metabolomic profiling in selaginella lepidophylla

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Molecular Plant Volume 6 Number 2 Pages 369385 March 2013 RESEARCH ARTICLE

Metabolomic Profiling in Seaginea epidophya

at Various Hydration States Provides New

Insights into the Mechanistic Basis of DesiccationTolerance

Abou Yobia, Berard W.M. Woeb, Wexi Xuc, Day C. Alexaderc, Liig Guoc, Joh A. Ryalsc,Melvi J. Oliverd and Joh C. Cushmaa,1

a Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 895570330, USA

b Department of Biological Sciences, University of Nevada, Reno, NV 895570314, USA

c Metabolon Inc., 800 Capitola Drive, Suite 1, Durham, NC 27713, USA

d US Department of AgricultureAgricultural Research Service, Plant Genetic Research Unit, University of Missouri, Columbia, MI 65211, USA

ABSTRACT Selaginella lepidophylla is oe of oly a few species of spike mosses (Selaginellaceae) that have evolved des-

iccatio tolerace (DT) or the ability to resurrect from a air-dried state. I order to uderstad the metabolic basis of

DT, S. lepidophylla was subjected to a five-stage, rehydratio/dehydratio cycle, the aalyzed usig o-biased, global

metabolomics profilig techology based o GC/MS ad UHLC/MS/MS2 platforms. A total of 251 metabolites icludig 167

amed (66.5%) ad 84 (33.4%) uamed compouds were characterized. Oly 42 (16.7%) ad 74 (29.5%) of compouds

showed sigificatly icreased or decreased abudace, respectively, idicatig that most compouds were produced co-

stitutively, icludig highly abudat trehalose, sucrose, ad glucose. Several glycolysis/glucoeogeesis ad tricarboxylic

acid (TCA) cycle itermediates showed icreased abudace at 100% relative water cotet (RWC) ad 50% RWC. Vaillate,

a potet atioxidat, was also more abudat i the hydrated state. May differet sugar alcohols ad sugar acids were

more abudat i the hydrated state. These polyols likely decelerate the rate of water loss durig the dryig process as well

as slow water absorptio durig rehydratio, stabilize proteis, ad scavege reactive oxyge species (ROS). I cotrast,

itroge-rich ad-glutamyl amio acids, citrullie, ad ucleotide catabolism products (e.g. allatoi) were more abudat

i the dry states, suggestig that these compouds might play importat roles i itroge remobilizatio durig rehydratio

or i ROS scavegig. UV-protective compouds such as 3-(3-hydroxypheyl)propioate, apigei, ad arigei, were

more abudat i the dry states. Most lipids were produced costitutively, with the exceptio of cholie phosphate, which

was more abudat i dry states ad likely plays a role i membrae hydratio ad stabilizatio. I cotrast, several poly-

usaturated fatty acids were more abudat i the hydrated states, suggestig that these compouds likely help maitai

membrae fluidity durig dehydratio. Lastly,S. lepidophylla cotaied seve uamed compouds that displayed twofold

or greater abudace i dry or rehydratig states, suggestig that these compouds might play adaptive roles i DT.

Key words: abiotic/evirometal stress; mass spectrometry; metabolomics; oxidative/photooxidative stress; desiccatio

tolerace; Selaginella.

InTRODUCTIOn

Lycophytes represent a key vascular plant lineage that includes

the Lycopodiaceae (club mosses), the Isoetaceae (quillworts),

and the Selaginellaceae (spike mosses) that arose over 400

million years ago during the Silurian and dominated the

Earths flora from the Devonian through the Carboniferous to

the end of the Permian (Friedman, 2011). Dramatic decreases

in atmospheric CO2 concentrations occurred during these

Paleozoic epochs and the resulting decomposed flora now

provide a major source of energy to mankind in the form of

coal (Beerling, 2002; Gensel, 2008). Today, extant lycophytes

comprise a single monophyletic clade of just over 1000 species.

