Progress 10/01/02 to 09/30/07
Outputs
This award fostered interactions between Plant Biology and other departments that sponsor agriculture-related research, and encouraged new lines of research related to agricultural priorities. Hatch funds were awarded on a competitive basis to initiate interdisciplinary collaborations, to purchase multi-user instrumentation, and to provide bridge funding on several existing projects. A brief synopsis of progress on the individually funded projects follows. More detail may be obtained in the project's annual reports: Brambl, R. Work on small heat shock proteins in Neurospora crassa revealed details of the mechanisms of chaperone function. Molecular interactions of conserved domains of Hsp30 were discovered. Secondly, regulation of expression of mitochondrial genes was examined, resulting in identification of regulatory proteins and UTR sequences that bind the putative regulators. Gleason, F. Update of instrumentation aided identification of a plant compound that acts as
an insect attractant, and may lead to biological control of an exotic plant species. Koukkari, W. An equipment purchase enabled monitoring of circadian rhythms in plants. Investigators examined changes in responses to herbicides that occur over the course of a day. In a new collaboration with Olszewski, the daily rhythms in transpiration were monitored. Marks, D. An equipment purchase allowed documentation of developmental phenotypes of Arabidopsis mutants. Marks and others screened insertional mutants and activation-tagged lines for developmental phenotypes. This aided projects studying responses to light, regulation of growth and pattern formation, and regulation of starch accumulation. May, G. Genetic loci in maize that control resistance to corn smut were mapped, and their evolutionary relationships were studied. McLaughlin, D. Fungi involved in wood and litter decomposition were identified and ectomycorrhizal fungal diversity in local oak savannahs was investigated, among other
projects. This work was carried out at a Long Term Ecological Reserch site, where the effects of fire in native ecosystem management are assessed. Olszewski, N. Gibberellin is a hormone that controls plant stature. A protein involved in gibberellin signaling was characterized. In collaboration with Gleason, this protein, called SPINDLY, was found to be part of a multi-protein complex. Also, Olszewski and collaborators investigated several plant viruses, including a newly discovered strain of the banana streak virus. Silflow, C. Molecular markers, including 80 SNPs developed in the project, were placed on the genetic map of Chlamydomonas reinhardtii. Using the markers as probes, a BAC library was screened, and the BACs were fingerprinted and assembled into contigs representing nearly 1/4 of the nuclear genome. This effort will aid understanding of chloroplast and flagellum function. Snustad, P. In collaborative research with J. Carter in Horticulture, all tubulin genes of Arabidopsis
were cloned. Several beta-tubulin genes show altered expression in response to cold temperatures, and may play a role in chilling tolerance.
Impacts
When the proposal for this award was submitted, it was reasoned that Hatch funds would be used most efficiently if they were allocated competitively to leverage future extramural funding. We viewed this to be a success based on the on-going collaborative projects that have been sponsored by these short-term grants. Six new extramural projects have been funded to date, based on preliminary work executed under the Hatch awards. Several Hatch-supported research projects have been especially fruitful, based on peer-reviewed and other publications. The work executed under the Hatch award contributed to advances with impact in many areas, including the following: 1. Tolerance to temperature stress in crops 2. Biological control of invasive, exotic species 3. Herbicide resistance and susceptibility 4. Control of plant development patterns and stature (height) 5. Plant disease resistance 6. Cataloging of fungal diversity, and assessment of ecosystem management strategies
Publications
- Brambl R. (2003). Post-transcriptional regulation of mitochondrial gene expression. In The Mycota III: Biochemistry and Molecular Biochemistry (second ed.). Springer-Verlag (In press).
- Cheng Z. (2000). Temperature-regulated and tissue-specific expression of Arabidopsis tubulin genes. Ph.D. thesis, University of Minnesota.
- Cheng Z, Snustad DP, and Carter JV. (2001). Temporal and spatial expression patterns of TUB9, a b-tubulin gene of Arabidopsis thaliana. Plant Molecular Biology 47:389-398.
- Gleason F, and Olszewski N. (2002). Isolation of the gene for the B12-dependent ribonucleotide reductase from Anabaena sp. strain PCC7120 and expression in Escherichia coli. Journal of Bacteriology, 184:6544-6550
- Hartweck LM, Scott CL, Olszewski NE. (2002) Two O-Linked N-Acetylglucosamine transferase genes of Arabidopsis thaliana L. Heynh. have overlapping functions necessary for gamete and seed development. Genetics 161: 1279-1291.
