Funct Integr Genomics (2011) 11:275291
DOI 10.1007/s10142-010-0206-z
ORIGINAL PAPER
Carbohydrate metabolism and cell protection mechanisms
differentiate drought tolerance and sensitivity in advanced
potato clones (Solanum tuberosum L.)
Sylvain Legay & Isabelle Lefvre & Didier Lamoureux & Carolina Barreda &
Rosalina Tincopa Luz & Raymundo Gutierrez & Roberto Quiroz & Lucien Hoffmann &
Jean-Franois Hausman & Merideth Bonierbale & Danile Evers & Roland Schafleitner
Received: 28 September 2010 / Revised: 1 December 2010 / Accepted: 18 December 2010 / Published online: 28 January 2011
# Springer-Verlag 2011
Abstract In potatoes and many other crops, drought is one
of the most important environmental constraints leading to
yield loss. Development of drought-tolerant cultivars is
therefore required for maintaining yields under climate
change conditions and for the extension of agriculture to
sub-optimal cropping areas. Drought tolerance mechanisms
have been well described for many crop plants including
Native Andean potato. However, knowledge on tolerance
traits suitable for commercial potato varieties is scarce. In
order to describe drought tolerance mechanisms that sustain
potato yield under water stress, we have designed a growth-
chamber experiment with two Solanum tuberosum L.
cultivars, the more drought tolerant accession 397077.16,
and the sensitive variety Canchan. After 21 days of drought
exposure, gene expression was studied in leaves using
cDNA microarrays. The results showed that the tolerant
clone presented more differentially expressed genes than
Electronic supplementary material The online version of this article
(doi:10.1007/s10142-010-0206-z) contains supplementary material,
which is available to authorized users.
S. Legay : I. Lefvre : D. Lamoureux : L. Hoffmann :
J.-F. Hausman : D. Evers (*)
Department Environment and Agrobiotechnologies (EVA),
Centre de Recherche Public-Gabriel Lippmann,
41, rue du Brill,
4422 Belvaux, Luxembourg
e-mail: evers@lippmann.lu
C. Barreda : R. T. Luz : R. Gutierrez : R. Quiroz : M. Bonierbale :
R. Schafleitner
Germplasm Enhancement and Crop Improvement Division,
International Potato Center,
La Molina 1895, Apartado 1558,
La Molina, Lima 12, Peru
the sensitive one, suggesting greater stress response and
adaptation. Moreover, it exhibited a large pool of upregu-
lated genes belonging to cell rescue and detoxication such
as LEAs, dehydrins, HSPs, and metallothioneins. Tran-
scription factors related to abiotic stresses and genes
belonging to raffinose family oligosaccharide synthesis,
involved in desiccation tolerance, were upregulated to a
greater extent in the tolerant clone. This latter result was
corroborated by biochemical analyses performed at 32 and
49 days after drought that showed an increase in galactinol
and raffinose especially in clone 397077.16. The results
depict key components for the drought tolerance of this
advanced potato clone.
Keywords Drought . Microarrays . Potato . Raffinose
family oligosaccarides . Solanum tuberosum
Introduction
Water deprivation is considered as an important factor
severely impacting optimal plant development. Since
several years, global warming and more erratic rainfalls
have been observed and drought has not only affected semi-
arid tropics but also temperate regions. With the increase of
food demand, crop surfaces tend to reach sub-optimal soils
and climatic areas. Potato, the fifth most important food
crop in the world, with an annual production approaching
300 million tons, is considered to be drought-sensitive. The
important yield loss upon drought is due to decreased net
photosynthesis as a consequence of stomatal closure and
overall reduction of photosynthetic surface under water
stress. Drought stress affects the osmotic potential of cells;
additionally, oxidative stress occurs at many cellular levels
276
such as thylakoids, peroxysomes and mitochondria (Cruz
de Carvalho 2008). At the photosystem level, decrease of
CO2 assimilation rates leads to reduced regeneration of
NADP+ in the Calvin cycle (Cruz de Carvalho 2008)
resulting into electron leakage at the photosynthetic
electron transport chain with concomitant generation of
reactive oxygen species (ROS). Moreover, decreased CO2
concentration in the leaves triggers a reduction of the
carboxylation function of RuBisCO and favors the oxygen-
ation leading to higher ROS production (Cruz de Carvalho
2008).
Studies in model plants such as Arabidopsis thaliana
and crop plants such as rice, barley, maize, and potato
suggest several mechanisms that are activated in response
to drought exposure. Drought generally alters metabolite
contents in plant cells. Under water stress, metabolites are
transiently accumulated and degraded after rewatering
(Valliyodan and Nguyen 2006). Amino acids such as
proline and proline analogs or glycine betaine accumulate
in the cells and prevent water stress-induced osmotic
imbalance. Also sugars and sugar alcohols, and particularly
the raffinose family oligosaccharides (RFOs) play a role in
the protection against desiccation (Taji et al. 2002).
Osmotic imbalance also has a strong impact on protein
folding and activity; in this context, late embryogenesis
abundant (LEA) proteins and heat shock proteins (HSPs)
are known to be induced during various abiotic stresses and
act as chaperone molecules to stabilize the conformation of
proteins and other molecules. In response to redox
disequilibrium and oxidative damage caused by desicca-
tion, plants may trigger the synthesis of a set of detoxica-
tion enzymes, such as superoxide dismutase, ascorbate
peroxidase, peroxiredoxin, or glutathione-S-transferase, to
name a few (Cruz de Carvalho 2008).
As for signaling events, several pathways are involved in
drought response. Abscisic acid (ABA)-dependent signal-
ing pathways are implicated in the response to various
abiotic stresses including drought and regulate stomatal
control, transcription regulation, plant growth, and devel-
opment. Studies on Arabidopsis reveal ABA-dependent and
ABA-independent pathways acting on distinct transcription
factor families, i.e., dehydration responsive element/C-
repeat (DREB/CBF) implicated in ABA-independent path-
ways and MYC and MYB family transcription or ABA-
responsive element binding factors that are responsive to
ABA (Shinozaki et al. 2003; Bartels and Sunkar 2005).
In contrast to model plants, drought stress responses and
particularly plant reactions that lead to drought tolerance
have been less well characterized in potato. Molecular and
biochemical responses of potato to drought have previously
been reported (Watkinson et al. 2006; Schafleitner et al.
