drought responses of leaf tissues from wheat cultivars of differing drought tolerance at the...
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Molecular Plant • Volume 5 • Number 2 • Pages 418–429 • March 2012 RESEARCH ARTICLE
Drought Responses of Leaf Tissues from WheatCultivars of Differing Drought Tolerance at theMetabolite Level
Jairus B. Bownea, Tim A. Erwina, Juan Juttnerb, Thorsten Schnurbuschb, Peter Langridgeb,Antony Bacica,c,d,e and Ute Roessnera,e,1
a Australian Centre for Plant Functional Genomics, School of Botany, The University of Melbourne, 3010 Victoria, Australiab Australian Centre for Plant Functional Genomics, University of Adelaide, Waite Campus, Glen Osmond, 5064 SA, Australiac Metabolomics Australia, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Rd, The University of Melbourne, 3010 Victoria, Australiad ARC Centre for Excellence for Plant Cell Walls, School of Botany, The University of Melbourne, 3010 Victoria, Australiae Metabolomics Australia, School of Botany, The University of Melbourne, 3010 Victoria, Australia
ABSTRACT Drought has serious effects on the physiology of cereal crops. At the cellular and specifically the metabolite
level, many individual compounds are increased to provide osmoprotective functions, prevent the dissociation of
enzymes, and to decrease the number of reactive oxygen species present in the cell. We have used a targeted GC–MS
approach to identify compounds that differ in three different cultivars of bread wheat characterized by different levels
of tolerance to drought under drought stress (Kukri, intolerant; Excalibur and RAC875, tolerant). Levels of amino acids,
most notably proline, tryptophan, and the branched chain amino acids leucine, isoleucine, and valinewere increased under
drought stress in all cultivars. In the two tolerant cultivars, a small decrease in a large number of organic acids was also
evident. Excalibur, a cultivar genotypically related to Kukri, showed a pattern of response that was more similar to Kukri
underwell-watered conditions. Under drought stress, Excalibur and RAC875 had a similar response; however, Excalibur did
not have the same magnitude of response as RAC875. Here, the results are discussed in the context of previous work in
physiological and proteomic analyses of these cultivars under drought stress.
Key words: abiotic/environmental stress; metabolomics; drought; wheat.
INTRODUCTION
Various countries around the world experience drought in dif-
ferent ways but, in all cases, it leads to dramatic annual yield
losses in crops and has consistently detrimental physiological
effects on the crop plants throughout (Boyer, 1982).
Morphological adaptations to drought stress, such as leaf
waxiness and trichome density, can decrease the amount of
water lost to the environment, providing longer-lasting pro-
tection against water-deficit stress (Richards et al., 1986;
Vogelmann, 1993; Grammatikopoulos and Manetas, 1994;
Save et al., 2000). Under water-deficit and drought stress, most
plants will close their stomata to decrease their transpiration
rate, further limiting the water lost to the environment—a
process mediated by the signaling molecule abscisic acid, pro-
duced in the roots. Limiting transpiration slows the production
of reactive oxygen species (Rizhsky et al., 2002; Mahajan and
Tuteja, 2005; Monneveux et al., 2006), which can cause DNA
nicking, amino acid and protein oxidation, and lipid peroxida-
tion (Reddy et al., 2004), which disrupts the lipid bilayer
structure and reduces the function of membrane-bound
enzymes (Sairam et al., 1997; Mahajan and Tuteja, 2005; Miller
et al., 2010). More severe or extended water deficit will cause
further changes, including loss of turgor and decreased cell
growth (Steudle, 2000).
Plants will also undergo a range of molecular changes that
enable them to better cope with the short-term effects of
water deficiency and allow for more rapid response times.
Osmotic adjustment (OA) has been shown in a number of
different cereals (e.g. barley (Gonzalez et al., 2008), canola
(Norouzi et al., 2008), and maize (Hajlaoui et al., 2010)), and
1 To whom correspondence should be addressed at address a. E-mail
[email protected], tel. +61 3 90353635.
ª The Author 2011. Published by the Molecular Plant Shanghai Editorial
Office in association with Oxford University Press on behalf of CSPB and
IPPE, SIBS, CAS.
doi: 10.1093/mp/ssr114, Advance Access publication 29 December 2011
Received 29 September 2011; accepted 6 December 2011
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may contribute to photosynthetic and stomatal adjustment
mechanisms in these crops. OA can be provided by many com-
pounds, including proline, polyols (mannitol and inositol), and
quaternary ammonium compounds such as glycine betaine
(Wang et al., 2003; Yancey, 2005; Bartels and Phillips, 2010).
