By Janaki Krishna

Water availability is the primary limiting factor of global crop yields.
Consequently, considerable effort is devoted to converging breeding and
technological research to develop crops with improved performance under
water-limiting conditions. Transgenic plants tolerant to abiotic stress are
being developed through the introduction of multiple genes; however,
successfully producing stress tolerant crops remains a challenging task.

Tolerance to abiotic stresses is improved by the expression of bacterial
cold shock proteins (CSP). A small set of E. coli cold shock proteins (CSPs)
exhibits a prototypical cold shock domain (CSD) during cold treatment.
Expression of related cold shock proteins from bacteria?CspA from
Escherichia coli and CspB from Bacillus subtilis?promotes stress adaptation
in multiple plant species.

Both CSPs and CSDs are found in bacteria and eukaryotes, including plants.
In bacteria, CSD proteins (7 ? 10 kD) contain sufficient nucleic acid
binding activity to function as RNA chaperones. RNA chaperones?often called
"protein chaperones"?are proteins that aid RNA folding by preventing or
resolving misfolded species of RNA, in contrast to proteins that assist
protein or RNA folding by catalyzing steps along the folding pathway or by
stabilizing the final folded protein or RNA structure. RNA chaperones in
bacteria favor active transcription, translation, and/or ribosome assembly.
Research on CSPs and CSDs confirms that the endogenous function of CSPs in
plants depends on RNA binding/chaperone activity through the CSD, and CSPs
regulate stress responses through a post-transcriptional mechanism.

Recently, Monsanto Company researchers showed that bacterial CSPs can confer
improved stress adaptation in multiple plant species by demonstrating
improved stress tolerance in both E. coli and maize, and reported further
'proof of concept' in dicots and monocots?Arabidopsis, rice, and maize.
Expression of bacterial CSPs improves cold tolerance in transgenic
Arabidopsis when compared to nontransgenic controls. Similar experiments
expressing CspA and CspB in transgenic rice show improved plant growth rates
in plants exhibiting an increased tolerance to a number of abiotic stresses
like cold, heat, and water deficit.

Further abiotic stress testing of transgenic CspA and CspB in maize
demonstrates that transgenic expression by CspB improves vegetative growth
performance. Twenty-two CspB transgenic events were evaluated under water
limited field trials using commercial grade corn in an environment that
received no rainfall during the target period. The best performing events
show a growth rate increase of 12% and 24% under water deficit conditions.
The CspB-expressing plants also demonstrate significant improvements in
chlorophyll content by 2.5%, increasing the photosynthetic rates by 3.6%
across all events. Transgenic maize plants expressing CspA under greenhouse
conditions were also tested.

Increases in plant growth rates, chlorophyll content, and photosynthetic
efficiency are key indicators of plant productivity and are expected to
improve the overall yield of the plant. Therefore, the reproductive
performance of CspB-expressing maize plants was also evaluated by harvesting
all kernel-bearing ears from six replicates (34 plants per replicate) for
each of six events selected for harvest, based on the magnitude of their
improved vegetative performance. An across-event analysis demonstrates
significant improvements were made in the number of plants showing an
increased number of kernels per plant. These improvements are congruent with
the expected results based on the timing of limited-water treatment, which
was provided during the late vegetative stages and early immature ear
development, and was relieved with sufficient water during the pollination
and grain-fill periods.

Grain yield trials were also carried out under water deficit stress and
non-stress conditions on 10 CspA- and 10 CspB-positive events that had
demonstrated superiority in vegetative performance. Grain yield data were
collected from four field sites where water was limited during the late
vegetative phase of development, a treatment similar to the initial water
deficit trial. An across-event analysis demonstrates that CspA transgenic
lines contribute to a yield increase of 4.6% under water stress, with the
two best performing events showing 30.8% and 18.3% improvement. CspB
transgenic plants show 7.5% improved yield averages over controls; the best
two performing events, CspB-Zm events 1 and 2, demonstrate yield
improvements of 20.4% and 10.9%, respectively. These are the same two events
that demonstrate significant improvements in leaf growth, chlorophyll
content, and photosynthetic rates, indicating that these improvements in
vegetative productivity translate into improvements in reproductive
performance and grain yield.

To further investigate the ability of the CspB gene to provide tolerance to
maize under water deficit conditions, CspB-Zm event 1 was deployed into
three hybrid backgrounds and evaluated under two distinct stress treatment
conditions at five replicated locations. The two treatments result in a
decrease of overall yield of approximately 50%, relative to well-watered
treatments. When compared to controls, the CspB transgenics demonstrate
improved yields by at least 0.5 t/ha across 12 out of 15 reproductive stress
treatments. The multi-year analysis with CspB-Zm event 1 shows the stability
of yield advantages across locations under water-limited conditions, which
proves the utility of this technology across the US maize growing regions.

The performance of CspB-Zm event 1 was also assessed by combining yield
performance data from three hybrid test crosses collected over four years.
The event yielded a 10.5% average benefit across the four years. CspB-Zm
event 1 was also tested under western dryland maize conditions without
supplemental water. Under these conditions when compared to the
non-transgenic control, the CspB transgenic event contributed a yield
benefit of up to 0.75 t/ha or 15%.

In summary, constitutive expression of E. coli CspA and bacterial RNA
chaperones can confer abiotic stress tolerance in transgenic Arabidopisis,
rice, and maize. This technology also provides a stable yield improvement
under water limiting conditions.

Sources:
Castiglioni P et al. 2008. Bacterial RNA chaperones confer abiotic stress
tolerance in plants and improved grain yield in maize under water-limited
conditions. Plant Physiol 147, 446-455

Goldstein J, Pollitt NS, Inouye M. 1990. Major cold shock protein of
Escherichia coli. Proc Natl Acad Sci USA 87, 283-287

Karlson D, Imai R. 2003. Conservation of the cold shock protein of
Escherichia coli. Plant Physiol 131, 12-15

Nakaminami K, Karlson DT, Imai R. 2006. Functional conservation of cold
shock domains in bacteria and higher plants. Proc Natl Acad Sci
USA103,10122 -10127
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