1 To whom correspondence should be addressed. [email protected],

tel. +1 775 784 1918, fax +1 775 784 1419.

The Author 2013. 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/sss155, Advance Access publication 13 December 2012

Received 9 August 2012; accepted 6 December 2012
mailto:[email protected]:[email protected]


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370 Yobi et a. Rehydration/Dehydration Metabolomics

Within the spike mosses, the single genus Seaginea con-

tains about 700 species that now exploit a diverse array of arc-

tic, temperate, tropical, and semi-arid habitats (Banks, 2009).

Although most spike moss species are susceptible to desicca-

tion, a few species have evolved the ability to survive vegeta-

tive tissue drying, defined as the near complete loss (80%95%)

of protoplasmic water (Tuba et al., 1998), and referred to as

desiccation tolerance (DT) (Oliver et al., 2000a). Such speciesinclude S. epidophya (Iturriaga et al., 2006), S. bryopteris

(Deeba et al., 2009), and S. tamarisina (Wang et al., 2010).

The DT trait is relatively common in algae, lichens, and mosses

(Alpert, 2006; Wood, 2007). However, among vascular plants,

DT is extremely rare; only about ~0.15% of species being are

known as resurrection plants (Oliver et al., 2000a; Porembski

and Barthlott, 2000; Proctor and Pence, 2002; Proctor and

Tuba, 2002).

In terms of response to desiccation, resurrection mosses

(e.g. bryophytes) rely on a combination of constitutive pro-

tection and inducible repair mechanisms that permit dehy-

dration to occur within minutes while maintaining their

photosynthetic apparatus relatively intact (Tuba et al., 1998;Oliver et al., 2000b, 2005). In contrast, DT angiosperms

require a much longer time to dehydrate and still remain via-

ble, presumably to allow sufficient time for mobilizing adap-

tive responses and accumulation of key metabolites, such as

sucrose, to survive in the desiccated state (Bernacchia et al.,

1996; Oliver et al., 2000a). In contrast, lycophytes represent

a plant lineage that lies between mosses and angiosperms

(Oliver et al., 2000a) and thus might be expected to exhibit

both constitutive and inducible adaptive mechanisms of DT.

Many monocot DT species lose their photosynthetic appara-

tus, at least partially, during the dehydration process (Farrant,

2000; Proctor and Tuba, 2002), whereas many eudicot species

do not (Georgieva et al., 2009). DT Seaginea species retain

their chlorophyll (and presumably their photosynthetic struc-

tures) during desiccation (Pandey et al., 2010).

Members of the Selaginellales are well known for their

use in traditional folk medicine. For example, S. epidophya

has been used as a diuretic, and as a treatment for urinary

and kidney infections, chronic gastritis, and gastric carci-

noma (Ruiz-Bustos et al., 2009; Robles-Zepeda et al., 2011).

Extracts of S. tamarisina (Ahn et al., 2006; Lee et al., 2011;

Ha et al., 2012), S. doedereinii(Liu et al., 2011), and S. bry-

opteris (Mishra et al., 2011) have been used as anti-cancer

therapeutics. Total flavonoids from S. tamarisina have also

been employed as an anti-diabetic treatment (Zheng et al.,2011b). Various Seaginea species have been recognized for

their potential therapeutic value as anitviral, antimicrobial,

or anticancer bioactivities. For example, several novel fla-

vones and biflavones have been characterized from S. epido-

phya (Aguilar et al., 2008), S. hrysoauos and S. bryopteris

(Swamy et al., 2006), S. abordei(Tan et al., 2009), S. moeen-

dorffii (Cao et al., 2010; Liu et al., 2010; Wang et al., 2011;

Wu and Wang, 2011), S. uninata (Zheng et al., 2011a), and

S. tamarisina (Zhang et al., 2011). Antimicrobial alkaloids

have also been characterized from S. moeendorfii (Wang

et al., 2009).

Despite their role in traditional medicine, comprehensive

metabolite studies on Seaginea species have been limited.

