Progress 05/12/04 to 05/11/07
Outputs
OUTPUTS: One central aspect of plant metabolism is the movement of metabolites within cells, between cells and throughout the plant. The purpose of this study is to investigate the transport of nucleobase compounds in plants. Nucleobases are nitrogen rich purines and pyrimidines that play central roles in plant metabolism. In plants, nucleobases are central to DNA and RNA metabolism, disease response, secondary compound synthesis and nitrogen transport. At least six distinct families of integral membrane proteins are known to, or suspected of, transporting nucleobases in plants. Our research involves characterizing genes named the nucleobase- ascorbate transporters (NAT) from the model plant Arabidopsis thaliana and in searching for new genes involved in nucleobase transport processes. Physical material generated from this research includes a series of new transgenic (genetically altered) Arabidopsis lines have been generated to monitor NAT gene expression throughout the plant life
cycle. In addition, novel genetic Arabidopsis lines were generated and characterized, which uncovered a new gene - for 1 (fluoroorotic acid resistant) - involved in the transport of uracil in plants. The main output of science based knowledge and data generated during this research includes a publication detailing the for1 mutation in Arabidopsis that was published in the peer reviewed journal, Journal of Experimental Botany. Other venues for disseminating finding include presentation of posters at scientific meetings and seminars delivered to other biologists at Yale and Indiana Perdue University and Fort Wayne (IPFW).
PARTICIPANTS: There are two primary investigators (PI) involved with the project: Neil P. Schultes (PI at The Connecticut Agricultural Experiment Station-CAES) and George S. Mourad (PI at IPFW). In addition, a number of undergraduate and Masters level students (B.M. Snook, J.T. Prabhakar and T. A. Mansfield (at IPFW) and two technicians Regan Huntley and Carol Clark (CAES) were involved in the project. George Mourad designed the genetic isolation and biochemical experiments and Neil Schultes designed and executed the molecular characterizations and molecular mapping in the for1 project. Neil Schultes designed the experiments in characterizing the NAT genes in Arabidopsis. Technicians Regan Huntley and Carol Clark performed molecular analysis including DNA construct assembly, DNA isolation, DNA sequence analysis, molecular marker characterization and transgenic plant generation and characterization. Professional development opportunities for the students involved in the project
included working in a laboratory environment on an independent project as well as interacting with PIs in experiment design and data interpretation. These opportunities are essential in training the next generation of scientists. Professional development opportunities were also available for the technicians involved in the project who were able to learn and perfect new techniques, including quantitative RT-PCR and real-time PCR. These new techniques are increasingly used in molecular analysis and soon will be standard procedures expected in molecular research.
TARGET AUDIENCES: The target audiences include scientific researchers in the plant biology community with an interest in primary and secondary metabolism and metabolite transport. Scientific based knowledge generated during this granting period was disseminated to the target audiences through publication in a peer review journal, poster presentations at scientific meetings and via seminar presentations at Yale University and IPFW. There was a request for the newly discovered and characterized novel Arabidopsis genetic lines carrying the for1 mutation by Dr. Marie-Pascal Doutriaux and the University of Paris, France for further analysis in her work.
Impacts
Our results generated new knowledge on how purines and pyrimidines are transported in plants. In particular our characterization of a novel uptake pathway for uracil - in finding and characterizing the for1 locus in Arabidopsis - will impact other researchers interested in primary and secondary plant metabolism and plant transport mechanisms. Additional data details the expression patterns of the NAT genes in Arabidopsis. We have analyzed all twelve NAT genes by a variety a number of independent molecular techniques and determined that each locus displays a tissue specific and developmental specific expression pattern. The novel genetic lines, new transgenic plants and molecular analysis generated and performed during this research has contributed to a more detailed picture of nitrogen use in plants. The long term goals of the research are to understand the flow of nitrogen-containing compounds within plants that will allow for future genetic manipulation to use
fertilizers more efficiently and to increase crop yield. Our data and material are currently used by other researchers in the field of expertise to advance overall understanding of plant metabolism.