2007; Mane et al. 2008; Vasquez-Robinet et al. 2008; Evers
et al. 2010); however, these investigations focused on
Funct Integr Genomics (2011) 11:275291
drought stress responses of native Andean potato clones
belonging to the Andigenum group of Solanum tuberosum
that are considered more tolerant to drought than improved
S. tuberosum varieties. In the present study, we have
identified differences in the expression of genes and
pathways in the bred S. tuberosum clone 397077.16 and
the S. tuberosum variety Canchan under drought. These two
clones were chosen as drought-tolerant and sensitive,
respectively, in order to differentiate between drought
tolerance and common drought stress responses. Plants
were exposed to drought in a growth chamber under
controlled conditions and drought responses were evaluated
in the leaves at the gene expression level. Results were
correlated with physiological and biochemical analyses in
order to get an accurate picture of the 397077.16 tolerance
mechanism. From these data and based on the drought
tolerance mechanisms established in model plants, we tried
to postulate a model of yield maintenance and drought
tolerance in potato under drought.
Material and methods
Plant material and stress treatment
Two S. tuberosum clones were chosen for this experiment.
Canchan (Bl1.2 [Black Libertas] Murillo III-80) was
released by CIP in 1990 with increased late blight tolerance.
397077.16 (392025.7 [LINEA 21 386614.16] 392820.1
[MONALISA 388213.1 (Y84.012 Y84.004)]) was
recently developed for higher yield maintenance under water
limiting conditions.
For each clone and treatment, 16 biological replicates
(plants) were randomly planted in rows in sand at day 0 in a
climate-controlled growth chamber in two 2.52.5 m contain-
ers with a substrate depth of 55 cm composed exclusively of
sand. Four rows were planted per container, but only the two
central rows were used for sampling and analysis. After
51 days of growth, drought was induced by reduced irrigation
to one container whereas the control plants in the other
container were continuously irrigated all over the experiment.
Air temperature ranged between 9.0C and 23.7C (minimum
and maximum average, respectively), average soil tempera-
ture was 18C (with a minimum of 16C and a maximum
of 19C). Average light radiation was 11.6 MJ m2 (with a
minimum of 5.7 MJ m2 and a maximum of 16.4 MJ m2)
with a photoperiod of 12 h/12 h.
Gravimetric substrate water content was measured at three
spots per row in substrate sampled from 0 to 20 cm depth each
10 days from day 40 to 130 after planting. Substrate water
content was determined gravimetrically (100g water/g
substrate). Repeated measurement analyses were performed
as explained by Wolfinger and Chang (1998).
Funct Integr Genomics (2011) 11:275291
Ground cover was measured by counting the number of
squares of an 8060 cm grid at 0, 20, 39, 49, and 66 days
after drought in six biological replicates.
For determination of root dry weight, three plants per
clone per treatments were sampled 49 days after drought,
dried during 24 h and subsequently weighted.
Osmotic potential was measured with a dew point
microvoltmeter HR-33 T (Wescor Inc.) in a psychrometric
chamber (Model C-52; Wescor Inc.) after calibration with
100, 290, and 1,000 mmol/kg NaCl standards.
For relative water content measurements (RWC), leaf
disks were taken from the third fully expanded leaf from
eight different plants per treatment per clone (eight
replicates), weighted (fresh weight) and rehydrated over
night in double-distilled water, blotted dry, and weighted
(turgid weight). Subsequently the leaf disks were dried at
80C for 24 h and weighted again (dry weight). RWC was
calculated as 100(fresh weight dry weight)/(turgid
weight dry weight).
Water use efficiency was evaluated in an additional dry
down experiment performed according to Kholova et al.
(2010). The dry down experiment was performed in 5-
l pots; the experimental unit comprised five plants and
water deficit was applied as soon as the roots colonized the
whole pots (~day 40 after planting).
For Fig. 1b and e, multiple comparison procedure between
means was performed with a Tukey test with confidence
limit of 95% (SigmaStat for Windows version 2.03). For
Fig. 1c and g, a t test (two-tailed with equal variance)
between drought and control condition was performed.
Yield analysis
One hundred thirty days after planting, yield was deter-
mined on 14 plants per clone under drought and well-
watered conditions. An aliquot of the tubers was oven-dried
for dry mass determination.
Sampling for gene expression and metabolite analysis
The third fully expanded leaves were harvested and pooled
from five plants on day 21 after drought onset for gene
expression and days 32 and 49 for metabolite analysis.
Three replicate blocks were sampled for each clone for
metabolite and gene expression analysis. The harvested
leaves were partitioned into two aliquots and were shock
frozen in liquid nitrogen and kept at -80C until RNA
isolation or lyophilisation for metabolite analysis.
RNA extraction and quality control
Total RNA was extracted from 100 mg of frozen leaves
using TRIzol reagent (Invitrogen) according to the manu-
277
facturers instructions. Traces of TRIzol were removed
using the RNeasy Mini elute Kit (Qiagen, Leusden, The
Netherlands) including DNase treatment (following the
manufacturers instructions). Quality control was performed
with the RNA Nano assay (Agilent Technologies, Diegem,
Belgium) using a 2100 Bioanalyzer with quality parameters
adapted to plant RNA profiles (Agilent Technologies).
RNA samples with a RIN (RNA integrity number) lower
than 7 were excluded from the experiment. RNA purity and
concentration were measured by the absorbance at 260 and
280 nm using a Nanodrop ND1000 spectrophotometer
(Thermo scientific, Villebon sur Yvette, France).
