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Molecular Plant Volume 5 Number 2 Pages 418–429 March 2012 RESEARCH ARTICLE Drought Responses of Leaf Tissues from Wheat Cultivars of Differing Drought Tolerance at the Metabolite Level Jairus B. Bowne a , Tim A. Erwin a , Juan Juttner b , Thorsten Schnurbusch b , Peter Langridge b , Antony Bacic a,c,d,e and Ute Roessner a,e,1 a Australian Centre for Plant Functional Genomics, School of Botany, The University of Melbourne, 3010 Victoria, Australia b Australian Centre for Plant Functional Genomics, University of Adelaide, Waite Campus, Glen Osmond, 5064 SA, Australia c Metabolomics Australia, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Rd, The University of Melbourne, 3010 Victoria, Australia d ARC Centre for Excellence for Plant Cell Walls, School of Botany, The University of Melbourne, 3010 Victoria, Australia e 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 valine were 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 under well-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 (Gonza ´ lez 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 at Bibliotheque de l'Universite Laval on July 14, 2014 http://mplant.oxfordjournals.org/ Downloaded from

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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

at Bibliotheque de l'U

niversite Laval on July 14, 2014

http://mplant.oxfordjournals.org/

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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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