Previous studies have characterized only a few, highly abun-

dant compounds such as lignin (White and Towers, 1967)

and trehalose (White and Towers, 1967; Adams et al., 1990;Iturriaga et al., 2000; Vzquez-Ortz and Valenzuela-Soto,

2004; Liu et al., 2008). A recent comparative study examined

the major metabolic differences between the desiccation-

sensitive (DS) S. moeendorffi and the DT S. epidophya.

Several sugars (e.g. glucose, sucrose), sugar alcohols (e.g.

inositol-1-phosphate, myo-inositol, mannitol), and betaine,

which act as osmoprotectants or hydroxyl radical scavengers,

were more abundant in S. epidophya at 100% and 50%

relative water contents (RWC) (Yobi et al., 2012). In addition,

a variety of -glutamyl amino acids, which are thought to

participate in reactive oxygen species (ROS) detoxification,

and possible nitrogen remobilization following rehydration,

were markedly higher in S. epidophya. A suite of second-ary compounds, such the flavonols apigenin, luteolin, and

naringenin, also accumulated to greater concentrations in

S. epidophya, where they are thought to mitigate ultravi-

olet light-induced free radical damage as well as aid in the

stabilization of membrane structures (Hoekstra et al., 2001).

Lastly, polyunsaturated fatty acids were significantly more

abundant in S. epidophya, possibly reflecting a need for

greater membrane fluidity in the dry state (Yobi et al., 2012).

The exact mechanisms used to withstand DT have not been

well investigated in lycophytes. To better understand the met-

abolic basis of DT at very low RWCs within the Selaginellaceae,

the metabolome of S. epidophya was compared over a

five-stage, rehydration/dehydration cycle using an unbiased,

high-throughput metabolomics platform. This temporal study

revealed that S. epidophya employs a combination of diverse

constitutive and inducible metabolic strategies to survive and

recover from desiccation of vegetative tissues.

RESULTS AnD DISCUSSIOn

Metabolomics approaches can provide a detailed

understanding of an organisms phenotype (Schauer and

Fernie, 2006; Cascante and Marin, 2008), and disposition

towards and response to environmental stresses (Sanchez

et al., 2008;

Shulaev et al., 2008;

Urano et al., 2010). Recentstudies have compared two closely related species of

Sporobous (Oliver et al., 2011) and Seaginea (Yobi et al.,

2012) in order to discern key metabolic differences that

might contribute to DT. In the current study, a total of 251

metabolites were identified to provide insights into both

constitutive and inducible metabolite abundance patterns

essential for the acquisition of DT in the resurrection

lycophyte, S. epidophya.


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Yobi et a. Rehydration/Dehydration Metabolomics 371

S. lepidophylla ad Dehydratio

In order to discern relative metabolite abundance changes in

S. epidophya during a rehydration/dehydration cycle, plants

were allowed to hydrate, then dehydrate, and their RWC was

tracked over a 24-h period. Upon hydration, water uptake was

relatively rapid; 70% RWC was achieved within 1 h and 100%

RWC after 24 h (Figure 1A). When hydrated, S. epidophya

plants displayed fully expanded green microphylls (Figure 1B).In contrast, water loss during dehydration was markedly

slower, with 70% water loss taking more than 4 h and the

plants retaining 7% RWC after 24 h of drying. The rate of water

loss in S. epidophya was similar to S. bryopteris, a related DT

species, which required about 6 h to lose 80%90% of its RWC

(Deeba et al., 2009; Pandey et al., 2010). During dehydration,

the microphylls curl to form a tight ball (Figure 1B), which is

an adaptive response to protect the plant against damage due

to high irradiance or temperatures, or both (Lebkuecher and

Eickmeier, 1991, 1992, 1993). Chlorophyll is maintained at least

partially on the inner surface of the microphylls, indicating

that S. epidophya is homiochlorophyllous (Tuba et al., 1998).

