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Editor’s Choice Series on the Next Generation of Biotech Crops
Bacterial RNA Chaperones Confer Abiotic Stress
Tolerance in Plants and Improved Grain Yield in
Maize under Water-Limited Conditions[W]
Paolo Castiglioni
1
, Dave Warner, Robert J. Bensen, Don C. Anstrom, Jay Harrison, Martin Stoecker,
Mark Abad, Ganesh Kumar
2
, Sara Salvador, Robert D’Ordine, Santiago Navarro, Stephanie Back,
Mary Fernandes, Jayaprakash Targolli, Santanu Dasgupta
2
, Christopher Bonin,
Michael H. Luethy, and Jacqueline E. Heard*
Monsanto Company, Mystic Research, Mystic, Connecticut 06355 (P.C., D.W., R.J.B., D.C.A., C.B.,
M.H.L., J.E.H.); Monsanto Company, Chesterfield, Missouri 63017 (J.H., M.S., M.A., R.D., S.N.,
S.B.,M.F.);MonsantoCompany,Malleswaram,Bangalore,560003India(G.K.,J.T.,S.D.);and
Monsanto Company, Cambridge, Massachusetts 02139 (S.S.)
Limited available water is the single most important
factor that reduces global crop yields, with far reach-
ing socioeconomic implications. In North America
alone, it is estimated that 40% of yearly maize (Zea
mays) crop losses are due to suboptimal water avail-
ability (Boyer, 1982). Agriculture currently accounts
for 70% of the fresh water used by humans. This rate of
water use can exceed local regeneration rates, often
relying on underground aquifers that are rapidly be-
ing depleted (Morison et al., 2008). The impending
scarcity of water available for agriculture will surely
increase overall costs of crop production and drive the
need for crops that use water more efficiently. While
tremendous progress has been made through breeding
and through cultural practices that improve maize
yields in water-limited environments, the potential for
additional large improvements still exists, and posi-
tive impacts on yield and increased yield stability
across a broad range of water availability is of great
value to farmers, consumers, and the environment.
Maize plants are sensitive to water-deficit stress
throughout the growing season. Stresses that occur
during the flowering stage, either just before floral
initiation or immediately after pollination, result in the
most significant reductions in end-of-season grain
yields (Claassen and Shaw, 1970; Boyer and Westgate,
2004). Water-deficit stress during the vegetative
growth phases typically leads to reductions in overall
productivity, resulting in grain loss through reduc-
tions in kernel numbers. Late-stage drought stress,
during the grain-filling period, can frequently lead to
reductions in yield by reducing kernel size as well as
increasing rates of kernel abortion, depending upon
the severity of the stress.
Improved plant performance under severe water-
limited growth chamber and greenhouse conditions
has been achieved through multiple transgenic ap-
proaches, including the use of osmotic protectants
such as the sugar alcohols trehalose (Romero et al.,
1997), mannitol (Tarczynski et al., 1993), galactinol
(Taji et al., 2002), and ononitol (Sheveleva et al., 1997).
Accumulation of zwitterionic compounds such as Pro
(Kishor et al., 1995) and Gly-betaine (Rathinasabapathi
et al., 1994), or of protein protectants such as HVA1 (Xu
et al., 1996), have all demonstrated an ability to confer
tolerance to a variety of abiotic stresses. To date, none
of the above approaches has been shown to provide
durable tolerance in an agriculture production setting.
Expression of a maize CAAT box transcription factor,
ZmNF-YB2, has been shown to confer drought toler-
ance and enhanced photosynthetic capacity under
drought stress with improvements in grain yield
observed across several growing seasons in maize
(Nelson et al., 2007). These studies clearly demonstrate
that plants are amenable to improved stress tolerance
through multiple mechanisms of action.
Biotechnology approaches facilitate our ability to
survey and capitalize on the extensive genetic diver-
sity that exists in nature and to improve on conserved
pathways important for adaptation to environmental
stress. Adaptation requires rapid recovery in growth
and maintenance of cellular function following stress.
We have looked to model systems such as bacteria
and plants for insights into adaptive stress response
pathways and commonalities among stress response
mechanisms broadly in search of candidates for crop
improvement. In this article, we demonstrate that ex-
pression of related cold shock proteins (CSPs) from
bacteria, CspA from Escherichia coli andCspBfrom
1
Present address: 1920 Fifth Street, Davis, CA 95616.
2
Present address: 700 Chesterfield Parkway, Chesterfield, MO
63017.
* Corresponding author; e-mail jacqueline.e.heard@monsanto.com.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Jacqueline E. Heard (jacqueline.e.heard@monsanto.com).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.118828
446 Plant Physiology, June 2008, Vol. 147, pp. 446–455, www.plantphysiol.org Ó2008 American Society of Plant Biologists
Bacillus subtilis, promotes stress adaptation in multiple
plant species. Interestingly, expression of CSP proteins in
maize is not associated with negative pleiotropic effects,
indicating that stress tolerance does not come at a cost to
crop productivity under well-watered conditions.
