Global Transcriptome Profiling Is a Poor Predictor of the Secondary ...

ISB NEWS REPORT AUGUST 2012
Global Transcriptome Profiling Is a Poor Predictor of the Secondary Effects of
Transgenes Influencing Abiotic Stress Tolerance
Zhulong Chan, Patrick J. Bigelow, Wayne Loescher, Rebecca Grumet
Engineering for salt stress resistance
Globally, the impact of salinity on agriculture is on the
rise. Increased demand for food, fiber, and fuel has led
to increased irrigation in semi-arid regions where there is
insufficient rainfall to leach excess salts from the soil, and
the cultivation of marginal salinity-impacted lands 1 has
expanded. This makes salt stress resistance an important
objective for many plant breeders, and a part of broader
efforts to increase crop resistances to abiotic stresses, like
heat, frost, drought, and salinity, via genetic engineering
(GE).
High salinity causes a combination of physiological
stresses caused by toxicity, mostly due to excess sodium,
along with dehydration, due to reduced water potential.
Plants cope with these stresses through a variety of
mechanisms, providing multiple options for genetic
engineering of increased salinity resistance. Plants can limit
sodium ion uptake, sequester excess ions, or accumulate
compounds that decrease the effects of those excess ions on
plant tissues. Candidate genes for each of these mechanisms
have been studied with the goal of increasing salinity
stress tolerance in crop species. While the potential benefits
to agricultural productivity could be substantial, possible
ecological concerns must also be considered.
Ecological questions about engineering abiotic stress
tolerance into crop species consider both the primary
effect of transgene expression, i.e., the desired phenotype
conferred by the transgene, and the possible secondary
effects of its expression. Increased salinity tolerance may
allow crop volunteers or hybrids with wild relatives to
invade natural or disturbed areas with high soil salinity,
a primary effect. In addition, secondary, non-target,
phenotypes resulting from transgene expression could lead
to changes in plant physiology, development, or fitness,
with ecological implications. First generation genetically
engineered crops express transgenes that directly encode
the desired trait, such as production of Bt toxin for insect
resistance or an additional or altered enzyme for herbicide
tolerance. Genetic engineering of crops for increased abiotic
stress resistance will require complex changes to stress
signaling cascades, regulatory networks, and metabolic
pathways. Compared to the prior GE traits, altering these
multipart and interconnected processes increases the
likelihood of secondary effects in engineered plants, which
raises questions for regulators conducting risk assessments
of abiotic stress tolerant crops prior to commercialization.
Global transcriptome profiling has been suggested as
a comprehensive method to examine transgenic plants for
secondary effects 2 . Previous studies comparing GE crops to
their conventional parent have shown greater transcriptional
differences between related conventional cultivars than
between the GE plant and its parent 3,4 . However, these
studies were performed with GE crops expressing simpler
first generation traits and not altered abiotic stress tolerance.
Our recent study compared secondary phenotypic and
transcriptomic effects resulting from three different
transgenic approaches to confer salt stress tolerance in the
model species Arabidopsis thaliana 5 .
Growth and Development
Three constitutively expressed transgenes were selected to
confer salinity tolerance by differing modes of action: an
abiotic stress related transcription factor [C-repeat binding
factor 3/drought responsive element binding factor 1a
(CBF3/DREB1a)], which activates a regulon of abiotic
stress response genes 6,7 ; a mannitol biosynthesis enzyme
[mannose-6-phosphate reductase (M6PR)] allowing the
production of an osmolyte and osmoprotectant 8 ; and a
plasma membrane Na+/H+ antiporter [Salt Overly-Sensitive
1 (SOS1)] which transports sodium ions out of plant cells 9 .
To examine the effect of transgene expression on plant
growth and development under a range of conditions, two
independent transgenic lines per transgene were grown
with their wildtype background under growth chamber,
greenhouse, and field conditions. CBF3 lines exhibited
dwarfism, as has been seen in previous studies 6,7 , and
were significantly delayed in reproductive development,
while SOS1 and M6PR lines were minimally impacted by
transgene expression regardless of growing environment.
These transgenic plants were slower to develop symptoms
of chlorosis and necrosis when grown under salinity stress
than WT genotypes. Overall the reduction in biomass due
to salinity stress was 50% smaller in transgenic lines than
wildtype and seed yield was 114% to 345% higher relative
to wildtype.