Metabolome Compositio of S. lepidophylla

To assess the metabolomic response of S. epidophya to a

rehydration/dehydration cycle, six biological replicate tissue

samples were collected from plants sampled at full dehydra-

tion 1 (7% RWC, DRY-1), partial rehydration (50% RWC, REH-

50), full rehydration (100% RWC, HYD), partial dehydration

(50% RWC, DEH-50), and full dehydration 2 (7% RWC, DRY-

2), and analyzed using non-biased, global metabolome tech-

nology based on GC/MS and UHLC/MS/MS2 platforms (Evans

et al., 2009). The combined platforms detected a total of

251 metabolites, of which 167 (66.5%) were named and 84(33.4%) were unnamed (Figure 2 and Supplemental Table 1).

After the named metabolites were mapped onto general

biochemical pathways, they were categorized into classes of

which amino acids were the most prevalent (19%), followed

by carbohydrates (16%), lipids (13%), cofactors (6%), nucleo-

tides (5%), peptides (4%), and secondary metabolites (3%).

Lastly, unnamed compounds in S. epidophya accounted for

one-third of all compounds surveyed (Figure 2).

Metabolomic Differeces durig a Rehydratio/

Dehydratio Cycle

Partial Least Squares-Discriminant Analysis (PLS-DA) wasused to build a comparative model of the five different

hydration states in S. epidophya. Metabolites missing three

or more out of six (50%) biological replicate values were

excluded, resulting in a total of 204 metabolites for compari-

son. The first three PLS-DA components explained 52.2% of

the variation (R2 = 0.50; Q2 = 0.40) and showed distinct clus-

tering among each of the five states, suggesting that various

metabolites likely accounted for the differences observed in

the model (Figure 3). The metabolomes of the two dry states

Figure 1. Rehydration/Dehydration Rates in S. epidophya.

(A) Rehydration time course (h) measured by tracking the percent gain

in relative water content (RWC) (blue line and squares) over time and

dehydration time course (h) measured by tracking the percent loss in

RWC over time (red line and diamonds). Data represent the mean of

three independent replicate experiments (n = 9). Error bars represent

standard error of the mean.

(B) Representative images of S. epidophya plants at initial dry state

(DRY-1), 50% rehydrated (REH-50), fully rehydrated (HYD), 50% dehy-

drated (DEH-50), and second dry state (DRY-2). Size bars = 2 cm.

Figure 2. Functional Categorization of 251 Named and Unnamed

Metabolites Detected in S. epidophya over Five Different RWCs.
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at 7% RWC were similar to one another. The metabolic pro-

file of the fully hydrated state at 100% RWC was distinct from

these dry states, and that of the 50% RWC rehydration state

was intermediate between the dry and fully hydrated plants.

Interestingly, the 50% RWC dehydration state was also clearly

distinct from those of the 50% RWC rehydration and the ini-

tial dry states, suggesting that distinct metabolic pathways

operate during rehydration compared with dehydration.

In comparing the S. epidophya metabolite profilesacross all five hydration states, a total of 114 (45.4%)

compounds were detected that showed significantly altered

abundance (p < 0.05), including 42 (16.7%, 27 named and

15 unnamed) that were more abundant in one or more of

the dry or partially dry states, and 74 (29.5%, 57 named and

17 unnamed) that showed a greater abundance in the fully

hydrated state. Two metabolites (i.e. pyruvate, galactose)

showed variable abundance patterns (Supplemental Table 1).

A total of 11 metabolites were significantly (p < 0.05) more

abundant in both dry states compared with the fully hydrated

state. In addition, 11 metabolites were more abundant only

in the initial dry state, whereas the abundance of only one

compound was higher in the second dry state. The precise

origin of these differences remains unclear, but might be due

to differential metabolic activity changes that occur between

the initial and subsequent rehydration/dehydration cycles.

In contrast, comparing the hydrated state with the two fully

dehydrated states, 26 metabolites showed significantly (p sucrose > glucose.