These compounds also surround the hydration shell around
delicate proteins and prevent their degradation under osmotic
stress (Galinski, 1993).
While there have been numerous genetic (Street et al., 2006;
Kato et al., 2008; Mathews et al., 2008; Keppler and Showalter,
2010; Ruan et al., 2010; Liu et al., 2011; Xue et al., 2011) and
transcriptional (Ozturk et al., 2002; Gorantla et al., 2005;
Talame et al., 2007; Deyholos, 2010; Dıaz et al., 2010; Yang
et al., 2010) studies of changes under drought stress, only
a handful of studies have examined drought and water-deficit
stress from a metabolomics perspective. Using NMR, Charlton
et al. (2008) found increased levels of proline, leucine, isoleu-
cine, valine, threonine, and homoserine, as well as myoinosi-
tol, malate, and c-aminobutyrate (GABA) in pea leaves (Pisum
sativum L.) under simulated field conditions, but noted that
leucine and isoleucine were not elevated when the same
plants were grown in a greenhouse. Alvarez et al. (2008), using
an LC–MS method for maize xylem sap, found increased pro-
line, malate, p-coumarate, and caffeate accumulated in the
sap; however, ferulate decreased under water stress. Sanchez
et al. (2011) investigated metabolic responses following differ-
ent levels of drought stress in a Lotus japonicus species, allow-
ing them to identify correlations between the stress level and
the magnitude of changes in the metabolite profiles. Most
changes were observed in the levels of organic acids, sugars,
and polyols. The authors also described a comparative study
analyzing the changes in the metabolite profiles of a number
of Lotus species, which indicated that some metabolite
changes are conserved between species but that there are also
numerous species-specific pathways affected upon drought
stress (Sanchez et al., 2011). In both studies, around 90% of
all detected significant changes were increases. Witt et al.
(2012) observed increased proline, putrescine, histidine, and
phenylalanine in maize leaves, while pyruvic and quinic acids
decreased in response to drought treatment.
Izanloo et al. (2008) studied the same three wheat cultivars
that were used in the present study under a cyclic drought
stress regime. In those experiments, the treated plants were
subjected to water-deficit stress to the point of visible wilting,
and then were re-watered to field capacity. This was repeated
a number of times to mimic the field conditions of the South
Australian climate in a growth room. Excalibur was the most
responsive cultivar, and had a quicker recovery from drought
stress, while RAC875 had a more moderate rate of osmotic ad-
justment, and thus a slower recovery from stress. RAC875 also
had higher chlorophyll content and higher leaf waxiness.
Kukri and Excalibur were seen to use similar amounts of water
in the control plants (well watered), while RAC875 used
slightly less. Prior to the imposition of drought stress, Excalibur
used 35% more water than the other two cultivars; however,
this may have been due to a later heading time (12 d later than
RAC875, 14 d later than Kukri). Of the two experiments
conducted in that study, Experiment II, which had a milder
water-deficit stress imposed, was the most similar to this study.
Under these conditions, RAC875 was the only cultivar to show
a significantly higher grain yield under the cyclic stress.
In the present study, a similar cyclic drought treatment on
the same wheat cultivars as studied by Izanloo et al. (2008)
was conducted. Over the course of two watering cycles (Ford
et al., 2011), flag leaf samples were taken for metabolomics
analysis (this work) and mature leaves for proteomics analysis
(Ford et al., 2011). The quantitative proteomic analysis
revealed that both tolerant cultivars (Excalibur and RAC875)
had a greater number of proteins responding to the drought
stress compared to Kukri. Also, proteins in RAC875 seem
to respond much more quickly to the onset of drought than
in the other cultivars and it was concluded that RAC875 seems
to have the highest capacity for a cellular-level response
amongst the three cultivars. Proteins found to respond to
drought were involved in photosynthesis and the Calvin cycle,
cell redox homeostasis, glycolysis and gluconeogenesis, trans-
port and protein folding mechanisms, protein degradation,
and amino acid metabolism. Additionally, a number of known
stress-related response proteins were found to be altered (Ford
et al., 2011). Here, we used a targeted GC–MS approach to
monitor 103 individual small molecules from leaf tissue of
the drought-stressed wheat plants, predominantly amino
and organic acids and sugars (see Supplemental Tables 1–3).
The aim of this study was to find compounds that may be
implicated in conferring the ability to maintain yield even
under drought conditions in the field by the two tolerant cul-
tivars (Excalibur and RAC875) and determine any differences
between them, and to examine the changes in metabolite
responses between drought-stressed and control plants for
all three cultivars. By comparing this with physiological
changes presented in Izanloo et al. (2008), we can begin to
refine our model of drought stress in crop species.