- Izhaki A, Swain SM, Tseng TS, Borochov A, Olszewski NE and Weiss D. (2001). The role of SPY and SPY's TPR domains in the regulation of gibberellin action throughout the life cycle of Petunia hybrida plants. The Plant Journal 28: 181-190.
- Kathir P, LaVoie M, BrazeltonWJ, Hass NA, Lefebvre PA, and Silflow CD. (2003). A molecular map of the Chlamydomonas reinhardtii nuclear genome. Eukaryotic Cell. 2:362-79.
- Kleidon J, Plesofsky N, and Brambl R. (2003). Transcripts and transcript-binding proteins in mitochondria of Neurospora crassa. Mitochondrion. (In press).
- Martinson KB, Sothern RB, Koukkari WL, Durgan BR, and Gunsolus JL. (2002). Circadian response of annual weeds to glyphosate and glufosinate. Chronobiol Intl. 19(2): 405-422.
- Olszewski NE, Sun TP. and Gubler F. (2002) Gibberellin signaling: biosynthesis, ctabolism and response pathways. Plant Cell 14: S61-S80.
- Plesofsky N. (2003). The heat shock proteins and their chaperone functions. In The Mycota III: Biochemistry and Molecular Biochemistry (second ed.). Springer-Verlag (In press).
- Plesofsky N and Brambl R. (2002). Analysis of interactions between domains of a small heat shock protein, Hsp30 of Neurospora crassa. Cell Stress Chaperones 7:374-386.
- Sothern RB, Tseng TS, Orcutt SL, Olszewski NE and Koukkari WL. (2002) GIGANTEA and SPINDLY genes linked to the clock pathway that controls circadian characteristics of transpiration in Arabidopsis. Chronobiology Int.19: 1005-1022.
- Swain SM, Tseng TS, and Olszewski NE. (2001). Altered expression of SPINDLY affects gibberellin response and plant development. Plant Physiol 126: 1174-1185.
- Swain, SM, Tseng TS, Thornton T, Gopalraj M and Olszewski NE. (2002) SPINDLY is a nuclear-localized repressor of gibberellin signal transduction expressed throughout the plant. Plant Physiol. 129: 605-615.
- Thornton T, Krepel L, Hart G, and Olszewski N. (1999). Genetic and Biochemical Analysis of Arabidopsis SPY. In Plant Biotechnology and In vitro Biology in the 21st Century, Altman A, Ziv M, Izha, S. eds., Kluwer Academic Publishers, New York, pp. 445-448.
- Thornton T, Swain S, and Olszewski N. (1999). Gibberellin signal transduction presents: The SPY who O-GlcNac'd me. Trends in Plant Science 4:424-428.
- Tseng TS, Swain S, and Olszewski NE. (2001). Ectopic expression of the TPR domain of SPY causes defects in gibberellin response. Plant Physiol 126: 1250-1258.
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Progress 01/01/01 to 12/31/01
Outputs
The Hatch funds awarded to the Plant Biology Department supported collaborative projects with researchers in COAFES as well as individual research projects. A collaborative project involved the laboratories of David Marks, Min Ni, Neil Olszewski, and John Ward in Plant Biology and Jerry Cohen in Horticulture. These researchers combined efforts in screening for Arabidopsis insertional mutants. Stocks of seeds carrying insertional mutations were purchased from the Arabidopsis Biological Resource Center. Plants were grown on soil under white light, on sterile plates under red light, and on plates in the presence of the gibberellin-biosynthesis inhibitor paclobutrazole. Plants growing on soil were screened for starch over-accumulation and aberrant trichome patterning. Activation-tagged lines were screened for altered hypocotyl length under red light and for germination in the presence of paclobutrazole. Mutants were identified that exhibit elevated starch levels, enhanced
sensitivity to red light, and the ability to germinate in the presence of paclobutrazole. Olszewski and Florence Gleason purchased a new controller and flow cell to update the FPLC system in Gleason's laboratory. Gibberellins (GAs) are phytohormones that regulate many aspects of plant development. Olszewski's laboratory is studying the GA signal-transduction pathway in Arabidopsis. SPINDLY (SPY) is a negative regulator of GA signaling. Olszewski is studying proteins that interact with SPY and an interaction between SPY and GIGANTEA (GI), a protein involved in responses of plants to light. Through the analysis of single mutants and mutant combinations, they have shown that SPY and GI participate together in the photoperiodic induction of flowering and the induction of photomorphogenesis by red and far-red light. The Silflow laboratory cloned the VFL6 gene of Chlamydomonas. Mutations in VFL6 result in variable flagellar number, abnormal motility, and recessive meiotic lethality.