Transcriptome
Microarray analysis was performed to assess global changes
in gene expression due to the expression of the transgenes.
Under non-salt treated conditions, the transgenes CBF3 and
M6PR resulted in greater changes to the plant transcriptome
than SOS1. The overexpression of CBF3 altered expression
of 1350 genes (>2-fold change, P

ISB NEWS REPORT AUGUST 2012
In M6PR lines, however, not only were abiotic stress
response genes affected, but also pathogen and oxidative
stress related genes. SOS1 had the lowest number of
changes in transcript level (619) of the three transgenes,
but its expression did result in significant down-regulation
of oxidative stress responsive genes. Very few expression
changes occurred in common for all three transgenes.
In the presence of salinity stress, wildtype lines WS and
Col had a large number of transcripts with altered expression
levels (1093 and 2552 respectively), while the number of
transcripts affected by salt stress in the transgenic lines
was significantly lower (between 100-500). There was also
considerable overlap in the transcripts induced by salinity
stress in the WT lines and those induced by expression of
the transgenes CBF3 and M6PR in the absence of salt stress
(33% and 47% respectively), suggesting preconditioning by
the transgenes for growth under salt stress. Both M6PR and
CBF3 affected approximately 1000 genes under salt stress,
a reduction in transgene effect on gene expression compared
to non-salt stressed conditions. SOS1 affected 845 genes,
but unlike in M6PR and CBF3 lines, this was an increase in
transgene effect from non-salt stressed conditions and there
was little overlap between transcripts affected by salt in WT
and those affected by overexpression of SOS1. Also SOS1
plants down-regulated a number of pathogen-related genes
when under salinity stress, a difference not observed in nonsalt
treated SOS1 plants or in the other transgenes under
either growing condition.
Gene expression and plant performance
Although all three transgenes confer increased salinity
tolerance, the effects on plant phenotype, performance,
and transcriptome differed significantly, and varied
depending on the presence or absence of salinity stress.
The extent of transcriptomic change, however, did not
correlate with the effect on plant performance (Fig. 1).
For example, the greatest phenotypic differences were
observed for CBF3 plants, which had an intermediate
level of transcriptomic change. M6PR plants had the
greatest level of transcriptomic change in the absence of
salinity stress, but had little to no change in phenotype
(growth, development, and seed production) in the growth
chamber, greenhouse and field. The presence of salinity
stress reduced the number of transcriptional differences
both between the CBF3 and M6PR plants, relative to WT,
but increased the phenotypic differences. SOS1 plants had
the least transcriptomic differences relative to WT, but had
intermediate effects on plant performance in the presence of
salt stress. These results indicate that extensive alterations
in a plant’s transcriptome do not necessarily led to equally
extensive changes in phenotype and performance. This also
has important risk assessment implications for the use of
global transcriptome profiles to predict secondary effects of
transgene expression.
Figure 1. Lack of relationship between magnitude of transcriptional
effects of the CBF3 (triangles), M6PR (diamonds) and SOS1 (squares)
transgenes and effect of the transgenes on vegetative (dry weight, black
symbols) and reproductive (seed production, grey symbols) performance
in the growth chamber. Open symbols, 0 mM NaCl; closed symbols,
100 mM NaCl. Performance values are averaged over the two lines for
each transgene. Magnitude of transcriptional effects was number of significantly
changed transcript levels for the transgenic lines vs. wild type.
Performance difference refers to above-ground dry weight or seed yield
for the transgenic lines relative to wild type, expressed as a percent of
wild type.
Implications for agricultural biotechnology
The lack of a predictive link from changes in a plant’s
transcriptome to changes in plant phenotype or performance
has implications for regulators of genetically engineered
crops. Transgenic crops are assessed for possible risks,
ranging from human and animal health to the environment.
Food safety questions are quantified and measured using
standard toxicological methods, but the environmental
concerns are not as easily standardized. The wide range
of possible environmental interactions makes these
concerns more difficult to render into testable hypotheses.
However, to assess scientifically the environmental risks
from releasing a genetically engineered crop, problem
formulation and risk assessment protocols have been
developed to break the environmental risks into their
component parts; identify the possible consequences of
environmental release; and evaluate the probability of
those consequences occurring 10 .