RNA chaperones are ubiquitous and abundant pro-
teins found in all living organisms and viruses. RNA
tends to be kinetically trapped in misfolded forms,
and RNA binding proteins, acting as chaperones, can
resolve these structures, ensuring accessibility for its bio-
logical function. In bacteria, RNA chaperones are be-
lieved to play a general role in sustaining active growth
by favoring active transcription, translation, and/or ri-
bosome assembly. In E. coli, cell growth is arrested by
cold shock, and the arrest is associated with a significant
reduction in protein synthesis (Etchegaray et al., 1996;
Etchegaray and Inouye, 1999). A small set of proteins
rapidly accumulates during the cold shock (for review,
see Gualerzi et al., 2003; Horn et al., 2007). These pro-
teins have been designated as CSPs and can account for
as much as 10% of the newly synthesized protein of
cold-shocked cells (Goldstein et al., 1990). One such
example of a CSP protein is the E. coli protein CspA,
which contains a prototypical cold shock domain (CSD)
that is composed of 65 to 70 amino acid residues and
has been reported in bacteria, archaea, and eukaryotes,
including plants (for review, see Karlson and Imai,
2003; Weber and Marahiel, 2003; Horn et al., 2007). In
bacteria, the CSP proteins are 7 to 10 kD in size and
contain the nucleic acid binding activity sufficient for
their function as RNA chaperones. The CSD contains a
polynucleotide binding function, and in E. coli CspA is
reported to act as an RNA chaperone where it binds to
and, when necessary, converts double-stranded RNA
into single-stranded RNA with low sequence selectivity
(Jiang et al., 1997). The chaperone function of CspA, as
well as other CSP proteins, is thought to be important
for stimulating growth following stress acclimation and
during periods of high metabolic activity. A recently
proposed model describes CSPs working in conjunction
with a DEAD box helicase to rescue misfolded mRNA
molecules and maintain proper initiation of translation
(Hunger et al., 2006). This model is consistent with pre-
viously reported evidence for the colocalization of CSPs
with ribosomes in transcriptionally active cells, where
CSP proteins are proposed to be implicated in coupling
transcription with translation (Mascarenhas et al., 2001;
Weber et al., 2001; for review, see El-Sharoud and
Graumann, 2007).
Several reports described below support the hypoth-
esis that the endogenous function of CSPs in plants
relies on RNA binding/chaperone activity through the
CSD and that these proteins, similarly to bacteria,
regulate stress responses through a posttranscriptional
mechanism. Mussgnug et al. (2005) describe the func-
tion of NAB1, a CSD-containing RNA chaperone in
Chlamydomonas that links its RNA binding/chaperone
activity with a target protein important for high light
acclimation. NAB1 plays a role in high light acclima-
tion by regulating the size of the light-harvesting
antennae of PSII through posttranscriptional control
of light-harvesting chlorophyll binding protein. Bind-
ing of NAB1 protein stabilizes light-harvesting chlo-
rophyll binding protein mRNA at the preinitiation
level via sequestration or masking and thereby sup-
presses translation. NAB1 activity accounts for ap-
proximately 50% of the translational repression of the
protein, suggesting a mechanism that fine tunes ex-
pression at a posttranscriptional level and allows for
very rapid response to occur under changing environ-
mental conditions. The CSD domain of NAB1 was
determined to be necessary and sufficient for specific
high-affinity binding to this target. This RNA masking
function was described previously in Xenopus oocytes
for another CSD-containing protein, FRGY2 (Matsumoto
et al., 1996; Manival et al., 2001).
In higher plants, Karlson et al. (2002) reported that
wheat (Triticum aestivum) expresses a homolog of E.
coli CSPA. The researchers found that this homolog,
WCSP1, contains two RNA binding domains and
increases in concentration during cold treatment.
Kim et al. (2007) investigated a Gly-rich RNA binding
protein from Arabidopsis (Arabidopsis thaliana), GRP2,
and demonstrated that this protein plays a role in salt
and cold stress adaptation. The researchers also found
that GRP2 can rescue cold-sensitive E. coli CSP qua-
druple knockout and exhibits other properties con-
sistent with an RNA binding/chaperone function.
AtCSP2 (synonymous with GRP2) is also associated
with floral transition and seed development (Fusaro
et al., 2007; Sasaki et al., 2007). The possibility that
CSPs functions in stress adaptation at least partially
mediated by its role in the development and protection
of reproductive structures should be explored. In
maize, floral transition and reproductive development
stages are the most sensitive to the water deficit stress
conditions in terms of yield impact.
In this article, we demonstrate that bacterial CSPs
can confer improved stress adaptation to multiple
plant species. The action of CSPs in plants through a
conserved stress adaptation mechanism common to
plants and bacteria is supported by data showing that
a functional RNA binding motif is required for the
improved stress tolerance in both E. coli and maize.
Stress tolerance at both vegetative and reproductive
stages is reported with enhanced yield stability ob-
served in maize under water-limiting conditions that
were either imposed at various stages during plant
development through controlled irrigation or occurred
naturally in the western dryland region of the U.S.
corn belt. Breadth of tolerance across environments
and germplasms are key elements in establishing the
value of transgenic strategies for crop stress tolerance
improvement and require years of rigorous field test-
ing to characterize the potential benefits.