Putative VFL6 clones were placed into a contig of overlapping clones. Several clones were transformed into mutant strains and the motility of the transformants was analyzed. One BAC clone restored the wild-type phenotype, indicating that it contained the VFL6 gene. Subclones of the BAC DNA are being tested in transformation experiments to localize the VFL6 gene. The Snustad laboratory investigated the effects of T-DNA knockout mutations in the two g-tubulin genes of Arabidopsis. Heterozygous double-mutant progeny of crosses were identified, self-pollinated, and F2 progeny were analyzed by PCR. The results showed that progeny with multiple insertions are under-represented and the double homozygote is lethal. Higher plants have multiple genes encoding heat-shock proteins (hsps). Robert Brambl is studying the heat-shock genes of plants by introducing them into a Neurospora strain that carries a deletion of the homologous fungal gene. By this approach, he can determine whether plant hsp
genes can substitute for the deleted fungal gene. The results will provide information about the roles of plant hsps in resistance to heat-stress.
Impacts
The collaborative project involving Cohen, Marks, Ni, Olszewski and Ward resulted in the isolation of several interesting mutant lines of Arabidopsis. The project improved communication between the participating labs and provided an efficient collaborative mechanism for the labor-intensive screens. Further characterization of the mutants isolated during this screen should result in the identification of genes that play roles in the responses of plants to light and hormones. The project in the Olszewski laboratory focused on an Arabidopsis mutant, SPINDLY, which is defective in the gibberellin signaling. Plant responses to hormones and light are key targets for the production of improved crops. Therefore, knowledge of the mechanisms underlying the responses to hormones and light should facilitate the production of improved crops. Cilia and flagella are highly conserved organelles found in animals, protists, and lower plants. These organelles grow from and are oriented
by basal bodies, which are embedded in the cytoplasm. Information obtained in the Silflow lab on basal bodies in Chlamydomonas should be applicable to other organisms due the evolutionary conservation of basal body structure. Cloning the VFL6 gene is a key step in elucidating the molecular function of its protein product and in determining whether the gene has functional homologs in other organisms. Research in the Snustad and Brambl laboratories will provide a better understanding of the mechanisms of intracellular transport and the resistance of plants to heat shock, respectively.
Publications
- Haas, N.A., Kathir, P., Brazelton, W.J., LaVoie, M., Lefebvre, P.A. and Silflow, C.D. 2002 Construction of BAC contigs associated with molecular markers in the Chlamydomonas nuclear genome. Manuscript in preparation.
- Tseng, T.-S. and Olszewski, N.E. 2002 The Arabidopsis SPINDLY and GIGANTEA proteins interact and participate in light responses. Manuscript in preparation.
- Plesofsky, N. & Brambl, R. 2002 A structural analysis of a small heat shock protein, Hsp30 of Neurospora crassa. (Submitted to J. Biol. Chem.)
- Swain, S.M., Tseng, T.-S., Thornton, T., Gopalraj, M. and Olszewski, N.E. 2002 SPINDLY is a nuclear-localized repressor of gibberellin signal transduction expressed throughout the plant. Manuscript in revision for Plant Physiol.