RESULTS
Over the time course, control plants maintained a steady
relative water content (RWC), averaging 97% across all
three cultivars (see Figure 1). In the stressed plants, there
was a steady decline in the first three time points, Kukri and
Excalibur showing a similar rate to each other (72.6–54.4
and 74.5–50.5%, respectively) while RAC875 maintained a
higher RWC throughout (87.1–67.9%). At the second wilting
point (time point D), Kukri showed a similar RWC to the pre-
vious wilting time point (C), while RAC875 and Excalibur had
increased RWC (14.5 and 16.7%, respectively) above their level
at time point C.
Since metabolite regulation is a major mechanism used to
maintain osmotic potential during water stress, we employed
a targeted GC–MS-based high-throughput approach to mea-
sure a wide range of metabolites simultaneously. We
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identified 103 metabolites including 26 sugars, 17 primary
amino acids, and 45 organic and fatty acids (for a complete list
of metabolites measured, see Supplemental Tables 1–3). Ana-
lyzed metabolites were broadly classified into amino acids, or-
ganic acids, sugars, and others.
Cultivar Specific Differences Prior to Stress Treatment
Comparing RAC875 and Excalibur to Kukri at time point A in
the well-watered controls (Supplemental Table 1), pipecolate
was the only compound that was significantly different in
Excalibur (3.9-fold higher). RAC875 had two to threefold
increases in isocitrate, oxalate, digalactosylglycerol, and
1-monooctodecanoglycerol as well as increased xylose
(3.2-fold), glycerol (3.4-fold), oxalate (3.6-fold), isocitrate
(3.9-fold), and decreases in asparagine (2.1-fold), leucine
(2.7-fold), and tryptophan (4.1-fold). Lysine was increased
by 2.5-fold in Excalibur and 21.5-fold in RAC875, but neither
increase was statistically significant.
Metabolite Changes of Control Samples over the Time
Course of Sample Collection: Developmental and
Cultivar-Specific Changes
The majority of samples in the well-watered controls showed
only a few changes as the age of the plants increased. Supple-
mental Table 2 shows the full list of metabolites and their
x-fold changes relative to the control time point A.
In Kukri, all time point B samples were missing. Only a few
amino acids decreased significantly between two and three-
fold over the three time points (C, D, and E) compared
to the first time point (A) (Supplemental Table 2). The most
pronounced amino acid change was proline, which was at
a higher level at time point E (6.6-fold), but this was not sta-
tistically significant. Of the analyzed organic acids, significant
increases over time were observed only for 2-oxogluconate
(6.3- and 6.8-fold at time point D and E, respectively). There
were a number of statistically significant; however low in
magnitude, decreases over time in the organic acids (between
1.3- and 3-fold; Supplemental Table 2). At time point D and E
compared to A, 2-oxoglutarate, itaconate, citrate, and aconi-
tate decreased between three and eightfold; however, most of
these changes were not statistically significant. Only a few sug-
ars changed in control Kukri plants over the time course of
the experiment, with the most pronounced increases seen
for fructose and glucose at time points D and E between four
and 15-fold (Supplemental Table 2). Additionally, glycerol
decreased at all time points compared to A, with the biggest
decrease of eightfold at time point D. Interestingly, adenosine
decreased at time points C and D between 22- and 26-fold.
A few other compounds decreased significantly, but with
low magnitude (Supplemental Table 2).
RAC875 and Excalibur have very similar changes throughout
all time points when compared to time point A. Very few
amino acids changed significantly in both cultivars; for exam-
ple, there was a decrease in GABA at time points B and D in
both Excalibur and RAC875, while the increased tryptophan
at time point D (eightfold) was only seen in RAC875 (Supple-
mental Table 2). The only striking changes in the organic acids
were decreases in a number of compounds at time point D
when compared to A in both cultivars, including caffeate,
glycerate, succinate, threonate, and threonate-1,4-lactone
(Supplemental Table 2). Other organic acids that decreased
only in RAC875 included quinate, aconitate, citrate, and shiki-
mate, while, in Excalibur, only isocitrate, muconate, and
malate (Supplemental Table 2). There were no striking changes
in sugars in RAC875 throughout all time points; however, two
sugars increased in Excalibur at time points D and E compared
to A—glucose (four and sixfold, respectively) and fructose (five
and sevenfold, respectively) (Supplemental Table 2). This is
a similar pattern to that seen in Kukri. Also as in Kukri, aden-
osine decreased at time points B, C, and D both in RAC875 and
Excalibur, with stronger decreases in RAC875 (around 15-fold)
than in Excalibur (between five and eightfold). As opposed to
Kukri, where the level of adenosine increased at time point E
compared to A, in RAC875 and Excalibur, it did not increase but
rather recovered to nearly the same levels as at time point A.