Publications
- Mourad, G. S., Snook, B. M., Prabhakar J. T., Mansfield, T. A., Schultes, N. P. (2006) A fluoroorotic acid-resistant mutant of Arabidopsis defective in the uptake of uracil. J. Exp. Botany 57:,3563-3573
|
Progress 01/01/06 to 12/31/06
Outputs
This research project investigates genes governing the transport of purines and pyrimidines within and between plant cells. During the past year, we have centered our research into the For1 locus of Arabidopsis. Plants homozygous for the for1-1 mutation are resistant to elevated levels of 5 fluoroorotic acid (5FOA). 5FOA is a toxic analogue of uracil. A number of diverse experiments indicate that For1 is involved in the uptake of uracil in plants. These experiments include growth on the toxic uracil analogues 5FOA and on 5 fluorouracil, as well as on other toxic nucleobase analogue compounds 5-fluoro-2'-deoxyuridine, 5-bromo-2'-deoxyuridine and 8-azaguanine. Since radio-labeled nucleobase uptake experiments had shown that for1-1 plants were defective in the uptake of uracil and as for1-1 had been mapped to chromosome V, we investigated the possibility that For1 corresponded to one of the known loci encoding for nucleobase transporters on chromosome V. These loci
include nucleobase-ascorbate transporters (NAT) AtNAT9 (At5g25420); AtNAt5 (At5g49990); AtNAT6 (At5g62890); purine related transporter AtPRT1 (At5g03555); purine permease PUP11(At5g41160) and Azaguanine-like transporter AtAZG2 (At5g50300). Through detailed genetic mapping experiments using molecular markers and by direct sequencing of select PCR-amplified loci from for1-1/for1-1 genome, we were able to confirm that For1 gene does not correspond to any of these loci. Two other loci located on chromosome II had been shown to encode for transporters that move uracil across biological membranes (uracil permeases AtUPS1 and AtUPS2) (Desimone et al. Plant Cell 14, 847-856 2002; Schmidt et al. J. Biol. Chem. 279, 44817-44824 2004). We monitored the gene expression levels for the uracil transporter loci through semi quantitative reverse transcriptase polymerase chain reaction in wild type and for1-1/for1-1 seedlings to determine if For1 plays a role in the transcriptional regulation of uracil
transporters. Our results revealed that For1 does not influence the steady state RNA levels of the two uracil transporter genes. In a related project we are investigating two loci that are involved in the uptake of guanine. Initial results indicate that T-DNA insertion mutant lines in loci At3g10960 (AtAZG1) or At5g50300 (AtAZG2) are resistant to growth on high levels of 8-azaguanine. We are pursuing these investigations and continuing to map the for1 locus for eventual map based cloning. The For1 and Azg investigations represent an ongoing collaboration with Dr. George Mourad at Indiana University-Purdue University.
Impacts
Our research into the movement of nitrogen-containing nucleobases (purines and pyrimidines) has a direct impact on other research laboratories interested in primary and secondary plant metabolism. The research generated during this proposal is part of a larger research collaboration between our scientists in our laboratory and those in the laboratory of Dr. Mourad concerning research of nucleobase transporter genes in Arabidopsis thaliana. Understanding the flow of nitrogen-containing compounds within the plant will allow for future genetic manipulation to use fertilizers more efficiently and to increase crop yield.
