Progress 12/01/01 to 09/30/06
Outputs
Cytokinins are a group of plant growth regulators important at all stages of plant development. This project focuses on the metabolism of zeatin, the most important cytokinin in plant tissues. Transgenic maize plants with increased conversion of zeatin to O-glucosylzeatin (an inactive compound) were characterized. Most interestingly, tassels of the homozygous transformants showed the tasselseed phenotype, indicating that sex determination is regulated by cytokinins. Promoter-reporter gene constructs involving zeatin glucosyltransferases were used to study gene expression in Arabidopsis. The results indicate that the glucosyltransferases have overlapping as well as unique functions. The zeatin binding sites on one of the enzymes were identified through site-directed mutagenesis of amino acids potentially involved in such interactions.
Impacts
Cytokinins are essential to plant growth and development and therefore, it is important to study the effects of differences in cytokinin composition on development of crop plants. Our emphasis at present is on reproductive development in cereals and legumes, which has significant implications for yield improvement.
Publications
- Mok, M.C., Mok, D.W.S., Martin, R.C. 2006. Enzymes responsible for the metabolism of cis-zeatin. U.S Patent No. 7,148,405
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Progress 01/01/05 to 12/31/05
Outputs
Cytokinins are a group of plant growth regulators important in all stages of plant development. Recent efforts have centered on the effects of modifications in cytokinin metabolism on plant growth and development. We have continued our characterization of plants transformed with a gene increasing cytokinin O-glucosylation. The development of corn cobs, kernels, and tassels was studied in detail. Tassel development was significantly inhibited and many florets were empty, indicating the importance of the aglycone for male flower development. To determine the expression of these glucosyltransferase in plants, promoter-reporter gene constructs were generated.
Impacts
Cytokinins are essential to plant growth and development and therefore, it is important to study the effects of differences in cytokinin composition on development of crop plants. Our emphasis at present is on reproductive development in cereals and legumes, which has significant implications for yield improvement.
Publications
- Mok, M.C., Martin, R.C., Mok, D.W.S. 2005. Cytokinin glycosyltransferases: genes, substrates, molecular models and transgenics. Biologia Plantarum 49 Supplemental, S2.
- Pineda A.R. Martin, R.C., Malbeck, J. Vankova, R., Habben, J.E., Mok, D.W.S. Mok, M.C. 2005. Transgenic maize with inceased zeatin O-glucosylation. Biologia Plantarum 49 Supplemental, S7.
- Novakova, M. Dobrev, P. Motyka, V., Gaudinova, A. Malbeck, J., Pospisilova, J., Martin, R. C., Mok, D. W. S., Mok, M. C. 2005. Evalutaion of the possible role of cytokinin O-glucosylation in responses of tobacco plants to drought stress. Biologia Plantarum 49 Supplemental, S31.
- Mok, M.C., Martin, R.C., Dobrev, P. ,Vankova, R., Ho, S., Yonekura-Sakakibara, K., Sakakibara, H., and Mok, D. W. S. 2005. Topolins and hydroxylated thidiazuron derivatives are substrates of cytokinin O-glucosyltransferases with position specificity related to receptor recognition. Plant Physiol. 137: 1057-1066.
- Pineda, A., Martin, R.C., Malbeck, J., Vankova, R., Habben, J.E.,Mok, D.W.S., Mok, M.C. 2005.Transgenic maize with increased zeatin O-glucosylation. Plant Biology. http://abstracts.aspb.org/pb2005/public/P45/7979.html.
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Progress 01/01/04 to 12/31/04
Outputs
This project centers on the effects of modifications in cytokinin (a plant growth hormone) homeostasis on plant growth and development. Previously, genes involved in cytokinin O-glucosylation were isolated. Zea mays (corn and sweet corn) plants transformed with these genes were characterized to determine their effects of crop performance. Increasing the level of glucosylation resulted in pronounced changes in leaf architecture, chlorophyll formation, and rachis development. With the goal of cloning an additional cytokinin gene, an enzyme involved in conversion of zeatin to dihydrozeatin has been purified to near-homogeneity. This particular conversion is important since dihydrozeatin is resistant to cytokinin-degrading enzymes whereas zeatin is not. In addition, further progress was made in defining the constructs effective in suppressing crown gall disease upon transformation of plants. A patent was awarded for the general scheme of generating crown gall-resistant
plants.