Another strong decrease was seen in RAC875 in the levels of
1-monooctadecanoglycerol, which decreased around 11-fold
at time points B, C, and D but only fourfold at time point E
compared to A. The only increases observed were allantoin
in RAC875 at time points B, C, and E of around ninefold
and octadecanol in RAC875 (time point E, 4.8-fold), Kukri
(2.6-fold at time point B, 3.1-fold at time point C), and Excalibur
(2.2-fold at time points B and E).
Figure 1. Relative Water Contents at Each Time Point for Each ofthe Three Cultivars.
No measurement was taken at time point E for the control samples.Time point B samples of Kukri were missing from the experiment.Time points C and D in the drought samples were at the wiltingpoint of Kukri. Excalibur follows the decline in RWC that Kukridoes over time points A–C, but behaves more like RAC875 at timepoint D.
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Most Amino Acids Increased Following Drought Stress in
Kukri, RAC875, and Excalibur
To determine the responses of each cultivar upon water-
deficit stress, we compared the level of each metabolite at
each time point of the stressed plants with the level of the
same metabolite at the respective time point of the control
plant.
The majority of amino acids increased in all cultivars
throughout the stress time course when compared to the
controls (Figures 2, 3, and 4, and Supplemental Table 3). Most
pronounced increases were observed for proline and trypto-
phan, which increased in all three cultivars between three
and 31-fold (proline) and three and 100-fold (tryptophan)
(Figures 2, 3, and 4, and Supplemental Table 3). Also, the
branched chain amino acids (BCAAs) isoleucine, leucine, and
valine all showed a similar pattern of response (Figures 2, 3,
and 4, and Supplemental Table 3). Under water-deficit stress,
the levels of the Kukri BCAAs increased between two and
sevenfold. The BCAAs in Excalibur increased dramatically
between two and 10-fold across all time points. In RAC875,
these compounds varied between two and ninefold across
all time points. Methionine, tyrosine, phenylalanine, gluta-
mine, and beta-alanine were other amino acids that changed
in all three cultivars, mainly at time points D and E. Changes
ranged between two and 19-fold (Figures 2, 3, and 4, and
Supplemental Table 3). Interestingly, homoserine increased
dramatically at time point D in all cultivars following water
deficit between five and 13-fold when compared to the con-
trol. Lysine showed an interesting pattern by increasing in
Kukri only at time points C, D, and E (five, three, and twofold,
respectively); however, it decreased in RAC875 at time point D
by sixfold, and did not show any significant changes in Excal-
ibur. Asparagine increased only in RAC875 at all time points
between two and 10-fold.
Most Organic Acids Decreased Following Drought Stress
at Time Point C in RAC875 and Excalibur
Most organic acids that changed were decreased throughout
the treatment series for all three cultivars, with only a few
exceptions. In Kukri, approximately 7% of all analyzed organic
acids decreased significantly and more than twofold across the
four time points, whereas RAC875 and Excalibur showed
decreases in 15.6 and 20%, respectively, that were significant
and greater than twofold across multiple time points. It is
important to note that a large proportion of organic acids
decreased at time point C in both RAC875 and Excalibur
(around 50% of all analyzed organic acids; see Figures 2, 3,
and 4, and Supplemental Table 3), although not greater than
fivefold. Only a handful of organic acids increased significantly
throughout the stress series, including 2-oxobutanoate
in Kukri at time point C (eightfold), in RAC875 in time
points A, B, and C (four, nine, and fivefold, respectively),
and in Excalibur at time points B and C (11- and threefold,
respectively).