ARABIDOPSIS COLD TOLERANCE
The expression of bacterial CSPs was shown to im-
prove cold tolerance in transgenic Arabidopsis seedlings
Bacterial RNA Chaperones Confer Stress Tolerance to Plants
Plant Physiol. Vol. 147, 2008 447
germinated and grown at low temperatures on stan-
dard agar media in petri dishes (Fig. 1). Transgenic
Arabidopsis seedlings expressing CspA and CspB were
tested for improved growth under low temperature
conditions, using nontransgenic seedlings as controls.
Following a 6-week treatment of 8°C under constant
light, seedlings were visually scored for relative growth.
At the end of the treatment period, a visual improve-
ment in growth was noted for both transgenes, rela-
tive to their negative controls. This proof-of-concept
for CSPs, demonstrating improved cold tolerance in a
dicot plant species, was followed by similar abiotic
stress experiments in transgenic rice (Oryza sativa).
RICE COLD, HEAT, AND
WATER-DEFICIT TOLERANCE
Transgenic rice plants expressing CspA and CspB
manifest improved stress tolerance for a number of
abiotic stresses, including cold, heat, and water defi-
cits. Improved tolerance was documented by demon-
strating improved plant growth rates of transgenic
plants relative to their nontransgenic controls, as mea-
sured by plant height (Table I).
On average, the cold and heat treatments led to 35%
and 31% reductions, respectively, in the final plant
height of nontransgenic control plants. In the cold
treatment, three of eight CspB-positive events demon-
strated significantly greater plant heights (P,0.05) at
the end of the recovery period when compared to
nontransgenic control plants. In the heat treatment, six
of eight CspB-positive events demonstrated signifi-
Figure 1. Transgenic Arabidopsis seedlings demonstrate improved
growth under constant light conditions at 8°C for 6 weeks. Transgenic
Arabidopsis seedlings expressing CspA or CspB are displayed on the
right and exhibit more growth under these conditions relative to non-
transgenic controls. A, CspA-negative control. B, CspA-positive trans-
genic. C, CspB-negative control. D, CspB-positive transgenic. The positive
growth effect was only observed under the chilling stress conditions
and not at 25°C (data not shown). Experimental details are described in
Supplemental Materials and Methods S1.
Table I. Transgenic rice plants expressing CspA or CspB demonstrated improved growth
characteristics under cold, heat, and water-deficit treatments
CspA transgenics were subjected to a cold stress of 3 d at 10°C and a heat stress of 50°C for 3 h, followed
by a 14-d recovery period. CspB transgenics were subjected to a cold stress of 1 h at 8°C and a heat stress of
53°C for 1 h, followed by a 14-d recovery period. CspB transgenics were also subjected to water-deficit
treatments by growth under 25% soil saturation for 15 d. Final plant height (defined as the distancebetween
the soil and the upper-most point of the leaf blade) for individual seedlingswas determined at the end of the
recovery period. Results shown for each event are the mean values for 10 transgene-positive or control
plants per treatment. Events have been designated as Os1, Os2, etc., to indicate the crop species O. sativa
(Os). Experimental details are described in Supplemental Materials and Methods S1. Nt, Not tested.
Gene-Event
Plant Height
Cold Treatment Heat Treatment Drought Treatment
cm
CspA-Os1 28.8 63.1a26.7 65.0 Nt
CspA-Os2 29.5 62.9a26.2 63.5aNt
CspA-Os3 15.8 62.9 25.2 61.9aNt
CspA-Os4 26.1 63.8 20.8 61.2 Nt
CspA-Os5 27.2 62.3a23.2 61.8 Nt
CspA-Os6 29.6 63.5a29.3 65.0aNt
CspA-Os7 24.6 63.4 24.7 62.8 Nt
Nontransgenic 20.6 61.7 18.5 63.5 Nt
No treatment 37.9 68.6 37.9 68.6 Nt
CspB-Os1 28.8 62.9 34.5 62.1a17.4 62.2
CspB-Os2 30.2 63.2 32.4 61.5aNt
CspB-Os3 30.4 62.2a28.8 64.2 Nt
CspB-Os4 32.1 63.4a33.3 63.9aNt
CspB-Os5 29.5 63.6 34.0 62.1a18.1 61.6a
CspB-Os6 27.1 63.4 33.8 63.7aNt
CspB-Os7 23.8 62.9 25.7 64.3 18.5 62.2a
CspB-Os8 33.8 63.5a34.8 61.7a19.5 62.0a
Nontransgenic 23.9 63.7 25.5 63.0 12.8 63.2
No treatment 36.7 64.0 36.7 64.8 24.6 61.6
a
Indicates significant (P,0.05) improvement relative to nontransgenic control.
Castiglioni et al.
448 Plant Physiol. Vol. 147, 2008
cantly greater plant heights (P,0.05) at the end of the
recovery period when compared to nontransgenic
control plants.
A water-limited treatment led to a 50% reduction in
the final plant height of nontransgenic control plants
relative to well-watered controls. CspB conferred im-
proved tolerance to water deficits as demonstrated by
greater final plant heights in transgene-positive plants.