Labeling and hybridization
Reverse transcription of 20 g RNA per sample was
performed using Superscript II kit (Invitrogen) during 2 h
(see Supplemental data). Reaction was stopped by addition
of 10 L 0.5 M EDTA. Untranscribed RNA was degraded
by addition of 10 L 1 M NaOH and incubation at 65C
during 15 min. Sixty microliters of 1 M TrisHCl were
added for neutralization. aa-cDNA purification was per-
formed using the QIAquick PCR purification kit (Qiagen,
Leusden, The Netherlands). Washing steps were performed
using 500 L of a solution of 5 mM KPO4 pH=8.5 and 80%
EtOH; elution was done using two times 30 L of 4 mM
KPO4 pH=8.5 (final volume 60 L). Precipitation of aa-
cDNA was performed (see Supplemental data for details)
during 1 h at 80C. After centrifugation at 16,000g during
20 min, the pellet was washed using cold 75% EtOH and
vacuum dried for 10 min. The pellet was solubilised in
4.5 L daily prepared 0.1 M Na2CO3. A volume of 4.5 L
of dye (previously resuspended in DMSO) was added and
the labeling reaction took place during 1 h at room
temperature. To remove uncoupled dye, the QIAquick PCR
purification kit was used and the elution was performed
twice with supplied elution buffer EB (final volume of
65 L). Dye incorporation was controlled measuring the
absorbance at 260 nm (cDNA), 550 nm (Alexa555) and
650 nm (Alexa647), to allow an adequate adjustment of the
Alexa555- and Alexa647-coupled cDNA quantities.
Probes were precipitated during 1 h at 80C (see
Supplementary data). After centrifugation at 16,000g at
4C during 20 min, the probes were washed with cold 75%
EtOH, dried, resuspended in 60 L hybridisation buffer (4
SSC, 0.01% SDS) and denatured at 95C for 6 min. Slides
were prehybridized with blocking buffer (see Supplemental
data) during 15 min, washed by immersion (15 times) in
five consecutive milliQ water solutions. Finally, slides were
denaturated in boiling milliQ water during 2 min and dried
by centrifugation at 30g for 3 min. Hybridisation in
individual chambers was done at 55C during 16 h. Slides
were washed (see Supplemental data) and dried by
278
centrifugation at 30g for 3 min. For each condition, three
biological replicates and two technical replicates (dye
swap) were performed.
S. tuberosum 12.3 K RLP array Version IV slides
provided by the Austrian Institute of Technology GmbH
were used for hybridization. This array is composed of
5,013 cDNA clones and based on two different cDNA
libraries: on one hand, a full-length cDNA library of S.
tuberosum var. yungay challenged by Phytophthora infes-
tans for 4 days and, on the other hand, a set of stress-related
clones ordered from the University of Arizona Genomics
Institute. For additional information, see http://www.ncbi.
nlm.nih.gov/geo/query/acc.cgi?acc=GPL10120.
Microarray data acquisition and statistical analysis
Slides were scanned using a Professional 4200A microarray
scanner (Molecular Devices Corporation, Union City, CA,
USA). Two laser channels were used (532 and 635 nm) at
different settings for the photomultiplier tube (PMT gain).
The two PMT gains were automatically adjusted with the
Genepix Pro 6.0 software (Molecular Devices) to reach a
global intensity ratio close to 1 with pixel saturation equal
to 0.005%. Spots with a diameter smaller than 60 m and
spots with signal intensity below background intensity plus
two standard deviations were excluded from further
analysis. MA plots (log ratio (stress/control) versus mean
(log(stress),log(control))) were performed to check the
quality of the arrays.
To analyze files generated by Genepix Pro 6.0
software, data were imported into Acuity 4.0 software
(Molecular Devices) and normalized with the print-tip
Lowess method. The fold change between stress and
control conditions was expressed by the following formula:
log ratio (stress/control)=log2 ((signal median 635back-
ground median 635)/(signal median 532background me-
dian 532)). A one sample t test was first performed for
the six slides using the formula below:
x  m0
t p
s= n
where x is the mean of the six replicates of the spot, 0
is the mean of the population (in our case 0=1 in linear
scale or 0=0 in log2 scale), s is the standard deviation of
the three replicates and finally n is the sample size. In
order to retain spots with repeatable results, only probes
with a p value below 0.05 were selected. As a second step,
probes with a log ratio below 0.5 and above 0.5 were
selected to generate the final list and will be discussed
hereafter.
Complete lists of differentially expressed genes are
available as supplementary data.
Funct Integr Genomics (2011) 11:275291
Quantitative RT-PCR validation
In order to validate microarray results, qPCR was
performed on 15 genes selected from the microarray dataset
and literature. In this set, some putative candidate genes for
drought tolerance were included: e.g., a dehydrin TAS14,
DREB1, and ANAC083 transcription factors, a galactinol
synthase, a chloroplastic small HSP to name a few. Some
genes previously described to be drought responsive were
also included (Evers et al. 2010; Schafleitner et al. 2007):
a 1-pyrroline-5-carboxylate synthetase (P5CS), a gene
coding for a raffinose synthase, an UDP-4-glucose epimer-
ase, proton gradient regulation 5 (PGR5), and a drought-
responsive SNF-1-like protein kinase. The best housekeep-
ing genes among the set previously described by Nicot et
al. (2005) were chosen using the geNorm tool (http://
medgen.ugent.be/~jvdesomp/genorm/). For this experiment,
the most stable housekeeping genes were a cyclophilin, an
elongation factor 1 alpha and an adenine phosphoribosyl
transferase (Supplementary data).
Transcription of total RNA was performed with random
hexamers using TaqMan Reverse Transcription Reagent
(Applied Biosystems Inc., Foster City, CA, USA) following
the manufacturers instructions. Primers were designed with
primer express 2.0 (Applied Biosystems) with the following
criteria: primer size between 18 and 25 base pairs, GC content
between 40 and 60%, amplicon size from 50 to 110 base pairs,
avoiding runs of G and C at 3-end, avoiding T at 3-end, Tm
of primers in the 5961C range. Matching primer sets were
validated using NetPrimer (http://www.premierbiosoft.com/
netprimer/index.html) for unexpected secondary structures
(G should be below 5 kcal/mol with default settings). In
order to test the specificity of primers, an in silico BLAST
engine was used. Gene expression was performed using a
7500 Fast system (Applied Biosystems). Primer specificity
was checked by presence of a single peak in the melting
curve with decreasing amount of cDNA (from 10 to
0.001 ng and no cDNA) and PCR efficiency was verified.
Reverse transcription was performed using Taqman Reverse
Transcription Reagents (Applied Biosystems) with 1 g
RNA, 1 reverse transcription buffer, 5.5 mM MgCl2,
500 M dNTP mix, 2.5 M random hexamer, 0.4 U/L
RNaseI, 1.25 U/L reverse transcriptase in a 50 L final
volume. PCR was performed using Mesa Green Low Rox
Real-time PCR Kits (Eurogentec, Lige, Belgium) with the
following final concentrations in 25 L final volume: 1
MasterMix, 300 nM forward and reverse primers, 0.4 ng/L
cDNA. Thermal cycling conditions were: initial 5 min
denaturation at 95C, followed by 50 cycles of 15 s at
95C and 1 min at 60C, and a final dissociation step.