Sucrose alone, or in combination with other sugars, such as

raffinose-series oligosaccharides and cyclitols, or with macro-

molecules, such as late embryogenesis abundant (LEA) pro-

teins (Wolkers et al., 2001; Shimizu et al., 2010), likely protects

vegetative tissues of resurrection species upon drying through

the formation of anhydrous glass or by replacement of water

to prevent damaging interactions, such as membrane fusion

(Hoekstra et al., 2001). S. epidophya synthesizes high consti-

tutive concentrations of sucrose (and trehalose and glucose),

and thus resembles DT mosses that maintain constitutively

high sucrose levels that do not further increase during drying

(Smirnoff, 1992). In contrast, in a DT fern (Farrant et al., 2009)

and all angiosperm species examined to date, sucrose accu-

mulation is induced following the imposition of dehydration

stress (Ghasempour et al., 1998; Whittaker et al., 2001; Peters

et al., 2007; Toldi et al., 2009; Oliver et al., 2011).

Figure 3. Metabolites at Different Hydration States in S. epidophya

as Defined by Partial Least-Squares-Discriminant Analysis (PLS-DA)

Constructed from 204 Metabolites.

The plot shows the three components that explains 52.2% of the

metabolite signatures (R

2

= 0.50; Q

2

= 0.40). Upside-down triangles(red) represent initial dry state (DRY-1), squares (brown) represent

50% rehydration (REH-50), asterisks (green) represent fully hydrated

(HYD), crosses (cyan) represent 50% dehydrated (DEH-50), and dia-

monds (moss green) represent the second dry state (DRY-2).
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Trehalose, a non-reducing disaccharide, is known to accumu-

late in high amounts in lycophytes (Adams et al., 1990; Iturriaga

et al., 2000; Liu et al., 2008) as well as in many lower-order DT

organisms (Elbein et al., 2003). However, a critical role for this

disaccharide in DT is questionable for several reasons. First, sev-

eral resurrection ferns and angiosperms accumulate trehalose in

only very small amounts (Ghasempour et al., 1998; Farrant et al.,

2009). Second, a Saharomyes erevisiae mutant defective in

trehalose biosynthesis can maintain DT, indicating that treha-

lose is neither necessary nor sufficient for DT (Ratnakumar and

Tunnacliffe, 2006). However, trehalose has been shown to act

synergistically with LEA proteins to stabilize client enzymes by

reducing their aggregation (Goyal et al., 2005). While trehalose

alone might not be sufficient for DT, it might act cooperatively, as

do other sugars, as a chemical chaperone to enhance the molec-

ular shield function of LEA proteins in reducing desiccation-

induced aggregation of proteins (Chakrabortee et al., 2012).

Interestingly, the osmoprotectant glucosylglycerol

(2-O-alpha-D-glucopyranosyl-sn-glycerol) was also detected

in S. epidophya and was more abundant in the hydrated

state relative to the dry state(s) (Supplemental Table 1).

Glucosylglycerol is typically found in cyanobacteria (Klhn

and Hagemann, 2011). This compatible solute can stabilize

various enzymes against thermal denaturation as well as

improve enzyme stability eightfold following lyophilization

and rehydration (Sawangwan et al., 2010). The constitutive

and concerted production of sucrose, trehalose, and glucosyl-

glycerol are very likely critical to the preservation of diverse

enzyme activities during and following desiccation.

Sugar Alcohol Metabolism

Sugar alcohols or polyols are known to play important roles

in water-deficit and salinity-stress adaptation and tolerance

(Nuccio et al., 1999; Chen and Murata, 2002; Rontein et al.,

Figure 4. Metabolites Associated with Energy Metabolism (Glycolysis and TCA Cycle) in S. epidophya Profiled during a Rehydration/

Dehydration Cycle.