At the end of the water-limited treatment, three of four
CspB events tested were significantly taller (P,0.05)
than their nontransgenic controls. One event demon-
strated significant improvements under all three stress
treatments, and several other events showed positive
trends across multiple treatments. Overall, this dem-
onstrated that expression of CspB in rice results in
tolerance to multiple abiotic stresses, and this toler-
ance occurs with fairly high frequency in the individ-
ual stress treatments.
For CspA-positive rice plants, cold and heat treat-
ments also resulted in improved growth, as measured
by greater final plant height. The treatment impact on
plant height at the end of the recovery period resulted
in a 46% and 51% reduction in plant height of non-
transgenic control plants for the cold and heat treat-
ments, respectively. Four of seven CspA events
demonstrated significantly greater final plant height
than their nontransgenic controls in the cold treat-
ment, while three of seven CspA events demonstrated
significantly greater final plant height compared to
their nontransgenic control plants under the heat
stress treatment. CspA rice plants were not evaluated
in a water-deficit treatment.
Thus, the cold tolerance we demonstrated in trans-
genic Arabidopsis, a dicot, was also observed in rice, a
monocot. Furthermore, the improved stress tolerance
was extended beyond the cold tolerance that we
observed in Arabidopsis and shown to also include
heat and water-deficit tolerance. The rice water-deficit
results, in particular, present a compelling outcome
that drove further abiotic stress testing of transgenic
CspA and CspB in maize.
MAIZE VEGETATIVE WATER-DEFICIT TOLERANCE
An important factor in the success of a field testing
program is the ability to accurately collect phenotypic
data under predictable and consistent water deficit
conditions in a managed stress environment. This has
been achieved through a field testing network located
in rain-free environments, allowing precise control
over irrigation levels. Herein, we have reported im-
proved vegetative and reproductive performance in
field-grown maize exposed to water deficits that were
introduced during periods of vegetative growth and
ovule and kernel development.
We have demonstrated that transgenic expression of
CspB in maize plants contributes to improved vegeta-
tive performance. Twenty-two CspB events were eval-
uated in water-limited field trials using commercial
grade hybrid corn in environments that received no
rainfall during the target period for the water-deficit
treatment, a span of 10 to 14 d immediately prior to
flowering. The water-deficit treatment resulted in an
average reduction in growth rates to 50% of the well-
watered rate. Using an across-event analysis, the CspB
transgenics demonstrated a 3.6% increase in leaf ex-
tension rates relative to nontransgenic controls (Table
II). The best performing events demonstrated growth
rate increases of 12% and 24%. This growth rate
improvement under water-limited conditions indi-
cated that the CspB transgene was having a substantial
positive impact on plant productivity during the veg-
etative phase of plant growth and development. The
CspB-expressing plants also demonstrated significant
improvements in chlorophyll content and photosyn-
thetic rates (Table II). Across all events, chlorophyll
content was increased by 2.5%, with the top two events
exhibiting increases of 4.4% and 3.3%. The improve-
ments to the photosynthetic rates were 3.6% across all
events, with increases of 8.5% and 7.7% for the top two
performing events. Similar observations have been
made with transgenic maize plants expressing CspA
under greenhouse conditions (data not shown). These
measures of vegetative performance are key indicators
of plant productivity and would be expected to en-
hance the overall yield potential of the crop. When
plants were grown under nonstressed, fully irrigated,
or rain-fed conditions in both the greenhouse and
field, we did not detect any appreciable difference
between CspA- or CspB-expressing lines and the
isogenic control for plant growth rate or plant height
measured at different stages of development (data not
shown).
Reproductive performance was evaluated for CspB
plants by harvesting all kernel-bearing ears from six
Table II. Stable transgenic maize seedlings demonstrate improved
growth, chlorophyll content, and photosynthetic rates under
water-deficit stress test
Water-deficit conditions were created in the field by reducing irriga-
tion for a 14-d period during the late vegetative stage of development,
immediately prior to flowering. The treatment reduced the relative
growth rates during the treatment and the average end of season yields
by approximately 50% of well-watered levels. Relative differences in
measures were determined by comparison with appropriate nontrans-
genic controls. Experimental details are described in Supplemental
Materials and Methods S1.
Gene Event LER Chlorophyll Photosynthesis
% increase
CspB-Event 3.6%a2.5%b3.6%c
Grouping (n5756) (n5432) (n5432)
CspB-Zm 12%a4.4%a8.5%b
Event 1 (n536) (n572) (n572)
CspB-Zm 24%a3.3%b7.7%c
Event 2 (n536) (n572) (n572)
a
Indicates significant (P,0.05) improvement relative to nontrans-
genic control.
b
Indicates significant (P,0.10) improvement rel-
ative to nontransgenic control.
c
Indicates significant (P,0.20)
improvement relative to nontransgenic control.
