Applications of recombinant DNA technology - Principles of Gene Manipulation - page 312

Table 14.8 Mode of action of herbicides and method of engineering herbicide-resistant plants.

Herbicide Pathway inhibited Target enzyme Basis of engineered resistance to herbicide

Glyphosate Aromatic amino acid

biosynthesis

5-Enol-pyruvyl shikimate-
3-phosphate (EPSP)
synthase
Acetolactate synthase
(ALS)
ALS

Overexpression of plant EPSP gene or introduction of
bacterial glyphosate-resistant aroA gene

Sulphonylurea Branched-chain amino

acid biosynthesis
Branched-chain amino
acid biosynthesis
Glutamine biosynthesis

Introduction of resistant ALS gene

Imidazolinones Introduction of mutant ALS gene

Phosphinothricin Glutamine synthetase Overexpression of glutamine synthetase or introduction

of the bar gene, which detoxifies the herbicide
Introduction of mutant gene for Q B protein or
introduction of gene for glutathione- S -transferase,
which can detoxify atrazines
Introduction of nitrilase gene, which detoxifies
bromoxynil

Atrazine Photosystem II Q B

Bromoxynil Photosynthesis

these processes, and developing herbicides that are
selective for weeds is very difficult. An alternative
approach is to modify crop plants so that they
become resistant to broad-spectrum herbicides, i.e.
incorporating selectivity into the plant itself rather
than relying on the selectivity of the chemical. Two
approaches to engineering herbicide resistance have
been adopted. In the first, the target molecule in the
cell either is rendered insensitive or is overproduced.
In the second, a pathway that degrades or detoxifies
the herbicide is introduced into the plant. An ex-
ample of each strategy is considered below.
Glyphosate is a non-selective herbicide that
inhibits 5-enol-pyruvylshikimate-3-phosphate (EPSP)
synthase, a key enzyme in the biosynthesis of
aromatic amino acids in plants and bacteria. A
glyphosate-tolerant Petunia hybrida cell line obtained
after selection for glyphosate resistance was found to
overproduce the EPSP synthase as a result of gene
amplification. A gene encoding the enzyme was sub-
sequently isolated and introduced into petunia plants
under the control of a cauliflower mosaic virus
(CaMV) 35S promoter. Transgenic plants expressed
increased levels of EPSP synthase in their chlo-
roplasts and were significantly more tolerant to
glyphosate (Shah et al. 1986). An alternative approach

to glyphosate resistance has been to introduce a
gene encoding a mutant EPSP synthase. This
mutant enzyme retains its specific activity but has
decreased affinity for the herbicide. Transgenic
tomato plants expressing this gene under the control
of an opine promoter were also glyphosate-tolerant
(Comai et al. 1985). Following on from this early
research, several companies have introduced gly-
phosate tolerance into a range of crop species, with
soybean and cotton the first to reach commercializa-
tion (Nida et al. 1996, Padgette et al. 1996). Currently,
nearly three-quarters of all transgenic plants in the
world are resistant to glyphosate (James 2000).
Phosphinothricin (PPT) is an irreversible inhibitor
of glutamine synthetase in plants and bacteria.
Bialaphos, produced by Streptomyces hygroscopicus,
consists of PPT and two alanine residues. When
these residues are removed by peptidases the herb-
icidal component PPT is released. To prevent self-
inhibition of growth, bialaphos-producing strains
of S. hygroscopicus also produce the enzyme phos-
phinothricin acetyltransferase (PAT), which in-
activates PPT by acetylation. The bar gene that
encodes the acetylase has been introduced into
potato, tobacco and tomato cells using Agrobacterium-
mediated transformation. The resultant plants were