Compounds in red indicate greater relative abundance in one or more dry states. Compounds in green indicate greater relative abundance in the

hydrated state. Dotted arrows indicate a gap in the pathway and double-headed arrows indicate a reversible reaction. Thex-axis represents the

percentage of relative water content (% RWC): initial dry state (DRY-1); 50% rehydrated (REH-50); fully rehydrated (HYD); 50% dehydrated (DEH-50); and second dry state (DRY-2). They-axis box plots indicate the scaled intensity mean (closed triangles) and median () values, top/bottom

ranges of boxes indicate upper/lower quartiles, respectively, and top/bottom whiskers indicate the maximum/minimum distribution of the data,

respectively. Open circles indicate extreme data points. The p- and q-values for the comparisons are presented in Supplemental Table 1. G-6-P,

glucose-6-phosphate; F-6-P, fructose-6-phosphate; M-6-P, mannose-6-phosphate; 3-PG, 3-phosphoglycerate; PEP, phosphoenopyruvate.
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2002). A variety of polyols showed significant (p < 0.05)

changes in abundance in S. epidophya undergoing

rehydration/dehydration, with peak accumulation occurring

in the fully hydrated state (Figure 5 and Supplemental

Table 1). For example, xylitol abundance was 1033-fold

greater at 100% RWC compared with the DRY-1 and DRY-2

states, respectively (Figure 5). Relative amounts of other

polyols were also significantly higher in the fully hydratedstate: ribitol was 16.520.0-fold higher, sorbitol was 5.820.0-

fold higher, erythritol was 3.39.0-fold higher, compared

with the DRY-1 and DRY-2 states, respectively, and glycerol

was 1.42.6-fold higher compared with the DRY-1 and DRY-2

states, respectively, and 5.0-fold higher in the REH-50 state.

Threitol was 3.05.0-fold higher compared with the DRY-1

and DRY-2 states, respectively; however, these differences

were not significant. In addition, related oxidation products

of these sugars included gluconate and erythronate, which

were 2.816.5-fold and 2.02.5-fold more abundant,

respectively, in HYD compared with the DRY-1 and DRY-2

states. Lastly, N-acetyglucosamine, a monosaccharide derived

from glucose and an important structural sugar, was 2.0-fold more abundant in HYD compared with the dried states

(Figure 5).

In contrast, mannitol and galactose were significantly

(p < 0.05) more abundant in the DRY-1 state (2.0-fold and

1.9-fold, respectively) compared with the fully hydrated state

(Supplemental Table 1). In addition to its probable role as

an osmolyte, mannitol has been shown to act as a hydroxyl

radical scavenger (Shen et al., 1999). Inositol 1-phosphate

was 1.21.8-fold more abundant in all dehydration states

compared with the fully hydrated state, but these differ-

ences were not significant (Supplemental Table 1). In contrast

to other DT species (Farrant et al., 2009; Oliver et al., 2011),

raffinose-series sugars (e.g. galactinol, raffinose, stachyose)

were not detected and probably do not participate in DT in

S. epidophya. Investigation into the metabolomes of other

lycophytes is needed to determine whether this observation

holds true for all members of this ancient order within the

plant kingdom. Raffinose-series sugars have been shown

to act as ROS scavengers in Arabidopsis (Nishizawa et al.,

2008; Nishizawa-Yokoi et al., 2008). The lack of detectable

raffinose-series sugars in S. epidophya indicates that this

function might be fulfilled by other sugars or other diverse

compounds with ROS-scavenging activities.

The accumulation of polyols in the fully hydrated state in

S. epidophya indicates several important roles for thesesugars. Because polyols, especially glycerol, reduce the sur-

face tension of water (Tiwari and Bhat, 2006) and display

strong water-binding activity via hydrogen bonding (Kataoka

et al., 2011; Weng et al., 2011), they might decelerate the

rate of water loss during the drying process (Figure 1A).