Relative expression was calculated using the model
described by Vandesompele et al. (2002) taking into
account multiple reference genes and gene-specific PCR
Funct Integr Genomics (2011) 11:275291
efficiency (for details see Evers et al. 2010). In order to
assess normal distribution and equality of data, QQ plot
and Levenes test were performed, respectively, using SPSS
vers. 16. A two-tailed Student t test was performed, using
SigmaStat vers. 2.03, for analysis of statistical significance
between control and drought conditions in each genotype.
Hierarchical clustering was performed with complete
linkage criterion on the different biological replications using
Cluster3 (http://bonsai.ims.u-tokyo.ac.jp/~mdehoon/software/
cluster/) and TreeView 1.1.4r3 (http://jtreeview.sourceforge.
net/)
Principal component analysis based on correlation
matrix was performed using R (http://www.r-project.org/)
and the FactoMiner package (http://cran.r-project.org/web/
packages/FactoMineR/index.html) using rescaled data.
Carbohydrate and polyol extraction and measurement
Water-soluble carbohydrate and polyol extraction was
performed following the method described by Oufir et al.
(2008) on 100 mg (10 mg) of freeze-dried leaves sampled
32 and 49 days after drought. Carbohydrate analysis was
performed following the method described by Oufir et al.
(2008). Trehalose, arabinose, xylose, melibiose, stachyose,
maltose, verbascose, cellobiose, cellotriose contents in the
leaf extracts were below the detection limit (data not shown).
Polyol analysis was performed following the method
described by Oufir et al. (2008). The contents of the polyols
ononitol, pinitol, sorbitol, mannitol in the leaf extracts were
below the detection limit (data not shown).
Carbohydrate data were analyzed by two-way ANOVA,
with treatment and duration of treatment as main effects.
Multiple comparison procedure between means was per-
formed with a Tukey test with confidence limit of 95%
(SigmaStat for Windows version 2.03).
Results and discussion
Agronomical and physiological differentiation of drought
tolerance traits and susceptibility
Drought was applied 51 days after planting (0 day after
drought (AD)) by reducing irrigation to 50% of the control.
This caused a rapid drop of substrate water content in the
root zone (Fig. 1a), stabilizing some 23 days AD. Differ-
ences in substrate water content between control and
drought plots were significant (P<0.05) 40 days AD and
for the rest of the trial. Substrate water content decreased
until plants were senescent in the drought plot (49 day AD).
The senescent plants extracted less water from the root zone
leading to a slight increase of substrate water content at the
end of the drought treatment. In the control plot, a decrease
279
of substrate water content was also observed between 40
and 90 days after planting due to the plant water uptake
(Fig. 1a). Yield potential under control conditions was
about 30% higher for Canchan than for 397077.16; how-
ever under drought, Canchans yield was almost 90% less
on dry weight basis than under well-watered conditions,
while yield loss in 397077.16 was less than 50%, resulting
in a higher yield under stress of 397077.16 compared to
Canchan (Table 1). Tuber dry matter content increased under
drought in both clones, from 19.3% to 25.4% in 397077.16
and from 19.8 to 22.3% in Canchan (data not shown).
Drought significantly (P<0.05) reduced ground cover
compared to control treatment. This difference was evident
20 days AD and onwards (Fig. 1b). The dynamic of the
response by varieties was similar, when the autocorrelation
was removed. Canchan plants had a more important ground
cover under well-watered conditions than those of
397077.16 but not under drought exposure. Under water-
limiting conditions, canopy growth was faster in 397077.16
than in Canchan until day 39 AD, but after this period,
ground cover of Canchan reached higher levels than in
397077.16 under control conditions (Fig. 1b). After day 49
AD, 397077.16 already became senescent and ground
cover declined. Canchan had much larger root dry weight
than 397077.16, but interestingly root dry weight signifi-
cantly increased under drought in 397077.16, while this
was not the case in Canchan (Fig. 1c). Moreover, root dry
weight/ground cover ratio increased by 173% in 397077.16
while only by 29% in Canchan (Fig. 1d).
Stomatal conductance was similar in both clones under
control conditions during the whole duration of the
experiment and was around 1 cm S1 (Table 2). Stomatal
conductance started to decrease slightly but significantly
under drought in Canchan on day 12 AD, while in
397077.16 at this time point the decrease of stomatal
conductance was much smaller, probably due to smaller
canopy size, and remained statistically insignificant. From
day 28 AD on until the end of the experiment, stomatal
conductance significantly decreased in both clones. From
day 39 after planting, stomatal conductance in the morning
was lower under drought in both clones compared to
control plants. Interestingly, additional measurements per-
formed during the afternoon (2 pm) at 34 and 39 days AD
showed a stomatal conductance significantly lower in
397077.16 than in Canchan (Supplementary data).
Leaf osmotic potential was constitutively more negative
in 397077.16 than in Canchan. Under drought, osmotic
potential declined in both clones (Fig. 1e), but remained
significantly more negative in 397077.16 while relative
water content did not change significantly (Fig. 1f). This
suggests a higher compatible solute accumulation in
397077.16 cells that helps to maintain cell turgor during
osmotic stress such as drought (Jongdee et al. 2002).
280
Fig. 1 a Gravimetric substrate water content from 0 to 20 cm depth. b
Ground cover measurement, Y-axis represents the number of counted
5 cm squares using a grid. Different letters significant ground cover
difference (P value <0.05) between drought and control plants. c Root
dry weight in g measured 49 days after drought. d Root dry weight/
Osmotic potential decrease under drought was similar
in both clones on day 41 AD and was stronger in
Canchan on day 60 AD, which can be explained by the
senescence occurring in 397077.16 or this clone might
already have reached the lowest negative osmotic
potential.
