Teaching Pedagogy for the Graduate Assistant, Mentoring Student Research, Research in Biology
CURRENT RESEARCH PROGRAMS:
The general research areas of my laboratory include plant biochemistry and plant physiology with an emphasis on enzymology and molecular biology. My main research interest is in plant natural products and the regulation of their biosynthesis. This research has been supported in part by research grants from the U.S. Department of Agriculture and by a current grant from the National Science Foundation. The focus of the current grant is to determine the biochemical function of putative flavonoid glucosyltransferase clones from Citrus paradisi (grapefruit) and to investigate structure/function relationships of these enzymes. An overview and summary is presented below. Students interested in a research experience are highly encouraged to contact me!
Flavonoid Biosynthesis: Function and Structural Characterization of Putative Flavonoid Glucosyltransferase Clones from Citrus paradisi
Flavonoids are a group of chemicals made by plants that are involved in flower color, fruit color, and some fruit flavors. Flavonoids sometimes cause insects to avoid eating plants, and some play important roles in attracting insect pollinators. Since plants are such a large part of the human diet, there has also been a lot of research into effects of flavonoids on human physiology. There are nine general types of flavonoids that are made using a common "core" biosynthetic pathway. Modifying reactions can also occur giving each compound specific chemical characters. Due to the variety that can be introduced by modifying reactions, over 5000 different flavonoids are found in plants. No single plant makes all of these; rather there is a specific pattern to the flavonoids made by any one plant type. Plants that can make a variety of flavonoids actually may accumulate one particular compound. For example, 70% of the dry weight of young grapefruit leaves and fruits comes from one single chemical - the bitter flavanone naringin. To make the bitter compound naringin, there is a step-wise addition of two sugars to a compound called naringenin. The first sugar added is a glucose and this reaction is catalyzed by a flavanone-specific 7-O-glucosyltransferase. In the next reaction, a rhamnosyltransferase adds a rhamnose sugar to a specific position on the glucose. So, adding two sugars actually makes the compound bitter! Grapefruit are also known to make other flavonoid glycosides and enzymes catalyzing these other reactions have been found in grapefruit leaves and other tissues. This makes grapefruit a nice model plant to study the structure and function of different flavonoid glucosyltransferase enzymes. Once the structure/function relationships for these enzymes are elucidated, it may be possible to develop plant varieties with enhanced or new abilities to produce flavonoids such as insect feeding deterrents, medicinal compounds, or desirable flavor and color components. It may also be possible to decrease the plant's production of undesirable compounds.
The overall goals of this research are to study regulation of biosynthesis of specific flavonoids and to elucidate factors that control synthesis and accumulation during plant development and growth. Emphasis is on regulation of 'derivatization' reactions, e.g. glucosylation reactions, involved in productions of characteristic compounds synthesized and/or accumulated in plants. These substituted and derivatized compounds are the biologically active compounds and, with the exception of anthocyanins, regulation of their production has not been rigorously investigated. Understanding factors regulating biosynthesis of these compounds and roles they play in the physiology and development of plants is critical. It is key to understanding potential outcomes of altering these factors during production of transgenic plants and evaluating physiological responses to "upstream" and "downstream" regulation of metabolic pathways. Another focus of our work is to elucidate structural aspects of flavonoid glucosyltransferases (GTs) that may confer substrate specificity, regiospecificity, and different biochemical regulation properties.
Specific goals include expanding knowledge of flavonoid glucosyltransferases (GTs) structure and function. Citrus paradisi (grapefruit) is well-known for the accumulation of flavanone and flavone glycosides so is a logical source for GTs acting on these compounds. GTs acting on flavonols and chalcones are also found in grapefruit. Isolation, partial purification, and some biochemical characterization of flavonoid GTs from young grapefruit leaf tissue has been done. There are additional biochemical questions that have yet to be answered. These include questions relating to structure/function analysis, identification of domains responsible for conferring acceptor substrate specificity, identification of domains conferring specificity on the structural position for glucose addition, and characterization of structural factors important for biochemical regulation of activity. The grapefruit enzymes, especially the flavanone-specific 7-O-GT involved in synthesis of the grapefruit bitter compound, naringin, are very active yet are present in very small amounts compared to many plant proteins. Because of the relatively low levels of the GT enzymes, an approach other than attempting to isolate sufficient quantities directly from leaf tissue was employed. The approach employed to further this work was to use cloning technologies to obtain candidate grapefruit flavonoid GT clones for subsequent heterologous expression and biochemical characterization. This has been done using several different methodologies.
We have designed RT-PCR primers using a highly conserved region within the PSPG box (the UDP-glucose binding domain) couples with SMARTRace PCR (Clonetech) to obtain 5’ and 3’ clones of putative GTS from very young leaf RNA. Several candidate clones representing partial sequences were identified, and primers designed to “walk” out to the ends to obtain complete sequence information. Bioinformatic and sequence analysis was performed to evaluate candidate clones for the presence of GT-identifying characters. Once identified as a putative GT, primers were designed and used in RT-PCR to obtain full-length clones. To date, we have 3 unique full-length clones in-hand. In addition, 2 other unique clones exist for which we have nearly full-length sequences. We are in the process of obtaining the rest of the information on them.