Impacts
Targeted changes leading to alterations in cytokinin metabolism can potentially lead to improved plant architecture and increased yield. Crop resistance to the crown gall bacterium is extremely important for cooler regions such as Oregon and Washington. Our scheme of generating resistance through transformation can be applied to essential horticultural crops such as apple and grape.
Publications
- REAM, L.W., MOK, MC, LEE, H. 2004. Plants having enhanced gall resistance and methods and compositions for producing same. US Patent No. 675974. Issued July 6, 2004.
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Progress 01/01/03 to 12/31/03
Outputs
Present investigations concern cytokinin metabolism and plant development. Tobacco, soybean and maize were transformed with a gene (ZOG1) mediating conjugation of active cytokinin to an inactive form. Developmental changes included modifications in plant height and branching habit. Gene constructs potentially conferring resistance to the crown gall bacterium were tested and found to be effective in suppressing disease symptoms after inoculation (collaboration with W. Ream, Dept. of Microbiology).
Impacts
Targeted changes leading to alterations in cytokinin metabolism can potentially lead to improved plant architecture and increased yield. Crop resistance to the crown gall bacterium is extremely important for cooler regions such as Oregon and Washington. Our scheme of generating resistance through transformation can be applied to essential horticultural crops such as apple and grape.
Publications
- VEACH, Y, MARTIN, RC, MOK, DWS, MALBECK J, VANKOVA, R AND MOK, MC. 2003. O-Glycosylation of cis-zeatin in maize. Characterization of genes, enzymes, and endogenous cytokinins. Plant Physiology 131:1374-1380.
- MOK, DWS, MOK, MC and MARTIN, RC. 2003. Nucleic acid molecules from Phaseolus lunatus encoding zeatin-O-glucosyltransferase. US Patent No. 6,600,091. Issued July 29, 2003.
- LEE, H, HUMANN, JL, PITRAK, JS, CUPERUS JT, PARKS TD, WHISTLER CA, MOK, MC, REAM, LW. 2003. Translation start sequences affect the efficiency of silencing of Agrobacterium tumefaciens T-DNA oncogenes. Plant Physiology 133:966-977.
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Progress 01/01/02 to 12/31/02
Outputs
trans-Zeatin is a major and ubiquitous cytokinin in higher plants. cis-Zeatin has traditionally been viewed as an adjunct with low activity and rare occurrence. Recent reports of cis-zeatin and its derivatives as the predominant cytokinin components in some plant tissues may call for a different perspective on cis-isomers. This project intends to elucidate the possible role of cis-zeatin in plant development, through characterization of genes and enzymes important to regulation of cis-zeatin levels. Results include the isolation of a second maize cisZOG (cis-zeatin O-glucosyltransferase) gene, characterization of biochemical properties of cisZOG1 and cisZOG2, differential expression of cisZOG1 and cisZOG2, and identification of substantial amounts of cis-isomers in maize tissues. The recombinant enzymes have similar properties, Kms of 46 and 96 mM, respectively, for cis-zeatin and a pH optimum of 7.5. Expression of cisZOG1 was high in maize roots and kernels, whereas
cisZOG2 expression was high in roots but low in kernels. cis-Zeatin, cis-zeatin riboside, and their O-glucosides were detected in all maize tissues, with immature kernels containing very high levels of the O-glucoside of cis-zeatin riboside. The results are a clear indication that O-glucosylation of cis-zeatin is a natural metabolic process in maize. Whether cis-zeatin serves as a precursor to the active trans-isomer or has any other unique function remains to be demonstrated
Impacts
Cytokinins are very important plant hormones, crucial for normal plant development; therefore, an understanding of the processes underlying the metabolism of cytokinins is of utmost importance. Furthermore, the genes/enzymes identified through this project can be used for plant growth modification in order to improve crop yield.
Publications
- Hanson, K.L. 2002. Isolation and characterization of homologues of the Phaseolus zeatin-O-glucosyltransferase gene from tomato. Thesis, Oregon State University Honors College.