Sugars and Other Compounds Follow a Similar Pattern of
Major Decreases at Time Point C in RAC875 and Excalibur
Similarly to the organic acids, most sugars decreased at time
point C in RAC875 (17 out of 26 greater than twofold) and
Excalibur (10/26), and also at time point E in RAC875 (12/26)
and to a lesser extent also in Excalibur (4/26) (Supplemental
Table 3). The same was seen for the compounds in the ‘other’
category, RAC875 with 7/10 and Excalibur 6/10 showing
greater than twofold decrease at time point C, and at time
point E in RAC875 4/10 compared to Excalibur, with 2/10 com-
pounds showing a decrease of more than twofold. In general,
these decreases were not large, ranging from two to fourfold;
however, most were statistically significant, with a P-value of
less than 0.05. Only two of the sugars, inositol and galactinol,
decreased across all cultivars and time points (between two
and sevenfold; Figures 2, 3, and 4, and Supplemental Table
3). Digalactosylglycerol decreased only at time points D and
E in all three cultivars, with the biggest decrease of 23-fold
at time point E in Excalibur when compared to the control sam-
ple. Fructose and glucose increased only in Kukri at time points
A and C and Excalibur at time points A and B, but recovered to
the level in the controls at time points D and E, while remain-
ing mostly unchanged in RAC875. This is interesting, since
fructose and glucose were increased at time points D and E
only in Kukri and Excalibur in the control samples compared
to time point A. Allantoin also increased in RAC875 at time
point A (fourfold) and in Excalibur at time point B (10-fold)
(Figures 2, 3, and 4, and Supplemental Table 3).
DISCUSSION
Izanloo et al. (2008) found that, under well-watered condi-
tions, the three cultivars had comparable grain yields and that,
under cyclic water stress, Excalibur depleted the soil water pro-
file faster than the other two cultivars. RAC875 showed the
highest grain yield in both mild and severe drought stress
experiments, while Excalibur only had higher yield than Kukri
in the severe drought stress experiment. Morphological and
physiological traits in the leaves of the three cultivars were also
likely to contribute to their ability to withstand drought stress.
The major source of water lost (95–99%) from plants in well-
watered conditions occurs through the pores of stomata
(Goodwin and Jenks, 2005); thus, increased leaf waxiness
allows less water to be lost after stomatal closure, increasing
tolerance to drought and increasing water-use efficiency
(Richards et al., 1986; Zhang et al., 2007). RAC875 has waxy
leaves, while Kukri does not. Excalibur has some wax, but rolls
its leaves under drought more rapidly than Kukri whereas
RAC875 shows almost no leaf rolling (Izanloo et al., 2008).
Excalibur was found to be more responsive to cyclic drought
stress, with higher osmotic adjustment and stomatal conduc-
tance, produced more tillers per se and aborted them under
drought stress, and showed rapid recovery after re-watering
(Izanloo et al., 2008). In comparison, RAC875 is a more
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Figure 2. Metabolic Pathway Map for Kukri.
Control samples are represented in blue, drought-stressed samples in red. Metabolite plots with a green border are discussed in the ‘Results’and ‘Discussion’ sections. Compounds with a blue border are metabolites that showed significant but minor (, fourfold) changes.
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Figure 3. Metabolic Pathway Map for Excalibur.
Control samples are represented in blue, drought-stressed samples in red. Metabolite plots with a green border are discussed in the ‘Results’and ‘Discussion’ sections. Compounds with a blue border are metabolites that showed significant but minor (, fourfold) decreases at timepoint C, similar to the changes seen in RAC875 at this time point.
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Figure 4. Metabolic Pathway Map for RAC875.
Control samples are represented in blue, drought-stressed samples in red. Metabolites with a green border are specifically referred to in the‘Results’ section. Compounds with a blue border are metabolites that showed significant but minor (, fourfold) decreases at time points Cand E. Most of these compounds behaved in a similar manner to Excalibur at time point C but not at time point E.
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conservative cultivar, producing fewer tillers, has a moderate
osmotic adjustment potential, had higher water-soluble car-
bohydrates to remobilize for grain filling, and a stay-green
phenotype that enabled it to continue grain filling under
drought stress (Izanloo et al., 2008). These traits allowed the
two tolerant cultivars (Excalibur and RAC875) to better avoid
damage from prolonged drought stress.
Here, we used GC–MS-based targeted metabolite profiling
to monitor 103 structurally identified metabolites throughout
the drought and re-watering regime. The most pronounced
changes were observed in the amino acid levels, of which
approximately half increased statistically significantly in all cul-
tivars and at most time points. One of the known markers for
water stress, proline, increased dramatically in all treated sam-
ples when compared to their respective controls. Under condi-
tions of drought stress, proline has been shown to increase in
several different plant species, including maize, wheat, pea,
rapeseed, and Lotus species (Rampino et al., 2006; Charlton
et al., 2008; Norouzi et al., 2008; Sanchez et al., 2011; Witt
et al., 2012) and is thought to provide an osmoprotective
function. Nanjo et al. (1999) showed that the osmoprotective
function is not due simply to hydrogen bonding, as exoge-
nously applied D-proline was unable to restore the osmopro-
tection, as did exogenous L-proline. Sharma and Dietz (2006)
posited that formation of proline may function as an electron
sink mechanism, and Alia et al. (1997) showed that proline can
reduce the amount of singlet oxygen present, which causes
lipid peroxidation of thylakoid membranes, providing evi-
dence that proline is an important contributor to cellular re-
dox balance (Szabados and Savoure, 2010).