Bacterial RNA Chaperones Confer Stress Tolerance to Plants
Plant Physiol. Vol. 147, 2008 449
replicates (34 plants per replicate) for each of six events
selected for harvest based on the magnitude of their
improved vegetative performance. CspB-positive plants
were compared to nontransgenic control plants grown
in an adjacent row. An across-event analysis demon-
strated significant improvements (P,0.05) in the
number of plants with kernel-bearing ears (14.0%)
and the number of kernels per plant (111.7%; data not
shown). There were no significant differences ob-
served for individual kernel weight. The nature of
the improvements, primarily more kernels on ears and
more plants with kernel-bearing ears, was consistent
with expectations based on the timing of the limited-
water treatment, which occurred during late vegeta-
tive stages and early immature ear development and
was relieved with sufficient water available to the
plants during pollination and grain fill periods.
MAIZE REPRODUCTIVE TOLERANCE AND YIELD
UNDER WATER-DEFICIT CONDITIONS
Grain yield trials were performed under water-
deficit stress and nonstress conditions on 10 CspA
and 10 CspB-positive events, most of which had
previously demonstrated improved vegetative perfor-
mance in either greenhouse screens or field trials.
Grain yield data was collected from four field sites
where water was limited during the late vegetative
phase of development, a treatment similar to the initial
water-deficit field trial. Mean yield at the water-limited
sites was 6.8 tons (t)/ha, representing an approxi-
mately 50% reduction in yield relative to the average
mean yield of crops in the Midwest. An across-event
analysis demonstrates that the CspA transgenic entries
provide a yield increase of 4.6% (P,0.2) under water
stress, with the two best performing events demon-
strating advantages of 30.8% and 18.3% (Table III).
Yield averages of CspB-positive plants as a group were
significantly greater than controls, by 7.5% (P,0.01).
A number of individual events exhibited significant
yield advantages as well; the best two performing
events, CspB-Zm event 1 and event 2, demonstrated
yield improvements of 20.4% and 10.9%, respectively.
These are the same two events that demonstrated
significant improvements in leaf growth, chlorophyll
content, and photosynthetic rates, providing evidence
that these improvements in vegetative productivity
will translate into improvements in reproductive per-
formance and grain yield.
Several years of field trials have been conducted
with a single CspB-expressing event, CspB-Zm event 1,
to further investigate the ability of this gene to provide
tolerance to water deficits during the late vegetative
and reproductive developmental stages. Field trials
were conducted under controlled water-deficit condi-
tions where two distinct stress treatments were con-
ducted by limiting water during these two stages of
development. This single CspB event was deployed in
three different hybrid backgrounds and evaluated un-
der these stress regimes at five replicated locations. The
two treatments resulted in decreases in the overall yield
of the experiment by approximately 50% relative to
well-watered treatments planted at the same locations.
The CspB-positive entries exhibited improvements in
end-of-season grain yield across the different hybrid
entries and under both water stress regimes when
compared to a conventional wild-type control of the
same genetic background (Table IV). Yield benefits in
these experiments ranged from 11% to as much at 21%
across yield values that averaged 6.4 to 8.5 t/ha. The
transgenic CspB event consistently out-yielded the
nontransgenic controls by at least 0.5 t/ha across 12
out of 15 reproductive stress treatments and 13 out of 15
vegetative stress treatments, highlighting the potential
agricultural benefits that this technology can deliver.
A multi-year analysis was also conducted with
CspB-Zm event 1 to assess the stability of the yield
advantages across locations under water-limiting con-
ditions. Locations that had experienced some level of
water stress, where yield reductions ranged from 20%
to 80%, were compiled and analyzed across years.
Figure 2 indicates the yield stability observed for the
transgenic entry and its control across 3 years of water
stress testing in a single hybrid background. Yield
advantages are evident across a wide range of envi-
ronments with varying degrees of water-deficit stress,
with yield levels ranging from as low as 2 t/ha to as
high as 16 t/ha, indicating that this technology could
have broad utility across the U.S. growing regions.
We have assessed the average performance of this
material over the past several years, combining yield
performance across three hybrid test-crosses that have
been under evaluation in each of the prior 4 years.
Across 4 years of testing, this CspB event provides an
average yield benefit of 10.5% across three hybrid test-
crosses under managed stress environmental testing.
The average yield advantage each year was 0.89, 0.48,
0.49, and 0.79 t/Ha, representing percentage increases
of 13.4%, 6.7%, 10.5%, and 11.3%, respectively.
This same transgenic CspB event was tested under
western dryland maize conditions without supple-
mental water. Hybrid entries were planted as 100-foot-
long 4-row plots to better simulate normal agronomic
Table III. CspA and CspB transgenic maize plants demonstrate
improved end-of-season grain yield under water-limiting conditions
Mean yield values (tons/hectare) of nontransgenic and transgene-
positive plots are shown for groupings of CspA and CspB events and
for two individual transgenic events from each construct. Experimental
details are described in Supplemental Materials and Methods S1.
Event-Event Pool Yield Yield Improvement
t/ha
CspA nontransgenic mean 6.38
CspA-event group mean 6.68 4.6% (P,0.2)
CspA-Zm event 1 8.35 30.8% (P,0.1)
CspA-Zm event 2 7.52 18.3% (P,0.1)
CspB nontransgenic mean 6.86
CspB-event group mean 7.38 7.5% (P,0.1)
CspB-Zm event 1 8.26 20.4% (P,0.1)
CspB-Zm event 2 7.61 10.9% (P,0.1)
Castiglioni et al.