Funct Integr Genomics (2011) 11:275291
ground cover ratio (49 days after drought). e Leaf osmotic potential
expressed in MPa. f Relative water content (in percent). g Water use
efficiency in g tuber DM per kilogram of water. Asterisk significant
difference (P value <0.05) between drought and control condition
In addition to the growth chamber trial, a dry-down experi-
ment was performed on pot-grown plants in the greenhouse in
order to evaluate transpiration and water use efficiency (gram
tuber dry matter per liter-transpired water, Fig. 1g). Water use
efficiency was twice higher (P<0.001) in 397077.16 under
control conditions as compared to Canchan and increased
Funct Integr Genomics (2011) 11:275291 281

Table 1 Tuber dry weight yield in gram per plant
Clone Tuber DW %Yield loss DW
Control Drought
Mean SD Mean SD
397077.16 93.65 34.91 49.21 21.55 47.45
Canchan 138.73 56.86 15.72 8.04 88.67
even more under drought compared to control (P<0.001) to
finally reach three times Canchans value (P<0.001).
In summary, under drought, tuber dry weight was higher
in 397077.16 (49.21 g plant1) than in Canchan (15.72 g
plant1), although yield potential (under control conditions)
was higher in Canchan (138.73 g plant1) showing that
397077.16 was able to produce more tuber mass under
drought conditions than the susceptible clone Canchan. At
the morphological level, drought tolerance of 397077.16
appears to be associated with reduction of the canopy size
under water stress, a trait that reduces overall transpiration,
while maintaining enough photosynthetically active leaf
area for yield development. Moreover, root dry weight was
smaller in 397077.16 than in Canchan, but 397077.16
displayed the ability for drought-induced root growth,
resulting in an increased root/shoot ratio and improved
water uptake capacity. At the physiological level,
397077.16 displayed a lower stomatal conductance under
drought especially during the afternoon; this trend is in
accordance with the water use efficiency, which was higher
in this clone than in Canchan under control conditions, and
increased even more under drought. A more negative
osmotic potential of 397077.16 further contributes to
optimized water distribution in the plant.
Screening for drought tolerance-related traits: investigations
on genes and metabolites
Microarray expression profiling, performed 21 days AD,
revealed 111 significantly differentially expressed genes in
Canchan under drought, 31 were downregulated and 80 were
upregulated. In 397077.16, the number of differentially
expressed genes was much higher with 158 repressed and
204 induced genes, indicating that drought response was more
pronounced in 397077.16 than in Canchan (Fig. 2a, b,
Supplementary data). A stronger upregulation of genes
involved in cell rescue, detoxication, transcription regulation,
and transport was observed in 397077.16 as compared to
Canchan, whereas primary metabolism (carbohydrate, amino
acid, and lipid metabolism), secondary metabolism and plant
defense genes tended to be downregulated in 397077.16. In
Canchan, protein metabolism pathway genes were rather
induced, while cell rescue and detoxication genes were only
slightly upregulated and plant defense-related pathways were
strongly repressed. The differential response on the expres-
sion level of several genes was confirmed by qPCR (Fig. 3).
Correlation between qPCR and microarray was excellent (y=
0.7596x+0.0736, R2 =0.962) and showed only a little bias at
extreme relative expression values (Supplementary data).
Hereafter, a more detailed description of the mechanisms
underlying yield maintenance and drought tolerance in
397077.16 will be given.
397077.16 displayed a better ability to adapt
photosynthesis and carbon metabolism to drought
Our results showed a downregulation of the light-
harvesting complex and an increase in transcripts coding
for subunits of the core reaction center. This observation
pinpoints to a mechanism that leads on one hand to a
decrease of the electron overload caused by an excess of
light collection (see below), and on the other hand, an
enhanced turnover of photosystem reaction subunits dam-
aged by ROS. More precisely, in 397077.16, photosynthesis-
related genes, belonging to porphyrin and chlorophyll
synthesis, were downregulated by drought exposure (see
Supplementary data). In the same way, genes coding for a
chloroplast light harvesting a/b binding protein of photosys-
tem I and II were repressed (see Supplementary data),
whereas three genes coding for structural polypeptides of

Table 2 Stomatal conductance (cm S1) measured at 10 am

Days after drought 397077.16 Control (10 am) 397077.16 Drought (10 am) Canchan control (10 am) Canchan drought (10 am)

Mean Standard deviation Mean Standard deviation Mean Standard deviation Men Standard deviation

12 days 1.14 0.51 1.02 0.24 1.77 0.55 1.02a 0.20

28 days 0.99 0.38 0.56a 0.22 1.08 0.44 0.49a 0.11
34 days 0.91 0.26 0.47a 0.22 1.10 0.46 0.62a 0.19
39 days 0.84 0.31 0.18a 0.11 1.08 0.29 0.34a 0.09
52 days 2.99 1.75 0.10a 0.08 2.10 1.20 0.14a 0.11
a
Significant difference between drought and control plants
282 Funct Integr Genomics (2011) 11:275291

Fig. 2 Summary of microarray results: differentially expressed genes were attributed to several functional categories in Canchan (a) and
397077.16 (b), selected with p value <0.05 and log2 ratio(drought/control) below 0.5 and above 0.5

6.4 (PsbW), 10 (PsbR), and 22 kDa (PsbS) were induced by
drought. PsbR and PsbW are involved in stabilization of the
oxygen-evolving complex, water cleavage and electron
transfer in photosystem II (Shi et al. 2000; Zhou et al.
2007). In Canchan, a similar gene expression pattern was
observed, but the fold changes were low and not significant.
Expression of three genes encoding rubisco activase
chloroplast precursors was repressed in 397077.16 suggest-
ing a decreased activity of rubisco carboxylase/oxygenase.
A similar effect has been described during long-term ozone
and drought stress in Pinus halpensis M. and could be
associated with decreased CO2 access to chloroplasts due to
stomatal closure (Pelloux et al. 2001) and thus mitigated
photorespiration. Upon drought exposure, several genes of
the glycolysis pathway as well as the citrate cycle-related
genes and cellulose synthase, which catalyzes the synthesis
of cellulose from UDP-glucose and sucrose, were repressed
(Supplementary data). Reduction of sugar metabolism gene
transcription probably is a way of adaptation to reduced
availability of hexoses under drought.