Tryptophan, an aromatic amino acid, increased across all
time points and cultivars, most dramatically in RAC875. Tyro-
sine and phenylalanine also increased over the course of the
experiment in all three cultivars, especially in Kukri and Excal-
ibur, but not as dramatically as tryptophan. These aromatic
amino acids are synthesized through the shikimate pathway
and serve as precursors for a wide range of secondary metab-
olites, such as indoleacetate, glycosides, lignin precursors, and
terpenoids (Korkina, 2007; Less and Galili, 2008). Levels of
tryptophan and phenylalanine also increased in maize under
drought stress (Witt et al., 2012). Dubouzet et al. (2007) found
that, in a transgenic rice line that accumulated large amounts
of free tryptophan, there were few adverse affects on plant
growth. As tryptophan is a target of oxidation, the free amino
acid may provide a buffer between ROS and proteins in the
chloroplast where both tryptophan and ROS are synthesized,
without incurring a (major) growth penalty. Proteomics anal-
ysis of the same plants used in this study has also shown a cor-
relation between increased protein levels involved in ROS
scavenging and oxidative stress metabolism (Ford et al.,
2011). Although singlet oxygen, superoxide, and peroxide
are generated normally by the photosynthetic machinery,
under conditions of oxidative stress, these compounds will
increase to levels that can inhibit de novo synthesis and
hence turnover of the D1 protein, which is necessary for the
repair of damage to Photosystem II (Nishiyama et al., 2001;
Allakhverdiev and Murata, 2004). ROS also cause oxidation
of proteins, including the D1 protein, which may cause confor-
mational changes that lead to its breakdown by proteases
(Silva et al., 2003). This could be the cause of the increase in
amino acids generally, especially in light of increased leucine
aminopeptidase and metacaspase seen in the proteomic study
of Ford et al. (2011).
Leucine, isoleucine, and valine, the branched chain amino
acids (BCAAs), were increased significantly in all three cultivars
at all time points apart from time point D in the tolerant
cultivars. Urano et al. (2009) stated that accumulation of
BCAAs, as well as a number of other amino acids, increased
under dehydration stress and was regulated at the transcrip-
tional level. Rizhsky et al. (2004) also saw increased BCAAs
under drought stress in Arabidopsis, but not to the same
extent. Less and Galili (2008), working with publicly available
data from Kilian et al. (2007), state that catabolic enzymes of
amino acids increase rapidly in response to abiotic stress and,
as such, have an important role in amino acid metabolism un-
der these conditions. In drought-stressed barley plants, a BCAA
transaminase is up-regulated (Malatrasi et al., 2006); the
authors suggested that this is likely to prevent toxic levels
of BCAAs in the cell whereas Taylor et al. (2004) suggest that
BCAAs may provide an alternative source of energy in sugar-
starved Arabidopsis. This supports the increased levels of
BCAAs over the time course of this experiment in the different
cultivars.
In extremely dehydrated states, non-reducing sugars and
oligosaccharides become important as a replacement for
water, even more so than proline, providing a hydration shell
around proteins (Hoekstra et al., 2001). Galactose and
mannose show a small (up to fourfold) increase over time in
the control samples, and follow a similar pattern in the
drought samples until the final time point, when the levels fall
back to that of their respective control samples in all cultivars.
The increases in these sugars may provide an initial defensive
state against further water loss.
As RAC875 is thought to respond to stress much more
rapidly, the down-regulation of photosynthesis could increase
oxidative respiration in order to provide cellular energy.
The cultivars that respond more slowly may reflect this with
the early increase in glucose and fructose compared to the
well-watered plants. Fructose and glucose can also have an
osmoprotective role and, when correlated with greater
raffinose concentration, have been shown to assist in dehydra-
tion tolerance in wheat seedlings (Bogdan and Zagdanska,
2006) and Urano et al. (2009) noted an increase in fructose un-
der dehydration stress in Arabidopsis. Digalactosylglycerol de-
creased only in the latter time points in all three cultivars. This
metabolite may either provide glycerolipid species for repair
of oxidative damage in photosynthetic tissue or alternatively
provide energy as carbohydrates during oxidative respiration.