Publications
- Mourad, G. S., Snook, B. M., Prabhakar J. T., Mansfield, T. A., Schultes, N. P. (2006) A fluoroorotic acid-resistant mutant of Arabidopsis defective in the uptake of uracil. J. Exp. Botany 57:,3563-3573
|
Progress 01/01/05 to 12/31/05
Outputs
This research project investigates a family of genes encoding plant transport proteins that are involved in the movement of purines, pyrimidines and possibly ascorbate across cellular membranes. We focus on a family of twelve genes called nucleobase-ascorbate transporters (NAT) in the model plant Arabidopsis thaliana. Metabolite transport is integral to nucleobase and ascorbate biochemistry. Both nucleobase compounds and ascorbate are involved in numerous biochemical pathways in plants. Specific transporters facilitate subcellular, intercellular and plant-wide transport of these compounds. One aim of the proposal is to complete NAT gene expression analysis by using four independent techniques. These techniques include analysis of NAT promoter-GUS transgenics plants, RT-PCR and RNA blot analysis and in situ analysis. During the previous year, we have generated transgenic Arabidopsis plants containing the promoter-GUS fusions for NAT loci At1g60030, At2g05760 and
At5g25420. We will analyze the resulting gene expression patterns with T2 plants and in total have plant-wide gene expression data for eleven of the twelve NAT loci. A second aim of the proposal is to obtain full-length cDNA clones of closely related loci At1g65550 and At5g25420. To date we have been able to document that both loci are expressed by isolating small partial cDNA fragments by nested RT-PCR protocols. However, we have been unable to isolate full-length cDNAs or overlapping cDNA molecules to generated full-length cDNAs by RT-PCR, 5 Prime and 3 Prime RACE procedures. This may be due to the extreme low expression levels of these genes. A third aim of the research is to characterize potential mutants in these loci. Through detailed molecular mapping studies we have been able to determine that the 5-fluoroorotic acid resistant allele, for1-1, does not correspond to any of the NAT loci in Arabidopsis. This latter work was performed in collaboration with Dr. George Mourad at
Indiana University-Purdue University.
Impacts
Our investigations of plant nucleobase-ascorbate transporter genes has an impact on a number of other research laboratories investigating similar research interests. The research generated during this proposal is part of a larger research collaboration between our laboratory and the laboratory of Dr. Mourad concerning research of uracil transporter-encoding genes in Arabidopsis thaliana.
Publications
- No publications reported this period
|
Progress 01/01/04 to 12/31/04
Outputs
This research project investigates a family of genes encoding plant transport proteins that are involved in the movement of purines, pyrimidines and possibly ascorbate across cellular membranes. We focus on a family of twelve genes called nucleobase-ascorbate transporters (NAT) in the model plant Arabidopsis thaliana. Nucleobases play important roles in plant biochemistry and are involved in DNA and RNA metabolism, disease response, secondary compound synthesis, plant hormone production and nitrogen transport. Ascorbate is a major carbon storage molecule in chloroplasts and plays central roles in the detoxification of oxygen radicals, cell wall expansion and long range carbon transport. Metabolite transport is integral to nucleobase and ascorbate biochemistry. Specific transporters facilitate subcellular, intercellular and plant-wide transport of these compounds. One aim of the proposal is to complete NAT gene expression analysis by using four independent techniques.
These techniques include analysis of NAT promoter-GUS transgenics plants, RT- PCR and RNA blot analysis and in situ analysis. We have generated four additional NAT promoter-GUS constructs corresponding to loci At1g10540; At1g60030; At2g05760 and At5g25420. These constructs are currently being transformed into Arabidopsis. We have analyzed all twelve NAT loci by RT-PCR technique and find that each locus displays a tissue specific expression pattern. A second aim of this proposal is to isolate full-length cDNA clones of loci At1g65550 and At5g25420. Although we have been able to confirm the expression of these loci by nested RT-PCR analysis, we have as yet been unable to isolate full-length cDNA versions of these loci. This may be due to the extreme low expression levels of these genes. In addition we are generating and characterizing Arabidopsis lines that carry T-DNA insertion mutations or deletion mutation of loci At1g65550 and At5g25420. These will be used for phenotypic analysis
and for assaying resistance to growth on toxic nucleobase compounds.
Impacts
Our investigations of plant nucleobase-ascorbate transporter genes has an impact on a number of other research laboratories investigating similar research interests. The research generated during this proposal is part of a larger research collaboration between our laboratory and the laboratory of Dr. Mourad concerning research of uracil transporter-encoding genes in Arabidopsis thaliana.
Publications
- No publications reported this period
|
|