On the biochemical level, glucose and fructose levels
were, indeed, reduced in both clones under long-term
drought, both glucose and fructose levels were even lower
in 397077.16 than in Canchan (Fig. 4). This strong decrease
of glucose and fructose contents was probably not the only
consequence of a reduction in carbon fixation during
drought stress due to stomatal closure and downregulation
of genes from the Calvin cycle, since some carbohydrates
have been shown to increase in numerous model systems
despite a strong reduction in photosynthetic activities (Xue
Funct Integr Genomics (2011) 11:275291
Fig. 3 Hierarchical clustering with complete linkage criterion
performed on qPCR relative gene expression value and individual
biological replicates after 21 days of exposure. T trend of significance
et al. 2008). Different factors may contribute to this
decrease in hexose content; carbohydrates are involved in
feedback regulation of leaf photosynthesis and development
in order to maintain the N/C status. Indeed, glucose and
fructose are thought to participate in gene expression
regulation such as also sucrose, starch, and several other
amino acids (Krapp et al. 1993; Paul and Pellny 2003).
283
(0.1> P value >0.05) between relative expression under control and
drought condition. Asterisk significant difference between relative
expression under control and drought condition (P value <0.05)
Moreover, evidences of the presence of some hexoses in
sieve-tube sap indicated that transport of some reducing
sugar to sink tissues is possible (van Bel and Hess 2008).
Another well-known stress responsive gene is sucrose
synthase (Susy) catalyzing the reversible cleavage of
sucrose into D-fructose and UDP-glucose. In Andean
cultivars, two isoforms of sucrose synthase, invertases and
284 Funct Integr Genomics (2011) 11:275291

Fig. 4 Carbohydrates and polyols (mol g1 DW) in 397077.16 and Canchan 32 and 49 days after the drought onset. Different letters indicate a
significant difference (P<0.05). Asterisks contents are under the threshold of detection
Funct Integr Genomics (2011) 11:275291
starch degradation enzymes were induced in response to
drought and might participate to the mobilization of
reserve carbohydrates (Schafleitner et al. 2007). In the
present experiment, sucrose synthase expression increased
only in Canchan late under drought stress, but not in
397077.16. Principal component analysis, based on corre-
lation matrix, performed on gene expression, 21 days AD,
and carbohydrate content, 32 days AD (Fig. 5) displayed a
negative correlation between sucrose synthase 2 expres-
sion and sucrose content, suggesting that this enzyme was
actually responsible for sucrose cleavage. Sucrose is
known to accumulate upon plant exposure to abiotic
stresses such as NaCl, drought and cold (Evers et al.
2007) and development of tubers requires large amount of
sugars, including sucrose, for tuber initiation and forma-
tion (Jackson 1999; Fernie and Willmitzer 2001). In our
study, sucrose content was higher in 397077.16 under
control and drought treatments and at all the time points
and might be a component of the lower osmotic potential.
Reduced sucrose content in Canchan might point to
increased stress susceptibility of this clone. In parallel,
transcripts encoding sucrose phosphate synthase, the key
enzyme in sucrose biosynthesis, decreased markedly in
both cultivars (Figs. 3, 6). However, other SPS, Susy
isoforms or alternative pathways should be responsible for
sucrose content maintenance in 397077.16 and might also
be implicated in yield maintenance.
In summary, the ability of 397077.16 to maintain sucrose
level under drought stress, while on one hand, glucose and
fructose contents decreased and on the other hand, susy and
Fig. 5 Principal component analysis performed on carbohydrate contents (32 days AD) and qPCR gene expression (21 days AD) data related to
sucrose and raffinose family oligosaccharide pathways across genotypes and treatments
285
SPS expression was repressed, supports the idea of a
modulation of sugar levels linked to carbon resources
reallocation and may be an interesting starting hypothesis
for further experiments on yield maintenance mechanisms
under drought conditions.
397077.16 and Canchan present different expression
patterns regarding protective compound synthesis
During abiotic stresses, plants can produce compounds with
protective properties, which help to face disorders caused
by osmotic and redox imbalance or reactive oxygen
species, to name a few. In the present study, we found in
397077.16 some genes coding for several key enzymes of
protective amines pathways such as polyamine pathways,
e.g., spermidine (for review see Groppa and Benavides
2008). These amines are involved in several stress responses
and are thought to have a protective role against ROS.
In the same way, 397077.16 displayed an increased
expression of asparagine synthetase. Asparagine has a high
C/N ratio and acts in a broad spectrum of plant functions.
This compound is the major form for nitrogen transport and
storage; moreover, asparagine has also been shown to
accumulate during osmotic stresses such as drought and
salinity, and acts as an osmolyte compound such as proline
(Lea et al. 2007). Several studies suggest that induction of
proline biosynthesis genes resulting in proline accumulation
during osmotic stress leads to increased tolerance (Zhang et
al. 1995; Igarashi et al. 1997; Bartels and Sunkar 2005).
However, P5CS, the key enzyme for proline synthesis, was
286
Fig. 6 Trend chart of carbohydrate, polyol and RFO pathway of both
397077.16 (39) and Canchan (C) under long-term drought exposure.
Trends of relative gene expression and contents are displayed
respectively in elipses and squares for each genotype. Red strong
not induced in 397077.16 and only slightly upregulated in
Canchan according to qPCR analysis (Fig. 3). A second
isoform of this gene, previously described to be induced by
drought in potato (Schafleitner et al. 2005), also did not
display any differential expression between drought-
stressed and control plants (data not shown). This result
suggested that P5CS is not required for drought tolerance in
397077.16. Also in previous studies on potatoes, this gene
was more expressed in susceptible than in tolerant clones
(Schafleitner et al. 2005; Vasquez-Robinet et al. 2008).
Similarly in barley, proline accumulation was recognized
rather as a marker of severe stress in leaves than as
compound contributing to drought resistance (Hanson et al.
1977) and in cassava, water deprivation can cause a proline
increase of 25-fold in susceptible cultivars but only
ninefold in tolerant ones (Sundaresan and Sudhakaran
1995). Another explanation for this could be, as suggested
by Deyholos (2010), that many transcriptomic studies have
been performed using sensitive cultivars and thus displayed
enhanced expression of proline synthesis genes. This would
indicate that proline accumulation is not a drought tolerance
trait in potato but would rather be related to stress response
suggesting a higher stress response in Canchan as compared
to 397077.16.
