Stress inducible promoters

1. Unlike the warm climates
that rule Asian countries,
North America is at the
mercy of harsh climates,
and frost. Productivity, and
yield is as such limited not
only by the crop's
production capacity, but
also temporal factors. In
particular, major crops
such as potatoes – freezing
sensitive – are cultivated in
cold climates which
substantially affects yield.
Between impossibility, and
actuality lies the potential
to genetically alter crops to
serve a defined function.
Where traditional breeding
has failed, biotechnology
has offered much potential
in the generation of crops
that are freezing tolerant.
Typically, this is realized
by conferring a gene -
AtCBF - from an organism
to a selected crop species.
AtCBF1-3, the CBF genes
found in Arabidopsis
thaliana, regulate the
activation of a myriad of
genes responsible for
freezing tolerance. In
particular, CBF genes
(AtCBF1, AtCBF2, and
AtCBF3), a transcription
factor found in Arabidopsis
thaliana, ciphers proteins
that bestows freezing
tolerance upon the plant
via induction of COR (cold
responsive genes).
Constitutive expression of
AtCBF was however found
to be associated with
adverse side effects.
Introduction of a transgene
is at times insufficient for
both resistance, and
productivity. The first
potential solution is to
inspect alternative
transgenes; the second
solution requires
modifications to the
existing system. In this

2. issue, Pino et al., (2007)
replaced a constitutive
promoter by a stress
inducible promoter to
enable production of the
transgene only under
stressful conditions. By
substituting a constitutive
CaMV 35S promoter with
a stress inducible rd29A
promoter, the authors
manage to bestow upon
Solanum tuberosum both
resistance, and
productivity.
It has been noted that
constitutive expression of
AtCBF – that confer
freezing resistance – leads
to retarded growth,
lowered biomass/foliar
mass, late flowering, and
abolished tuber formation.
The simplest solution to
this problem would be to
substitute the current
transgene with another.
However, it is quite
probable that the novel
transgene will result in the
same problem. In fact,
Kasuga et al. (1999)
utilized the DRE-binding
protein DREB1A, and the
CaMV 35S constitutive
promoter to confer
resistance to freezing,
drought, and salt stresses.
However, constitutive
expression of the transgene
resulted in growth
retardation as well. The
alternative is to modify the
existing system; in this
case, the latter is done by
inducing the transgene
only when required or
under cold stress. In fact, it
has been shown multiple
times that a transgene
associated with a stress
inducible promoter
compared to a constitutive
promoter possess fewer
negative traits such as low
yield, or biomass.
Replacing a constitutive
promoter with a stress-
inducible promoter results
in transgenic Solanum
tuberosum plants that are
both highly productive,
and resistant.
The experiments
conducted by Pino et al.,
(2007) are akin to those
conducted by Kasuga et al.
(1999). Kasuga et al.
(1999) attempted to
compare the phenotype of
the 35S:DREB1A, and the
rd29A:DREB1A lines.
They showed that the
35S:DREB1A line had
fewer seeds, and showed
stunted growth. The
rd29A:DREB1A lines had
mild growth retardation.
Further, Kasuga et al.
(1999) showed that 96.2%
of the rd29A:DREB1A
lines, and 77.9% of the
35S:DREB1A lines
survived after exposure to
cold temperatures. Oddly
enough, the
rd29A:DREB1A lines
outperformed the
35S:DREB1A lines for
many a stress (drought
stress:
35S:DREB1A=39.7-69.2%
survival,
rd29A:DREB1A=76.7%
survival; salinity:
35S:DREB1A=29.4%
survival,
rd29A:DREB1A=78.6%
survival).
Much like Kasuga et al.
(1999), Pino et al., (2007)
altered the genomic unit by
replacing the constitutive
CaMV 35S promoter with
the rd29A stress inducible
promoter. Constitutive
over-expression of AtCBF
is responsible for the side
effects; use of a stress
inducible promoter such as
rd29A can reduce adverse
side effects. Following
substitution of the
constitutive CaMV 35S
promoter for the stress
inducible rd29A promoter,
and transformation using
agrobacterium into S.
tuberosum cv. Umatilla,
explants – derived from
transformed plants – were
propagated in vitro. Tissue
culture was utilized to
regenerate the callus.
Relative to the 35S:AtCBF
lines (constitutive
promoter), the
prd29A:AtCBF lines
showed higher plant, and
tuber mass. Foliar biomass
in the prd29A:AtCBF lines

3. were unaffected by the
construct.
The paper by Pino et al.,
(2007) implies that
transgenic systems can be
further altered to obtain
desired features instead of
resorting to novel
transgenes. This also
implies that transgenes
alone do not control the
system, but rather that by
modifying a subsection of
a unit (that controls
transcription/translation),
one can tweak the plant’s
genome to perform in a
particular manner. In other
words, this also implies
that when generating
transgenic plants,
introduction of a novel
gene or alteration of
existing genes is one way
of modifying the system;
however, modifications
can be carried out on the
promoter, and the polyA
signal amongst others. In
this paper, the authors
attempt to modify the
promoter in addition to the
transgene, however, it is
quite possible that with
further modifications, this
system can be tweaked.
For instance, it is quite
well known that there
exists a negative
relationship between
resistance, and growth. The
Resource allocation theory
states that due to a
limitation in the resources
available, the plant must
partition said resource
between resistance, or
growth. In such cases, the
activation of a particular
gene under a particular set
of circumstances would
enable the plant to bypass
this limitation. If such is
the case, then one might
add that reducing energy
expended for production of
proteins might aid the
plant. In other words, the
turn-over rate, the efficacy
of the transgene, and the
time of production would
affect the phenotype of the
plant.
References
Kasuga, M., Liu, Q.,
Miura, S., Yamaguchi-
Shinozaki, K., and
Shinozaki, K. (1999).
Improving plant drought,
salt, and freezing tolerance
by gene transfer of a single
stress-inducible
transcription factor. Nature
Biotechnology 17, 287-
291.
Pino, M., Skinner, J., Park,
E., Jeknić, Z., Hayes, P.,
Thomashow, M., and
Chen, T. (2007). Use of a
stress inducible promoter
to drive ectopic AtCBF
expression improves potato
freezing tolerance while
minimizing negative
effects on tuber yield. Plant
Biotechnology J 5, 591-
604.