Metabolic Engineering to Study the
Regulation/Plasticity of, and to Modify Diterpene Metabolism
in Trichome Gland Cells
George J. Wagner
University of Kentucky
Plant trichome glands represent potential
"green-factories" for the biosynthesis of useful
chemicals (molecular farming). These factories require
only energy from the sun, carbon dioxide from the air, water,
and minerals as feedstocks. Before this potential can be
realized, however, the regulation and plasticity of carbon
flow in trichome glands must be better understood, and
protocols for engineering glands to produce desired chemicals
must be developed. The specific objectives of this
project are 1) to investigate the regulation/plasticity of
carbon flow in the biosynthesis of trichome-exudated
diterpenes of glands, and 2) to study the feasibility of
introducing heterologous genes into glands to facilitate
molecular farming. Exudating plant trichome glands are
specialized tissues that occur on the aerial surfaces of about
30% of higher plants. They produce exudates that serve
the plant in pest/insect resistance, temperature control, etc.
We isolated a gland-specific c-DNA library, which yielded a
P450 gene involved in the conversion of cembratriene-ol
(CBT-ol) to cembratriene-diol (CBT-diol), the major diterpene
of the experimental tobacco, T.I. 1068. This plant can
accumulate up to 17% of leaf dry weight as trichome exudate,
and CBT-diol accounts for 60% of exudate weight.
Knockdown of the P450 gene activity (using antisense and
co-suppression strategies) resulted in a 20-fold increase in
CBT-ol and a corresponding decrease in CBT-diol. Exudate
from high CBT-ol plants was more toxic to aphids, and high
CBT-ol plants had greatly reduced aphid colonization.
Thus, we have metabolically engineered the last step in the
biosynthesis of the major exudate diterpene and significantly
altered natural-product-based aphid resistance in this
plant. Knockdown strategies (antisense, co-suppression,
and RNA interference) are being applied to determine the
function of additional trichome-specific genes, and to
determine the impact of altering their activities on exudate
chemistry. Full-length genes of known function will be
introduced into host plants, trichome-specifically, to
determine the ability glands in these plants to accommodate
heterologous diterpene biosynthetic genes. A
trichome-specific promoter has been isolated that can serve in
planned transformation experiments designed to metabolically
engineer glands.
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