Kieffer et al. (2009) suggested increased galactinol to be
a mechanism for storage of photoassimilates as more complex
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sugars in poplar species under cadmium stress, since this
sugar is not required for development. Galactinol can provide
galactose for the raffinose family oligosaccharides, which can
be used for carbon storage and as compatible solutes
(Hannah et al., 2006), and has been seen to accumulate under
dehydration stress (Urano et al., 2009). Raffinose is difficult
to measure accurately by GC–MS due to its co-elution with
1-kestose.
At time point C, just prior to the first wilting point, both
Excalibur and RAC875 showed a similar decrease in organic
acid levels that were generally significant by t-test but were
not large in magnitude. Though the reason for this is unclear,
the increase in cytosolic pH may act in signaling pathways for
extreme stress, and the recurrence at time point E in RAC875
may show a cellular ‘memory’ for the drought stress. The lack
of a similar reaction in Kukri could go some way to explaining
its susceptibility to cyclic water-deficit stress.
The broad picture of changes in metabolites under drought
stress in these cultivars is an increase in amino acids for the
duration of the stress, while organic acid levels in the two tol-
erant cultivars Excalibur and RAC875 are only affected at or
near their wilting point. Kukri, the intolerant cultivar, did
not exhibit these changes even though it shares a part of its
pedigree with Excalibur. The results presented here provide
molecular evidence for the cultivar-specific differences seen
in Izanloo et al. (2008), clearly indicating different mechanisms
employed by the two tolerant cultivars under conditions of
cyclic drought stress. As discussed in Ford et al. (2011), the
proteomic analysis of these cultivars has provided potential
candidate genes for genetic manipulation of wheat cultivars
to enhance drought tolerance, and the metabolite data
further validate these results.
METHODS
Cultivars, growth, stress treatment, and sampling were as per
Ford et al. (2011) and are briefly provided below.
Triticum aestivum Cultivars
� Kukri: 76ECN44/76ECN36//RAC549;MADDEN/6*RAC177;
released in 1999 by the University of Adelaide;
� Excalibur: RAC177/‘Monoculm’//RAC311S; released in 1991
by the University of Adelaide;
� RAC875: RAC655/3/Sr21/4*LANCE//4*BAYONET; breeding
line from Roseworthy Agricultural Campus, SA.
All three cultivars have similar heading times, and carry the
semi-dwarfing gene Rht2. For a more detailed description
of these cultivars and the basis for their selection in these
experiments, see Izanloo et al. (2008).
Growth of Plants
T. aestivum cultivars Kukri, Excalibur, and RAC875 were grown
using the cyclic drought protocol established by Izanloo et al.
(2008) in consultation with wheat breeders at Australian
Grain Technologies and CIMMYT, under controlled growth
and soil conditions in a growth chamber (Waite Campus,
Adelaide University, Glen Osmond, South Australia). Plants
were grown in watertight bags containing 6 kg of Roseworthy
clay mixed with sand (1:1) then watered daily to a weight that
was equivalent to the field capacity (900 ml).
Stress Treatment
Upon emergence of the first flag leaf, the drought experiment
commenced. Water was incrementally withdrawn by daily
weighing of pots and watering to gradually reduce soil
moisture levels. Once Kukri reached wilting point (symptoms
apparent in the morning, volumetric soil water content
about 7%), the plants were watered to field capacity to
simulate the sporadic rainfall events that occur during the
growing season. Plants were then subjected to drought again
by withholding water and letting the soil dry until Kukri again
displayed symptoms of wilting. Control plants were watered
daily to field capacity.
Five time points were selected for the experiment (Figure 5).
The first three time points were at 5, 9, and 14 d (A, B, and C in
Figure 5) after the beginning of the drought stress. After this,
plants were re-watered to field capacity and, once Kukri plants
showed wilting, another sample was taken (day 24; D in
Figure 5). After this, plants were re-watered to field capacity
and a final sample was collected 24 h later (day 25; E in
Figure 5). Time point B samples in Kukri were not collected.
Sampling Procedure
Control plants and drought-treated plants were sampled at
the same time (midday) to account for diurnal fluctuations
in metabolite levels. Flag leaf tissue samples from the primary
tiller were immediately frozen in liquid nitrogen. Leaf tissue
was ground under liquid nitrogen using a ball mill in
a 50-ml falcon tube. Approximately 60 mg of tissue was
prepared for metabolomic analysis.
Metabolite Extraction and Derivatization
Metabolites were extracted based on a modified version of
the method used in Jacobs et al. (2007). Briefly, 500 ll of
Figure 5. Design of the Drought Stress Experiment.
