SCORE Program Director
Kuehn has had an ongoing, active research program at New Mexico State
University since 1970. His research interests have centered on several
topics in polyamine metabolism and roles in cellular homeostasis.
oxidase (PAO) catalyzes oxidative cleavage of polyamines spermidine
(spd) or spermine (spm) to produce diaminopropane (dap), H2O2, and
an aminoaldehyde derivative. In plants, dap is the precursor for biosynthesis
of norspermidine (nspd) and norspermine (nspm) via the enzyme, Schiff
base reductase decarboxylase (SBRD). Recently, the catabolism of spd
and spm by PAO has been proposed by several investigators to be a causative
agent, through product H2O2 and ensuing oxidative stress, which forces
animal cells into programmed cell death (apoptosis). The gene for PAO
from oat seedlings was recently cloned, sequenced, and characterized
in this laboratory. The availability of this newly isolated gene offers
unique opportunities to gain genetic evidence for a potential role(s)
of PAO and polyamine-catabolism in apoptosis in a plant model test
The hypothesis of this application is: (i) PAO has a causative role
in apoptosis in cells through H2O2 produced by its oxidation of spd
or spm, and (ii) the nspd and nspm produced from dap serve as suppressors
of apoptosis through feedback inhibition of PAO and reduction in H2O2
synthesis. The specific aims of this proposal are: (1) The PAO cDNA
gene sequence will be used to generate antisense PAO gene constructs
ligated to a copper-inducible promoter. These constructs will be used
to transform alfalfa plants with Ti-plasmid methods to analyze the
consequences of controlled PAO-deficiency on apoptosis in plant tissues.
(2) The cDNA gene sequence for the enzyme, SBRD, which catalyzes nspd
and nspm biosynthesis from dap, will be used to generate antisense
SBRD gene constructs ligated to a copper-inducible promoter. These
constructs will be used to transform alfalfa plants in order to analyze
the consequences of uncoupling generation of H2O2 by PAO from the biosynthesis
of nspd and nspm derived from dap by SBRD.
Aims (1) and (2) are direct tests of parts (i) and (ii) of the hypothesis.
(3) The PAO cDNA gene sequence from oat will be used as a gene
probe to attempt the isolation of the human PAO gene from kidney
and liver cDNA libraries. A characterized human PAO gene will make
possible the development of a molecular biological approach to
investigate the role of PAO in generating H2O2 and its alleged
role to elicit animal apoptosis. (4) The cDNA gene sequence of
a signal peptide for the PAO gene will be ligated to a gene coding
for a green fluorescent protein (GFP). The fusion protein produced
from this construction will be analyzed by fluorescent imaging
techniques in tissues of alfalfa and oat plants transformed with
this construction in order to identify the subcellular localization
of PAO. In situ labeling of PAO by immunogold antibody reagents
and electronmicroscopic analysis, will corroborate the localization
studies by the GFP fusion protein technique. These results will
aid in localizing the origin of events that initiate PAO-dependent
D. Kuehn and G.C. Phillips. 2005. Role of Polyamines
in Apoptosis and
Other Recent Advances in Plant Polyamines. Critical Reviews
D. Kuehn. 2005. Bridges to American Indian Students in Community
Colleges Program. Science 307:1685. [Invited Contribution
to Science Next
Wave at www.nextwave.org <http://www.nextwave.org/>
, March 18th issue.
Isolation and Sequence of the Gene for Polyamine Oxidase from
Avena sativa L. (Oat.)
Gardner-Johnson, Yvette J., A. Dharma, N. Dong, G.C. Phillips, D.
Benn. and G.D. Kuehn (2000)
Plant Physiology, in review.
Polyamines and Their Biological Consequences in Mammals.
Patoaka, J. and G.D. Kuehn (1999)
Acta Medica, in press.
of uncommon polyamines in cultured tissues of maize
Koc, E.C., S. Bagga, D.D. Songstad, S.R. Betz, G.D. Kuehn, and G.C.
In Vitro Cellular and Developmental Biology-Plant 34: 252-255.
Aminopropyltransferase is Responsible for Biosynthesis of Spermidine,
Spermine, and Multiple Uncommon Polyamines in
Osmotic Stress-Tolerant Alfalfa (Medicago sativa L.).
Bagga, S., J. Rochford, Z. Klaene, G.D. Kuehn, and G.C. Phillips
Plant Physiology 114:445-454.
of Chile Pepper Fruit Peroxidases During Ripening.
Biles, C.L., G.D. Kuehn, and M.M. Wall (1997)
Plant Physiology and Biochemistry 35:273-280.
Purification and Kinetic Characterization of Acyl CoA:alcohol
Transacylase from Developing Jojoba Cotyledons.
Garver, W.S., J.D. Kemp and G.D. Kuehn (1996)
Plant Physiology: Advanced Life Sciences 13:45-49.
High Performance Liquid Chromatography- Based Radiometric Assay
for Acyl-CoA:alcohol Transacylase from Jojoba.
Garver, W.S., J.D. Kemp, and G.D. Kuehn (1995)
Analytical Biochemistry 196:335-340.
Acid Matrices for the Affinity Purification of Glycoproteins
Hageman, J.H. and G.D. Kuehn (1992)
Practical Protein Chromatographys. Methods in Molecular Biology Series
(A. Kenney and S. Fowell, Eds.), The Humana Press, Inc., 11:45-71.
and Partial Characterization of Transglutaminase from Physarum
Klein, J.D., E. Guzman, and G.D. Kuehn (1992)
Journal of Bacteriology 174:2599-2605.
for the Occurrence of Polyamine Oxidase in the Dicotyledonous
Plant Medicago sativa L. (Alfalfa)
S., A. Dharma, G.C. Phillips, and G.D. Kuehn (1991)
Plant Cell Reports 10:550-554.
of the Large Subunit of Ribulose 1,5 bisphosphate/carboxylase/oxygenase
as a Substrate for Transglutaminase in
Margosiak, S.A., A. Dharma, A.P. Gonzales, D. Louie, and G.D. Kuehn
Plant Physiology 92:88-96.
of Norspermidine and Norspermine in Medicago sativa L. (Alfalfa).
Rodriguez-Garay, B., G.C. Phillips, and G.D. Kuehn (1989)
Plant Physiology 89:525-529.