We also prepared a directionally cloned EST (Expressed Sequence Tag) library to screen for additional flavonoid GT candidates. To date, single pass sequencing has been done on over 4000 clones. These sequences have been analyzed and we found yet another unique candidate flavonoid GT clone. In yet another approach, information on two secondary product glucosyltransferase clones from other citrus species were used to design primers to pull clones from grapefruit. One of these has been functionally characterized and the other is in progress (see below). We also examined the harvEST database, focusing on citrus EST sequences, to inform design of primers to try to pull out additional clones. This is a recent venture, and three more full-length grapefruit putative secondary product glucosyltransferase clones are in-hand.
To determine biochemical function, we modify the ends of our full-length clones and put them into a protein expression vector for heterologous protein expression or in a plant expression vector for transient production of recombinant protein in plant tissues. To date, we have expressed and finished testing 4 of the clones for flavonoid GT activity. For those not acting on flavonoids, we also test other phenolic compounds. We are optimizing protein expression conditions for the other clones and will screen them for flavonoid GT activity as well. This will be followed by more rigorous biochemical characterization to answer the questions described above.
2007-2009 Lab Staff
From left to right: Zhangfan Lin (M.S. student, current), V. “Siddhu” Mallampalli (M.S. student, current), Fei Han (undergraduate exchange student, current), Sean Bowen (Undergraduate ETSU Research Discovery student, 2007-08), Brett Pearson (Undergraduate ETSU Research Discovery student and University Honor’s student, current), Jala Daniel (M.S. student, current), Mat Halter (NSF REU student, 2008, entering graduate school 2009), Dr. McIntosh, Patricia Campbell (NSF REU student, 2007-08), Dr. Daniel Owens (NSF Postdoctoral Research Associate, current), Josephat Asiago (NSF REU student, 2007-08, now in Ph.D. program at Purdue).
Mansell, R.L. and C.A. McIntosh. (1991) 'Citrus spp.: In Vitro Culture and the Production of Naringin and Limonin', in Biotechnology of Medicinal and Aromatic Plants, vol 3, ed. by Y.P.S. Bajaj, Springer-Verlag Berlin, Heidelberg, New York pp 193-210.
Oliver, D.J. and C.A. McIntosh. (1995) 'The Biochemistry of the Mitochondrial Matrix.' in The Molecular Biology of Plant Mitochondria, ed. by S. Levings and I. Vasil, Kluwer Academic Publishers, The Netherlands. pp. 237-280.
McIntosh, C.A. (2000) 'Quantification of Limonin and Limonoate A-ring Monolactone During Growth and Development of Citrus Fruit and Vegetative Tissue by Radioimmunoassay', Chapter 6 in Citrus Limonoids: Functional chemicals in agriculture, food, flavor, and health, ed. by M. Berhow, S. Hasegawa, and G. Manners, ACS Symposium Series. pp. 73-95.
McIntosh, Cecilia A. (2006) “Translational Opportunities in Plant Biochemistry”, in Integrative Plant Biochemistry as We Approach 2010, Recent Advances in Phytochemistry, vol. 40, J.T. Romeo (ed.), Elsevier Press, New York. pp 307-318.
McIntosh, C.A., and R.L. Mansell. 1997. Three-Dimensional Analysis of Limonin, Limonoate A-ring Monolactone, and Naringin in the Fruit of Three Varieties of Citrus paradisi. J. Agric. Food Chem. 45:2876-2883.
Durren, R.L. and C.A. McIntosh. 1999. Flavanone-7-O-Glucosyltransferase Activity from Petunia hybrida. Phytochemistry 52:793-798.
Owens, D.K., T. Hale, L.J. Wilson, and C.A. McIntosh. 2002. Quantification of Dihydrokaempferol Production by Flavanone-3-Hydroxylase using Capillary Electrophoresis. Phytochemical Analysis 13:69-74.
Pelt, J., W.A. Downes, R. Schoborg, and C.A. McIntosh. 2003. Flavanone 3-Hydroxylase (F3H) Expression in Citrus paradisi and Petunia hybrida Seedlings. Phytochemistry 64:435-444.
RoySarkar, T., C.L. Strong, M.B. Sibhatu, L.M. Pike, and C.A. McIntosh. 2007. Cloning, Expression, and Characterization of a Putative Flavonoid Glucosyltransferase from Grapefruit (Citrus paradisi) Leaves. In Proceedings of the 3rd International Congress on Plant Metabolomics, Iowa State University, B. Nikolau (ed.), Springer Publishing, The Netherlands. pp. 247-259 (Invited submission)
Knisley, D., E. Seier, D. Lamb. D.K. Owens, and C.A. McIntosh. 2009. A Graph-Theoretic Model Based on Primary and Predicted Secondary Structures Reveals Functional Specificity in a Set of Plant Secondary Product UDP-Glucosyltransferases. Proceedings of the 2009 International Conference on Bioinformatics, Computational Biology, Genomics, and Chemoinformatics. (Invited, IN PRESS)
Owens, D.K. and C.A. McIntosh. 2009. Identification, Recombinant Expression, and Biochemical Characterization of a Flavonol 3-O-Glucosyltransferase Clone from Citrus paradisi. Phytochemistry (IN PRESS)
Daniel, J.J., D.K. Owens, and C.A. McIntosh. Determining Secondary Product Glucosyltransferase Expression During Citrus paradisi Growth and Development. (In Preparation)
This website was designed by Cecilia McIntosh
and Lois Hyder
Last Update: July 2009 07/17/2009