450 Plant Physiol. Vol. 147, 2008
conditions for this region, planting at appropriate
population densities for those regions, and were
paired with appropriate nontransgenic controls. Envi-
ronmental data was collected and seasonal weather
patterns, including rainfall accumulation, were uti-
lized to classify the dryland locations as to whether
each of the locations experienced water-deficit stress
during the season. In the final analysis, 12 of the loca-
tions planted across the western dryland were catego-
rized as having experienced water stress during the
late vegetative through reproductive developmental
stages and were utilized for analysis. As depicted in
Figure 3, yield benefits were observed in the same
three hybrid backgrounds that were evaluated under
controlled water-deficit conditions described in Table
IV. When compared to the nontransgenic control, the
CspB transgenic event provides yield benefits of up to
0.75 t/ha, or 15%. These dryland growing conditions
created a lower yielding environment (average yield of
the controls were 4.9 t/ha) than the controlled water-
deficit locations where the overall yields of the con-
trols ranged from 6.4 to 8.5 t/ha. The results from the
managed stress environment treatments were very
similar to the observations made under dryland grow-
ing conditions, highlighting the utility of the managed
stress environmental testing platform.
Demonstration of significant yield improvements
with transgenic CspB events under controlled drought
environments as well as under water-stressed western
dryland conditions represents a major advancement
in the field of abiotic stress research. Continued eval-
uation of these materials across additional genetic
backgrounds will be important ongoing work in the
development of products for the U.S. corn belt.
A FUNCTIONAL RNA BINDING SITE IS REQUIRED TO
CONFER YIELD BENEFITS TO FIELD-GROWN MAIZE
UNDER WATER STRESS
A well-documented functional aspect of CSPs, in-
cluding CspB, is their ability to bind single-stranded
DNA or single-stranded RNA that can be measured by
the protein’s ability to open a stem-loop or double-
stranded dual-labeled fluorescence probe in vitro. By
mutating a key residue in the RNA binding domain of
B. subtilis CspB (CspB_F30R), we have reduced the
ability of this protein to open a double-stranded stem
loop structure engineered into the probe (Fig. 4). There
was an approximately 10-fold reduction in fluores-
cence observed with the CspB_F30R mutant, suggest-
ing that this residue is important for maintaining RNA
chaperone activity. A similar result has been obtained
through mutation of the identical residue in the E. coli
CspE protein (Phadtare et al., 2002), and its activity in
Figure 2. A single transgenic CspB event demonstrates yield improve-
ments across multiple years of testing under water-stressed environ-
ments. Performance of CspB-Zm event 1 (black diamonds) and the
appropriate control (white circles) were plotted against the location
average yields across the past three seasons of testing under managed
stress environments. xaxis, Average yield (tons/hectare) at each loca-
tion; yaxis, mean yield (tons/hectare) of the entries, by location.
Experimental details are described in Supplemental Materials and
Methods S1.
Table IV. Yield results from managed irrigation water-deficit conditions
Yield results from replicated, multi-location evaluation of a single CspB transgenic event (CspB-Zm
event 1) under vegetative or reproductive water-deficit stress. Three hybrids expressing the CspB event were
evaluated using 20 replications of data across five locations for each stress treatment window. Experimental
details are described in Supplemental Materials and Methods S1.
Stress Class Entries Mean Yield
Positive
Mean Yield
Check Difference Difference
t/ha %
Vegetative Hybrid 1 (positive) 10.1 8.5 1.6 19
Reproductive Hybrid 1 (positive) 9.0 7.7 1.3 16
All stress Hybrid 1 (positive) 9.1 7.9 1.1 14
Vegetative Hybrid 2 (positive) 7.7 6.5 1.2 18
Reproductive Hybrid 2 (positive) 8.1 6.8 1.3 19
All stress Hybrid 2 (positive) 7.7 6.4 1.3 21
Vegetative Hybrid 3 (positive) 8.3 7.2 1.1 16
Reproductive Hybrid 3 (positive) 8.9 8.0 0.9 11
All stress Hybrid 3 (positive) 8.8 7.9 0.9 12
Bacterial RNA Chaperones Confer Stress Tolerance to Plants
Plant Physiol. Vol. 147, 2008 451
antitermination was similarly lost. The comparable
mutation in the F31 residue of the E. coli CspA protein
(Schro
¨der et al., 1995) resulted in a similar loss of func-
tion. Moreover, the protein containing the F30R muta-
tion no longer complements an E. coli mutant (BX04;
Xia et al., 2001) that lacks four native CSPs and is
highly sensitive to cold stress (data not shown). These
results suggest that this protein has lost its ability to
function as an RNA chaperone in E. coli.