Funct Integr Genomics (2011) 11:275291
increase of expression or content under drought exposure, pink slight
increase, light blue slight decrease, dark blue strong decrease, and
gray color no change in expression or content between control and
drought
Finally, in both clones, a gene coding for raffinose
family oligosaccharides such as a UDP-4-glucose epimer-
ase, galactinol synthase and raffinose synthase were
upregulated by drought exposure and even more in
397077.16, as supported by microarray and qPCR results
(Figs. 3, 6, Supplementary data). These results are in
accordance with the increased galactinol content as ob-
served during drought treatment (Figs. 4, 5, 6). This
increase was more important in the tolerant cultivar
397077.16 than in the sensitive Canchan, 3.63 and 1.87
times more, respectively. Galactinol is a precursor for RFO
synthesis. Despite the fact that raffinose synthase was
induced after 21 days of drought, raffinose content was
detectable only after 49 days of drought treatment in
397077.16; this could be explained by a delay between
gene expression and detection of metabolite accumulation
or posttranscriptional regulation. In Canchan, inositol, both
substrate and product of RFO synthesis, and galactose, a
product of sucrose synthesis from raffinose, increased after
49 days of drought treatment (Fig. 4). At this time point, the
significant decrease of the sucrose content in Canchan
leaves correlated with an increase in galactose, inositol, and
galactinol (Fig. 4). Altogether, these results indicated that
carbon flux was partially diverted from starch synthesis,
Funct Integr Genomics (2011) 11:275291
cellulose synthesis, and glycolysis into RFO metabolism or
synthesis, such as galactinol, in both cultivars, raffinose in
397077.16, and inositol and galactose in Canchan. The
difference in sugar alcohol accumulation patterns between
both clones suggests that galactinol and raffinose might be
linked to beneficial effects for yield maintenance. RFOs are
thought to act as osmoprotectant molecules and are thus
involved in tolerance to water deprivation; indeed, RFOs
such as stachyose and raffinose were shown to accumulate
during seed desiccation in maize and to enhance drought
tolerance in plants (Taji et al. 2002). However, in the
present study, the measured sugaralcohol levels were not
sufficient to explain any osmotic potential decrease in the
leaves. Additionally to their osmotic effect, sugaralcohol
are thought to protect cells against oxidative damage by
scavenging hydroxyl radicals (Nishizawa et al. 2008). The
higher galactinol levels and the accumulation of raffinose
only in 397077.16 are suggested to be involved in the
increased drought tolerance of this clone.
Drought-responsive structural modifications
Cell wall modifications-related genes were usually associated
with biotic and abiotic stress responses and, interestingly,
three genes coding for pectin methylesterase inhibitor
(PMEI) protein were induced by drought exposure in
397077.16; these proteins are usually associated to responses
against pathogen attack but overexpression of PMEI proteins
has recently been shown to increase both basal resistance to
pathogen and abiotic stress tolerance such as drought and
oxidative stress in Capsicum annuum L. cv. Nockwang (An
et al. 2009). 397077.16 also displays higher expression of
genes coding for non specific lipid transfer protein (nsLTP1
and nsLTP2) compared to Canchan. nsLPTs are able to bind
several lipids such as phospholipids and glycolipids, in-
volved in several pathways including cutin synthesis, beta-
oxidation, somatic embryogenesis, plant signaling, plant
defense against pathogens, pollen adherence (Carvalho and
Gomes 2007) and are induced during several abiotic stresses
such as cold, drought and high salinity (Trevino and
O'Connell 1998). According to Cameron et al. (2006), there
is a correlation between cuticular wax accumulation, drought
tolerance and nsLTP expression in tobacco.
397077.16 was able to face drought-induced cell damages
Drought stress can initiate a secondary oxidative stress
(recently reviewed by Miller et al. 2008), consequently
tolerance to drought must implicate mechanisms of cell
protection and repair, such as detoxication enzymes like
metallothioneins, thioredoxin, glutathion-S-transferase, and
glutaredoxins, which help to maintain oxidoreduction
balance and protect proteins and membranes from irrevers-
287
ible damages (Schafleitner et al. 2007; Vieira Dos Santos
and Rey 2006; Bartels and Sunkar 2005). In 397077.16,
many thioredoxins, metallothioneins, glutaredoxins, and a
gene coding for ascorbate peroxidase were induced. In
Canchan, only a few thioredoxins were slightly upregu-
lated; neither metallothionein nor ascorbate peroxidase
genes were found to be differentially regulated. According
to Nishizawa et al. (2008), transgenic plants overexpressing
genes cited above and possessing enhanced ROS-
scavenging capacity, displayed an increased tolerance to
abiotic stresses such as salinity and drought. Thus, higher
expression of detoxication-related enzymes, observed in
397077.16 (Fig. 2a, b) is suggested to be a component of its
drought tolerance.
Similar expression patterns as found for detoxication-
related genes were observed for putative structural
protective proteins such as chaperones, dehydrins and
late embryogenesis abundant proteins: they were stron-
ger or exclusively upregulated in the tolerant clone
397077.16. Among these genes were dehydrin (TAS14-
like), which belong to group 2 of LEA as well as
further members of this gene family, LEA-1,-5 genes.
LEAs have been shown to accumulate during seed
maturation, various abiotic stresses and confer drought
tolerance in tomato (Godoy et al. 1990; Rorat 2006).
Upregulation in 397077.16 also concerned small chloro-
plastic and mitochondrial heat shock proteins (sHSPs,
Fig. 3). Small HSPs are proteins that can form oligomers
of 1224 subunits and are to bind denaturated proteins
thus maintaining correct folding and preventing protein
aggregation (Waters et al. 2008).
Despite an efficient ROS detoxication and folding
maintenance system, some damaged and unnecessary
protein might be degraded (Vierstra 1993). This trend
was observed in Canchan, and even to a greater extent in
397077.16: transcription of the RD19A cysteine protease
increased in response to drought; this protease has been
described by Koizumi et al. (1993) to be responsive to
drought and high salinity but not to ABA treatment in A.
thaliana suggesting that this gene is responsive to
osmotic changes in the cells and involved in protein
turnover. However, in Canchan, many protease inhibitors
were strongly induced whereas they were repressed in
397077.16 (see Supplementary data). This latter result
was confimed by qPCR (Fig. 3, Supplementary data).