Three time points were measured over the initial reduction in soilwater availability to the wilting point of the most susceptible cul-tivar (Kukri) at time point C. D reflects the second wilting point forKukri, and time point E was taken 24 h after the re-watering at timepoint D. Watering to weight is indicated by dots.
426 | Bowne et al. d Drought Metabolomics in Wheat
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100% methanol and 20 ll of 0.2 mg ml�1 ribitol/norleucine
was added to the frozen ground leaf tissue and incubated
at 70�C for 15 min. Samples were centrifuged for 15 min at
14 000 rpm and the supernatant transferred to a new tube,
then 500 ll of water was added and 50 ll aliquoted to new
tubes for TBS derivatization. To the remaining sample in the
tube, 400 ll CHCl3 was added and the sample thoroughly
vortexed prior to centrifugation for 10 min. The polar phase
was then transferred to a new tube, 300 ll CHCl3 was added,
and the sample vortexed and centrifuged again. The polar
phase was transferred to a new tube, and 50 and 5 ll trans-
ferred to new tubes for TMS derivatization. All aliquots for
derivatization were dried under vacuum and then filled with
N2 gas and stored on silica gel. TBS and TMS dry residues were
derivatized as described in Jacobs et al. (2007).
GC–MS Analysis
The analysis of TMS and TBS samples was performed as per
Jacobs et al. (2007). Briefly, sample volumes of 1 ll
were injected onto the GC column using a hot-needle tech-
nique. The GC–MS system comprised an AS 3000 autosampler,
a Trace gas chromatograph Ultra, and a DSQ quadrupole mass
spectrometer (ThermoElectron Cooperation, Austin, USA). The
mass spectrometer was tuned according to the manufacturer’s
recommendations using tris-(perfluorobutyl)-amine (CF43).
Gas chromatography was performed on a 30-m VF-5MS col-
umn with 0.25-lm film thickness with a 10-m Integra guard
column (Varian, Inc., Victoria, Australia). The injection temper-
ature was set at 230�C, the MS transfer line at 280�C, and the
ion source adjusted to 250�C. Helium was used as the carrier
gas at a flow rate of 1 ml min�1.
Data Analysis
Chromatograms and mass spectra were evaluated using the
Xcalibur program (ThermoFinnigan, Manchester, UK). Mass
spectra of eluting TMS compounds were identified using
the commercial mass spectral library NIST (http://www.nist.
gov/mml/chemical_properties/data/electionlibcomp.cfm) and
the public domain mass spectra library of Max-Planck-Institute
for Plant Physiology, Golm, Germany (http://csbdb.mpimp-golm.
mpg.de/csbdb/gmd/msri/gmd_msri.html). Metabolites that had
a confirmed identity and were present in all three cultivars were
used for further data analysis, the remaining metabolites not
being present at a high enough abundance across the dataset
to be useful for statistical analysis. Mass spectra of eluting
TBS compounds were identified using an in-house TBS mass
spectral library.
Normalization of the data was performed by dividing
the response values for each metabolite in each sample by
the average value of the reference sample for the metabolite
in the sample batch run on that instrument. The reference sam-
ple was pooled leaf tissue from a selection of the entire sample
range (covering all cultivars, time points, and treatments) that
was extracted in bulk as 972 mg of tissue in 9 ml methanol.
Five of these reference samples were run per batch. Relative
response ratios (Roessner et al., 2001) were calculated based
on internal standard extracted per gram fresh weight for
each analyzed metabolite as described in Roessner et al.
(2006), and x-fold changes were presented relative to the time
point A control sample for each cultivar as well as to the
respective control time point for the drought samples.
All experimental data were tested for statistical significance
by Student’s t-test algorithms in Microsoft Excel, coupled
with a Bonferroni-type correction for false discovery rate
(Benjamini et al., 2001; Broadhurst and Kell, 2006). Pathway
maps were constructed in VANTED (Junker et al., 2006) and
normalized log-transformed data were mapped to these
pathway constructs.
SUPPLEMENTARY DATA
Supplementary Data are available at Molecular Plant Online.
FUNDING
This work was funded by grants to the Australian Centre for Plant
Functional Genomics (ACPFG), from the Australian Research
Council (ARC) and the Grains Research and Development Corpora-
tion (GRDC), the South Australian Government, the University of
Adelaide, the University of Queensland, and the University of
Melbourne.
ACKNOWLEDGMENTS
The authors would like to thank Prof. M. Tester for proofreading
the manuscript, S. Suren and T. Stark for GC–MS analysis, K. Geipel
for sample extraction, S. Morran for harvesting plants, and B. Lovell
for grinding samples. No conflict of interest declared.
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