By expressing the CspB_F30R mutant protein consti-
tutively in maize, we were able to assess the require-
ment of effective RNA chaperone activity to confer
yield benefits under water stress. Of 11 CspB_F30R
events tested, none was able to provide an increase in
yield under water-deficit conditions when compared
to nontransgenic controls, and results were effectively
neutral when an across-event analysis was performed
(data not shown). Field trial conditions were similar to
those described in previous studies. Transgenic maize
plants expressing CspA and CspB in the same field
trials continued to confer yield benefits (data not
shown). These data suggest that effective RNA chaper-
one activity of CspB is critical for providing tolerance
to water-deficit stress and that the mode of action of
CspB in maize occurs through its predicted function as
an RNA chaperone.
DATA SUMMARY AND CONCLUSIONS
Future progress toward more sustainable agricul-
tural practices will be accelerated through the system-
atic analysis of gene function important for yield
under water-deficit conditions. Model systems research
has provided a greater understanding of pathways of
plant stress acclimation that is further providing valu-
able insights into crop responses under environmental
stress conditions. Rapidly moving to large-scale anal-
yses of candidate gene function in crops, under stan-
dard agronomic practices and where the influences of
environmental factors and the ultimate impact on
yield can be assessed, is critical for developing im-
proved crops through biotechnology.
We have demonstrated that constitutive expression
of two members of a family of bacterial RNA chaper-
ones, E. coli CspA and B. subtilus CspB, was shown to
confer abiotic stress tolerance in transgenic Arabidop-
sis, rice, and maize. Importantly, the improvements in
water-limited field trials were not associated with a
yield penalty in high-yielding environments. Consis-
tent with the timing of the water deficit, the positive
yield impact of the transgenes was predominately on
kernel numbers, not on kernel weight (data not
shown). This technology has been observed under
different stress regimes and across environments as
clearly providing performance benefits under late
vegetative/flowering water deficit as well as during
the grain fill period. During these two periods, water-
deficit stress leading to three consecutive days of
wilting can lead to 30% to 50% reductions in grain
yields (Claassen and Shaw, 1970). The sensitivity of
maize yield at these developmental stages and the
frequency with which these conditions occur across
the corn belt are consistent with the observed higher
correlation of grain yield to kernel number and not
kernel weight (Duvick, 2005). A multi-year analysis
across the full range of stress conditions suggests that
this technology provides broad yield stability under
water-limiting conditions. In addition to the managed
stress environment testing, a CspB transgenic event
was tested under dryland conditions as well. Demon-
stration of consistent yield benefits under managed
water environments and dryland conditions is an
important advancement in the development of this
technology. These yield improvements further outline
Figure 4. Single-stranded DNA probe opening ability for B. subtilis
CspB and the CspB_F30R mutant. Probe opening assay for BsCspB,
BsCspBF30R, and controls. Negative control contained all elements
that Csp samples contained except protein. The average of three inde-
pendent replications is represented (6SD). The yaxis is labeled as the
average relative fluorescence units (RFU). Experimental details are
described in Supplemental Materials and Methods S1.
Figure 3. Yield results from Midwest evaluations under water-deficit
conditions. Three different hybrids carrying a single transgenic event
expressing CspB were evaluated in yield trials across the western dryland
market. Yield results were averaged across locations that experienced
water-deficit stress during the late vegetative or grain fill periods of the
season. Experimental details are described in Supplemental Materials
and Methods S1.
Castiglioni et al.
452 Plant Physiol. Vol. 147, 2008
the potential of this technology to provide a significant
agricultural benefit. As these hybrids were configured
with an elite transformation germplasm, a component
of future evaluation of this technology will be testing
of these transgenic events in germplasm that is adapted
for the marketplace.
The potential utility of CSPs as transgenes is
strongly supported by the demonstration that the
two CSPs tested, which share only 61% overall iden-
tity, were both capable of improving stress tolerance in
plants. As tested, we were not able to distinguish the
performance of CspA from CspB, and we speculate
that they are likely acting by similar mechanisms. The
myriad of CSPs known in nature, and available for
testing, and the possibility of improving on the current
expression pattern and coding regions of these trans-
genes present intriguing possibilities for even further
improvements.
Previous reports demonstrated that CSD protein
functionality in vivo is significantly impaired by mu-
tations that affect RNA binding. Nakaminami et al.
(2006), for example, reported that site-directed muta-
genesis of WCSP1 ribonucleoprotein (RNP) domains
abolishes the ability of the protein to complement the
E. coli cold-sensitive mutant BX04. Our observation
that maize plants expressing the CspB_F30R single
amino acid mutation do not exhibit improved yield
under water-deficit conditions further supports the
prediction that the biochemical function of these pro-
teins as defined in bacterial systems is also important
in plants. Testing the ability of other known CSP
mutants for their ability to confer tolerance to water-
deficit in plants will further define the structure-function
relationships and improve our understanding of the
mode of action of these proteins. Consistent with these
hypotheses, we have evaluated the subcellular loca-
tion of GFP protein fusions with CspA, CspB, and a
cotton (Gossypium hirsutum) CSP transiently expressed
in maize protoplasts. As depicted in Figure 5, we
observed GFP fluorescence in the cytosol, nucleus, and
nucleolar region, consistent with observations of sub-
cellular localization of plant and animal CSD proteins
(Nakaminami et al., 2006; Sasaki et al., 2007). By
comparison, expression of an Arabidopsis AP2-type
transcription factor is restricted to the nucleus and
nucleolar region, consistent with its function. Nucleoli
are largely comprised of proteins and the ribosomal
DNA sequences of chromosomes, surrounded by a
layer of condensed chromatin. Because nucleoli are
involved in the production and maturation of ribo-
somes, these suborganellar structures are extremely
important during phases of rapid growth and devel-
opment. Concentrations of CspA and CspB proteins in
these regions as well as in the cytosolic regions of the
cell are highly consistent with their proposed role in
RNA binding and participation in RNPs.
