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 4000 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. This research is
designed to gain an understanding of flavonoid biosynthetic regulation. "Core"
and "modifying" enzymes will be used to study factors regulating production of
flavonoid intermediates versus "end products", especially those controlling
synthesis/accumulation in tissues during plant growth and development. Once
understood, 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.
PROJECT SUMMARY
The overall goals of this research are to study biochemical and genetic
regulation of flavonoid biosynthesis. 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.
Specific goals include expanding knowledge of flavonoid
glucosyltransferases (GTs) to include those that act on flavonoid classes
other than flavonols and anthocyanins and/or that act on positions other than
the 3-OH group. 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, 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
used to further this work was to use cloning technologies to obtain candidate
grapefruit flavonoid GT clones for subsequent heterologous expression and
biochemical characterization.
This was done using two different techniques.
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. Bioinformatiic 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 3 unique
candidate flavonoid GT clones.
One of these was a full-length clone. The other two were not full-length,
and we are in the process of obtaining the rest of the information on these
clones (as briefly described above).
In addition to the work described above, we have modified the ends of 3 of
our full-length clones (and are working on the 4th) and put them
into a protein expression vector for heterologous protein expression. We have expressed and tested 1 of them
for flavonoid GT activity. We are optimizing protein expression conditions for
the others and will be screening them for flavonoid GT activity as well. This will be followed by more rigorous
biochemical characterization to answer the questions described above.
SELECTED PUBLICATIONS
BOOK CHAPTERS/INVITED REVIEWS:
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 Maxtrix.' 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) 'Quanitification 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. (In Preparation).
ARTICLES (selected; student authors are underlined)
McIntosh,
C.A. and R.L. Mansell.
1990. Biosynthesis of naringin in Citrus paradisi:
UDP-glucosyltransferase activity in grapefruit seedlings. Phytochemistry 29:1533-1538.
McIntosh,
C.A., L. Latchinian, and
R.L. Mansell. 1990. Flavanone-specific O-7 glucosyltransferase activity in
Citrus paradisi seedlings: Purification and characterization. Arch. Biochem. Biophys. 282:50-57.
McIntosh,
C.A. and D.J. Oliver. 1992.
NAD-Linked Isocitrate Dehydrogenase: Isolation, Purification, and
Characterization of the Protein from Pea Mitochondria. Plant Physiology 100:69-75.
McIntosh,
C.A. and D.J. Oliver. 1992.
Isolation and Characterization of the Tricarboxylate Transporter from Pea
Mitochondria. Plant Physiology
100:2030-2034.
McIntosh,
C.A. and D.J. Oliver. 1994.
The Phosphate Transporter from Pea Mitochondria: Isolation and Characterization
in Proteolipid Vesicles. Plant
Physiology 105:47-52.
McIntosh, C.A. 1997.
Partial Purification and Characteristics of Membrane-Associated NAD+-Dependent
Isocitrate Dehydrogenase Activity from Etiolated Pea Mitochondria. Plant Science 129:9-20.
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. 2005. 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.), Elsevier Publishers
(Invited Submission, In Press)
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Last Update: 09/19/2007