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Progress 01/01/01 to 12/31/01
Outputs
Zeatin and its derivatives are major constituents of plant cytokinins. Metabolic steps modifying the isoprenoid side chain, such as O-glycosylation, are expected to have a direct bearing on cytokinin-mediated processes. To examine this possibility, transgenic tobacco plants were generated harboring a gene (ZOG1) encoding a zeatin O-glucosyltransferase from Phaseolus lunatus under the control of a constitutive (35S) or an inducible (Tet) promoter. In cytokinin/auxin interaction experiments, Tet::ZOG1 leaf discs, in the presence of tetracycline, required 10-fold higher zeatin concentrations for the formation of shoots and callus than the controls. In 35S::ZOG1 plants, developmental changes included adventitious root formation on the lower stems, shorter stature, and axillary shoot growth. Thus increased zeatin O-glucosylation in detached, cytokinin-dependent tissues in Tet::ZOG1 leads to a shift in the response to exogenous zeatin indicative of cytokinin sequestering.
In whole plants, the effect simulates a reduction or a rise in cytokinin activity depending on the tissue and stage of development. In addition to studies on the effects of O-glucosyltransferase genes in transgenic plants, efforts have been focused on isolation of other genes involved in cytokinin glycosylation. This resulted in identification of genes specific for conversion of the stereoisomer of zeatin, cis-zeatin, to its O-glucoside.
Impacts
Isolation of genes involved in cytokinin metabolism and characterization of their effects on developmental patterns in transgenic plants will improve our understanding of plant growth and development as influenced by cytokinins. Moreover, targeted changes involving these genes can lead to improved plant architecture and increased yield.
Publications
- Martin, R.C., K.A. Cloud, M.C. Mok, and D.W.S. Mok. 2000. Substrate specificity and domain analyses of zeatin O-glycoslytransferases. Plant Growth Reg. 32:285-287.
- Mok, D.W.S., R.C. Martin, X. Shan, and M.C. Mok. 2000. Genes encoding zeatin O-glycosyltransferases. Plant Growth Reg. 32:289-293.
- Mok, M.C. and D.W.S. Mok. 2000. Cytokinins: biosynthesis, metabolism and perception. In Vitro Cell. and Devel. Biol.--Plant 36:102-107.
- Mok, M.C., A.I. Samuelsen, R.C. Martin, and D.W.S. Mok. 2000. Fe(III) reductase, the FRE genes, and FRE-transformed tobacco. J. Plant Nutr. 23:1941-1951.
- Martin, R.C., M.C. Mok, and D.W.S. Mok. 1999. Isolation of a cytokinin gene, ZOG1, encoding zeatin O-glucosyltransferase from Phaseolus lunatus. Proc. Natl. Acad. Sci. USA 96:284-289.
- Martin, R.C., M.C. Mok, and D.W.S. Mok. 1999. A gene encoding the cytokinin enzyme zeatin O-xylosyltransferase of Phaseolus vulgaris. Plant Phys. 120:553-557.
- Samuelsen, A.I., R.C. Martin, D.W.S. Mok, and M.C. Mok. 1998. Expression of the yeast FRE genes in transgenic tobacco. Plant Phys. 118:51-58.
- Samuelsen, A.I., F.R. Rickson, D.W.S. Mok, and M.C. Mok. 1997. A temperature-dependent morphological mutant of tobacco. Planta 201:303-310.
- Martin, R.C., M.C. Mok, and D.W.S. Mok. 1997. Protein processing and auxin response in transgenic tobacco harboring a putative cDNA of zeatin O-xylosyltransferase from Phaseolus vulgaris. Plant Journal 12:305-312.
- Martin, R.C., M.C. Mok, J.E. Habben, and D.W.S. Mok. 2001. A maize cytokinin gene encoding an O-glucosyltransferase specific to cis-zeatin. Proc. Natl. Acad. Sci. USA 98:5922-5926.
- Mok, D.W.S. and M.C. Mok. 2001. Cytokinin metabolism and action. Ann. Rev. Plant Phys. and Plant Mol. Biol. 52:89-118.
- Martin, R.C., D.W.S. Mok, R. Smets, H. Van Onckelen, and M.C. Mok. 2001. Development of transgenic tobacco harboring a zeatin O-glucosyltransferase gene from Phaseolus. In Vitro Cell. and Devel. Biol.--Plant 37:354-360.
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