New insights into the functions of plant CSPs sug-
gest that we are tapping into a conserved mechanism
of cellular function. Plant CSPs and other plant RNA
binding proteins have been shown to function as RNA
chaperones in Arabidopsis and wheat (Nakaminami
et al., 2006; Kim et al., 2007; Sasaki et al., 2007). CSD-
containing proteins from other eukaryotic organisms,
Chlamydomonas,Xenopus, and mammalian systems,
have also been demonstrated to play a role in RNA
metabolism, protein translation, and regulation of
gene expression, coupling the transcription of mRNA
to its translational fate (Matsumoto et al., 1996;
Matsumoto and Wolffe, 1998; Manival et al., 2001;
Mussgnug et al., 2005). Eukaryotic mRNPs likely play
a central role in integrating various cellular signals
by providing a physical context for the control of
mRNA translation, stabilization, and sequestration.
CSD-containing proteins are likely components of
mRNPs and important for this level of regulation.
Further work is required to establish the cause and
effect relationship linking RNA structure stabilization
and posttranscriptional control with stress acclimation
mechanisms at the cellular level. However, these ob-
servations as well as several other reports in the
literature, suggest that posttranscriptional control is
an important level of regulation, allowing plants to
adjust protein synthesis and fine-tune expression un-
der periods of long-term stress.
Based on the yield and stress benefits we have
observed from expression of CSD-containing proteins
in plants and our current understanding of the com-
mon RNA chaperone function of CSD proteins in pro-
karyotes and eukaryotes, expression of CSD proteins
Figure 5. CspA (A), CspB (B), and cotton CSP-like 1 (C) GFP fusion
proteins accumulate in the cytoplasm, nucleus, and nucleolus when
expressed in maize protoplasts. D, Results obtained with an AtCBF3
transcription factor:GFP fusion protein that localizes exclusively to the
nucleus. Experimental details are described in Supplemental Materials
and Methods S1.
Bacterial RNA Chaperones Confer Stress Tolerance to Plants
Plant Physiol. Vol. 147, 2008 453
from a variety of plant and bacterial sources represents
a promising approach for stimulating growth and pro-
ductivity in plants under conditions of abiotic stress.
The fact that expression of CSP proteins did not result
in detrimental effects on plant size, development, or
productivity as previously observed for other trans-
genic approaches further supports the utility of these
proteins. It is interesting to speculate why CSP func-
tionality was not selected for over the years of con-
ventional breeding. However, the low heritability of
yield, the challenges associated with controlled envi-
ronment testing, and the quantitative nature of the
trait are likely contributing factors.
The stress tolerance conferred by CSPs represents
a novel, compelling approach toward engineering
improved plant productivity in suboptimal growth
conditions. These studies have confirmed that this
family of proteins is capable of delivering broad stress
tolerance, which also translates to improvements in
grain yield under both managed stress studies and
marketplace environments. As water resources be-
come increasingly scarce and the global demands for
grain continue to increase, the ability to bring yield
stability across water-limiting environments presents
an important advancement in the area of stress toler-
ance research. The opportunity exists for the drought-
tolerant trait to be added to a growing set of
germplasm and trait options that mitigate environ-
mental stresses on the corn plant and provide the crop
with a better opportunity to reach its yield potential in
any environment.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers M30139 and U58859.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Materials and Methods S1.
AUTHOR CONTRIBUTIONS
AND ACKNOWLEDGMENTS
This work could not have been accomplished without the assistance from
Don Nelson, Tom R. Adams, Brendan Hinchey, and Adrian Lund. Valuable
advice and consultation was received from Philip Miller, Tom H. Adams, Stan
Dotson, Nordine Cheikh, Mark Lawson, Tom Peters, and Mike Stephens. We
would also like to acknowledge the many people who supported this work,
including the plant transformation, greenhouse staff, and farm network teams.
Mary Fernandes and Mark Abad contributed to the Arabidopsis work.
Ganesh Kumar, Jayaprakash Targolli, and Santanu Dasgupta contributed
to the rice work.
Paolo Castiglioni, Don Anstrom, Robert Bensen, Dave Warner, Martin
Stoecker, Jay Harrison, Christopher Bonin, Robert D’Ordine, Sara Salvador,
Santiago Navarro, Stephanie Back, Michael Luethy, and Jacqueline Heard
contributed to the maize work.
Received March 11, 2008; accepted April 21, 2008; published June 6, 2008.
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