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Progress 01/01/00 to 12/31/00
Outputs
The Hatch funds awarded Plant Biology were used in three different ways: to purchase research equipment that is available to multiple users, to support collaborative projects involving researchers in two colleges, and to provide seed monies for new initiatives. One major item of equipment purchased is a VayTek deconvolution and 3-D volume rendering and measurement package that was combined with software and computers purchased by the Imaging Center and a cooled color CCD digital camera purchased by Pete Lefebvre. This camera is mounted on a Nikon Eclipse 800 fluorescence microscope. The complete system is available for use in the Imaging Center. The funds awarded to the Koukkari lab were used to purchase a Macintosh G4 computer with monitor, extra RAM, a CD Burrner, and peripheral equipment for use in processing long-term video recordings of circadian leaf movements in plants. Twenty-four hour monitoring of circadian rhythms has been done in 13 different plant
species. This work has recently led to a new collaboration between Koukkari and Neil Olszewski. The funds awarded to the Marks laboratory were used to purchase a Kodak MDS 290 microscopy system which permits computer controlled capture of microscopic images. This has been used store photographic data on developmental defects in Arabidopsis mutants. Additional funds were used to obtain preliminary data the effects of over-expression of the GL1 gene of Arabidopsis on resistance to insects. The results obtained led, in part, to a recently funded $130,000 per year NSF grant. The funds designated to the Snustad laboratory were used to support collaborative research with John Carter in Horticulture. Researchers in the Snustad lab had previously cloned all 17 alpha-, beta-, and gamma-tubulin genes of Arabidopsis. Three of the beta-tubulin genes exhibited altered expression in plants exposed to cold temperatures. The present project involved studies on the cold-regulation of two of these
genes. Cis-acting regulatory elements and the effects of abscisic acid were investigated. Work in the Brambl lab focused on the function of heat shock (HS) genes in plants. Because plants contain a large number of HS genes, Brambl has used Neurospora as a system in which to study these important genes. He is introducing key HS genes from plants into an HS-deficient mutant of Neurospora and asking whether the plant genes can complement the fungal mutation.Another collaborative project involves work on the tobacco vein clearing virus of Nicotiana edwardsonii and the banana streak virus (BSV) in the Olszewski and Lockhart (Plant Pathology) labs. They have demonstrated that the genome of a newly discovered strain of BSV is integrated into the genome of the commercial cultivar Goldfinger. Funds awarded to the McLaughlin lab were used to support collaborative research with Peter Reich in Forest Resources. They are investigating the ectomycorrhizal fungal diversity above- and below-ground in
oak savannahs in unburned and frequently burned plots at the Long Term Ecological Research Site. Other graduate student projects on the Psathyrella mushrooms, the clavarioid fungi, and the lichen genus Stricta were also supported.
Impacts
Research on the response of plants to low temperature and the effect of heat shock genes could lead to plant varieties that are more tolerant to heat and cold. Work on the tobacco vein clearing virus and the banana streak virus could eventually result in cultivars that are free of or resistant to these viruses. The result obtained in studies on the over-expression of the Arabidopsis GL1 gene were in part responsible for the recent award of a $130,000 per year NSF grant to the M.D. Marks laboratory.
Publications
- Cheng, Zigang. "Temperature-Regulated and Tissue-Specific Expression of Arabidopsis Tubulin Genes." 1999
- Cheng, Zigang. "Temporal and Spatial Expression Patterns of TUB9, a b-Tubulin Gene of Arabidopsis thaliana." Plant Molecular Biology, 2000
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Progress 01/01/99 to 12/31/99
Outputs
Funds from this project have been used in a variety of ways including the analysis of a diverse genus of fungi that are responsible for wood and litter decomposition in temperate forests; characterization of a protein that is intimately involved in gibberellin (a plant hormone that partially controls plant stature) signaling; genetic mapping and evolutionary analyses of maize loci that control resistance to a common fungal pathogen, Ustilago maydis; physical mapping of the alga Chlamydomonas genome which will lead to a detailed understanding of photosynthesis and chloroplast function; isolation of a weevil attractant from Eurasian water milfoil; and characterization of beta-tubulin genes that may be important in a plant,s response to chilling. All of these projects are in the very early stages. Much of this research is a collaborative effort between faculty in the Department of Plant Biology and faculty in the College of Agriculture, Food, and Environmental Sciences
and the College of Natural Resources. Perhaps the project that has made most progress is the one investigating the mode of action of plant hormones. The SPINDLY gene in Arabidopsis thaliana has been found to play an important role in the plant's response to gibberellins. Work in Dr. Neil Olszewski and Dr. Florence Gleason's laboratories suggests that the SPINDLY protein is part of a large multiprotein complex. The result is surprising because work from other labs on a related protein from animals did not detect this complex. Currently we are trying to purify the complex to homogeneity with a long term goal of identifying the other components of this complex and determining their role in gibberellin signal transduction.
Impacts
Some of the research described above examines the biological bases of important agricultural advances. For example, the semi-dwarfing genes that enabled the Ogreen revolution, are in fact components of the gibberellin signaling system. Also, understanding the evolution of disease resistance genes maize will assist the informed management of genetic resources that are available to plant breeders.
Publications
- Thornton, T., Krepel, L., Hart, G. and Olszewski, N. (1999) Genetic and Biochemical Analysis of Arabidopsis SPY. In Plant Biotechnology and In vitro Biology in the 21st Century, Altman, A., Ziv, M., Izhar, S. eds., Kluwer Academic Publishers, New York, pp. 445-448.
- Thornton, T., Swain, S. and Olszewski, N. (1999) Gibberellin Signal Transduction Presents The SPY Who O-GlcNAc'd Me. Trends in Plant Science. 4:424-428.
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