Previous studies showed that protease inhibitors such as
Kunitz-type protease inhibitors are induced by drought
and ABA (Downing et al. 1992) while some other
protease inhibitors were induced by wounding, jasmonic
acid and ABA (Hildmann et al. 1992). Protease inhibitor
induction during drought exposure or wounding is
suggested to be part of a general mechanism of damage
prevention against proteases that are activated by protein
288
denaturation due to osmotic stress (Plant and Bray 1999;
Rabbani et al. 2003). In native potato, proteinase inhibitor
genes were rather induced in a drought susceptible as
compared to a tolerant clone, suggesting that upregulation
of these genes is more associated with a stress than a
tolerance response (Schafleitner et al. 2007).
These results suggest that induction of cell rescue-related
genes in 397077.16 enables this clone, on one hand to
mitigate cellular disorders caused by drought and on the
other hand to better recycle its proteome (Figs. 2a, b, 3).
According to these results, it can be assumed that improved
ROS detoxication and protein structure maintenance pre-
venting cell damage and metabolic disorder contributes to
tolerance of 397077.16.
Fig. 7 On left overview of the physiological, transcriptomic, and
biochemical results. For physiological and biochemical result, blue
and red arrows represent decrease and increase of the measurements,
respectively. For microarray result, blue and red arrows means that a
Funct Integr Genomics (2011) 11:275291
Co-regulation of drought tolerance mechanisms
in 397077.16
qPCR results displayed a common expression pattern for
genes coding for galactinol synthase, raffinose synthase,
UDP-4-glucose epimerase, dehydrin TAS14, chloroplastic
small HSP, 25.6 kDa HSP, DREB1, ANAC083, PGR5, and
SNF1-related protein kinase suggesting these genes (path-
ways) were coordinatly regulated (Fig. 3). Beyond these
end point mechanisms leading to better tolerance, an
efficient stress sensing and signal transduction apparatus
is needed to trigger an appropriate response to drought.
397077.16 showed an involvement of calcium signaling
pathway by differential expression of calmodulins and
majority of the genes belonging to the category was down or
upregulated. Two arrows represent more than 75% of the genes of
the respective functional category. On the right, suggested model for
drought tolerance and yield maintenance in 397077.16
Funct Integr Genomics (2011) 11:275291
calreticulin. Calreticulin is an endoreticulum Ca2+-binding
protein, which is involved in intracellular calcium homeo-
stasis and is also repressed in potato leaves during salinity
stress (Legay et al. 2009). Calmodulins are calcium sensors
that are implicated in many cellular processes and also in
the ABA-signaling pathway (Ma et al. 2009). This
signaling apparatus seemed to be more effective, in
397077.16, to trigger expression of transcription factors.
As example and compared to Canchan, 397077.16 dis-
played a higher expression of many transcription factors
such as NAC-like, DREB protein. In our study, a NAC083-
like transcription factor, which displays a strong similar-
ity with NAC1-like transcription factor, was also upregu-
lated. NAC-1-like transcription factor has been shown to
mediate the auxin-signaling pathway and to promote
lateral root development in A. thaliana (Xie et al. 2000).
Overexpression of DREB1a, -b, and -c strongly induced
galactinol synthase gene (Taji et al. 2002; Gilmour et al.
2004) or displayed common functional activities on
several genes such as RD29B, COR proteins and various
other dehydrins (Gilmour et al. 2004), as observed in the
present study. Enhanced induction of these various
transcription factors in 397077.16, could be important for
drought tolerance traits capture. Indeed, in Canchan, none
of these transcription factors was significantly differen-
tially regulated in response to drought exposure. Induction
of DREB1-like transcription factor and NAC083-like
transcription factor remained below the fold change cut-
off value in Canchan.
General model for drought tolerance in 397077.16
At the agronomical level, 397077.16 had an adapted shape
to drought with smaller leaves and ground cover, which
could be part of the reduced yield under well-watered
conditions but might also lead to enhanced tolerance to
water loss under drought.
As decribed in the previous sections, 397077.16s yield
maintenance under drought was driven by a muticomponent
system effective at different levels of the potato metabolism
(Fig. 7). This included better management of primary
resources such as light collection adjustment and slowdown
of primary metabolism. 397077.16 showed also an inter-
esting ability to face osmotic imbalance and cell damages
by enhanced expression of genes related to synthesis of
protective compounds, ROS detoxication, redox balance
and protein folding maintenance. These traits have been
already observed in plant models (Bartels and Sunkar 2005;
Deyholos 2010), but interestingly, in the present work, a
large part was highly expressed in the tolerant clone
and only slightly in Canchan. Thus, we might speculate
that tolerance was correlated to the expression level of
tolerance genes.
289
However, some differences were observed compared to
plant models. In many studies, higher or overexpression of
proline pathway related genes has been shown to enhance
tolerance, however, in our work it seems this pathway was
more related to stress response than tolerance. Furthermore,
due to the specificity of tuber crop such as potato, the
carbon remobilisation and the maintenance of high sucrose
content observed in leaves of 397077.16 may constitute one
of the crucial components of the drought tolerance and
yield maintenance. All together these data provide a first
draft of a drought tolerance model in 397077.16.
Conclusion
Physiological analysis showed higher water use efficiency,
stomatal control, root elongation, and yield maintenance in
397077.16, which reflects a better ability to maintain
photosynthesis and putative sucrose export to tubers under
drought exposure. The gene expression pattern, under
drought, differed a lot between the sensitive and the
tolerant clone. On one hand, the drought-susceptible
Canchan presented only a slight upregulation of tolerance-
related genes and at the same time displayed reactions that
are putatively implicated with drought susceptibility, such
as proline biosynthesis or protease inhibitor-related genes,
the latter being usually expressed in response to membrane
disruption as observed in biotic stress response. On the
other hand, in 397077.16, we observed increased expres-
sion of genes, which are involved in various functions such
as gene expression regulation, carbohydrate metabolism
and flux, reactive oxygen species detoxication, maintenance
of protein folding, lowered osmotic potential, and limitation
of transpiration. These genes might be key components of
the drought tolerance of 397077.16. To our knowledge, this
is the first report on drought tolerance traits in advanced
potato clones.
Acknowledgments The authors would like to thank Laurent
Solinhac for his excellent technical assistance, Drs Bodo Trognitz
and Fiederike Trognitz for the microarray slides and finally Dr Torsten
Bohn for his precious help on statistics. This work was financially
supported by the Ministry of Finance (Luxembourg).
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