "They have a nice flavor to us, but they are toxic or repellant to insects feeding on them." Some enzymes produce cyanide when plants are attacked, he said.
As we understand these genes and what their product enzymes do," Esen said, "through genetic engineering, we can produce plants that defend themselves without pesticides."
Learn more at http://www.biol.vt.edu/faculty/esen/glycosidaselab/
PR CONTACT: Sally Harris
540-231-6759, slharris@vt.edu
Contact: Asim Esen, 540-21-5894, aevatan@vt.edu