Thus, for regulators the most critical information
relates the phenotypic effects of transgene expression in

ISB NEWS REPORT AUGUST 2012
a crop to the baseline information about that crop and
associated agronomic and environmental factors such
as: cultural practices; related wild species; weediness;
and biotic and soil interactions. Together, these datasets
allow for the formulation of testable hypotheses and
finally a scientific determination of risk, where possible
consequences and their probability of occurrence are
combined. Our results show that large-scale changes
in transcript levels do not indicate equally sweeping or
even necessarily detectable changes in plant phenotype.
These results indicate that global transcriptome profiling
would be of questionable utility for regulators concerned
about non-target secondary effects from transgenes, and
if used, would need to be considered in terms of natural
transcriptome variability.
Global transcriptome profiling may be valuable in
the formulation of testable hypotheses, i.e., are there
specific categories of genes affected that may be predicted
to cause phenotypic effects under certain environmental
conditions? However, it does not, in and of itself, provide
an accurate assessment of the scope of secondary effects
nor eliminate the need for phenotypic analysis under a
range of environmental conditions. Indeed, requiring the
inclusion of transcriptome profiles in regulatory packages
for genetically engineered crops could detract valuable
time and resources from the phenotypic analyses critical
for ecological risk assessment of GE crops enhanced for
abiotic stress tolerance.
References
1. Munns R and Tester M. (2008) Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59, 651-681.
2. Ricroch AE, Berge JB and Kuntz M. (2011) Evaluation of genetically engineered crops using transcriptomic, proteomic, and metabolomic profiling techniques.
Plant Physiol. 155, 1752–1761.
3. Baudo MM, Lyons R, Powers S, Pastouri GM, Edwards KJ, Holdsworth MJ and Shewry PR. (2006) Transgenesis has less impact on the transcriptome of
wheat grain than conventional breeding. Plant Biotechnol. J. 4, 369–380.
4. Cheng KC, Beaulier J, Iquira E, Belzile FJ, Fortin MG and Stromvik MV. (2008) Effect of transgenes on global gene expression in soybean is within the
natural range of variation of conventional cultivars. J. Agric. Food. Chem. 56, 3057–3067.
5. Chan Z, Bigelow PJ, Loescher W and Grumet R. (2012) Comparison of salt stress resistance genes in transgenic Arabidopsis thaliana indicates that extent of
transcriptomic change may not predict secondary phenotypic or fitness effects. Plant Biotech J. doi: 10.1111/j.1467-7652.2011.00661.x.
6. Jaglo-Ottosen KR, Gilmour SJ, Sarka DG, Schabenberger O and Thomashow MF. (1998) Arabidopsis CBF overexpression induces COR genes and enhances
freezing tolerance. Science 280, 104-106.
7. 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. Nat. Biotechnol. 17, 287–291.
8. Zhifang G and Loescher W. (2003) Expression of a celery mannose 6-phosphate reductase in Arabidopsis thaliana enhances salt tolerance and induces
biosynthesis of both mannitol and a mannitol dimer. Plant Cell Env. 26, 275–283.
9. Shi H, Lee B, Wu SJ and Zhu JK. (2003) Overexpression of a plasma membrane Na+ ⁄ H+ antiporter gene improves salt tolerance in Arabidopsis thaliana.
Nat. Biotech. 21, 81–85.
10. Wolt JD, Keese P, Raybould A, Fitzpatrick JW, Burachik M, Gray A, Olin SS, Schiemann J, Sears M, and Wu F. (2010) Problem formulation in the environmental
risk assessment for genetically modified plants. Transgenic Res. 19:425-436.
Zhulong Chan
Graduate Program in Plant Breeding, Genetics and Biotechnology
Michigan State University, East Lansing, MI, USA
Current address: Wuhan Botanical Garden
Chinese Academy of Sciences
Wuhan 430074, PR China
zhulongch@hotmail.com
Patrick J. Bigelow
Graduate Program in Plant Breeding, Genetics and Biotechnology
Michigan State University, East Lansing, MI, USA
bigelowp@msu.edu
Wayne Loescher
Graduate Program in Plant Breeding, Genetics and Biotechnology, and Department of Horticulture
Michigan State University, East Lansing, MI, USA
loescher@msu.edu
Rebecca Grumet, corresponding author
Graduate Program in Plant Breeding, Genetics and Biotechnology, and Department of Horticulture
Michigan State University, East Lansing, MI, USA
grumet@msu.edu