Indeed, the ability of S. stapfianus to reduce the rate of water

loss during dehydration, compared to the DS S. pyramidiis,

appears to be a result of a similar metabolic priming of the

hydrated tissue with osmolytes (Oliver et al., 2011). Slowing

the rate of water loss might permit more time for the biosyn-

thesis of desiccation-adaptive proteins and metabolites that

are required for the establishment of DT (Alpert and Oliver,

2002). Conversely, biosynthesis of polyols might also slow the

rate of water absorption during rehydration. Notably, several

polyols such as sorbitol and erythritol exhibited increased

accumulation at the 50% RWC rehydration stage (REH-50),consistent with this hypothesis. A direct comparison of DT

S. epidophya and DS S. moeendorffii indicated that the

DT species accumulated significantly greater amounts of no

fewer than seven different polyols in the fully hydrated state

(Yobi et al., 2012). Furthermore, a recent comparison of DT

Sporobous stapfianus and DS S. pyramidais showed that

several polyols, including arabitol, erythritol, and mannitol,

were several-fold more abundant in fully hydrated leaves of

the DT species (Oliver et al., 2011). These observations sug-

gest that constitutive expression of polyols in the hydrated

state might be critical for many resurrection species to slow

the rate of water loss during dehydration as well as water

uptake during rehydration. Indeed, S. epidophya shows aslower rate of rehydration than does S. moeendorffii(Yobi

et al., 2012).

In addition to a role in water equilibrium, several poly-

ols can also act as osmoprotectants by stabilizing protein

(yeast hexokinase A) structure against thermal (Tiwari and

Bhat, 2006) or acidic (Devaraneni et al., 2012) denaturation.

Glycerol, sorbitol, and xylitol exhibit better stabilization

than glucose or trehalose through preferential hydration of

proteins in their denatured state (Xie and Timasheff, 1997)

by increasing surface tension (Kaushik and Bhat, 1998).

Both sorbitol and xylitol showed the greatest relative abun-

dance in S. epidophya compared with S. moeendorffii

(Yobi et al., 2012) and in this study (Figure 5), which is

consistent with such a protein-stabilizing function. Several

sugar alcohols, such as myo-inositol, mannitol, and sorbitol,

also possess the ability to scavenge hydroxyl radicals as a

way of reducing oxidative damage (Sanchez et al., 2008).

Thus, polyol accumulation might limit the oxidative stress

burden of DT plants during the early phases of dehydration

and rehydration. In addition to osmotic adjustment, the

accumulation of polyols might also facilitate other meta-

bolic functions such as consumption of NADH, which might

enhance redox control and reduce ROS production in the

cells (Shen et al., 1999). Lastly, sugar alcohols might stimu-

late the expression of stress-adaptive genes, as has beenobserved inArabidopsis plants engineered to express man-

nitol (Chan et al., 2011).

Amio Acid ad Peptide Metabolism

Of the 48 amino acids and derivatives detected, relative

amounts of 10 of them occurred in significantly (p < 0.05)

greater amounts in one or more of the dry states than in the

fully hydrated state, whereas only eight amino acids were
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significantly (p < 0.05) more abundant in the hydrated state

compared with one or more of the dry states (Supplemental

Table 1). Those more abundant in a dry state included

nitrogen-rich amino acids such as glutamine, glutamate,

arginine, aspartate, citrulline (Figure 6), asparagine,

3-(3-hydroxyphenyl)propionate, N-6-trimethyllysine, trans-

4-hydroxyproline, and ophthalmate (Supplemental Table 1).

In contrast, glycine, quinate, and alanine were significantly

(p < 0.05) more abundant in fully hydrated S. epidophya

compared with both dry and REH-50 states (Supplemental

Table 1). In comparison, quinate accumulation was induced

by dehydration (at 60% RWC) in DT S. stapfianus, but not in

DS S. pyramidais (Oliver et al., 2011).

The accumulation of nitrogen-rich amino acids (e.g.

asparagine, aspartate, arginine, glutamate, and glutamine)

partially resembles the desiccation response of the DT grass

S. stapfianus, in which several nitrogen-rich amino acids (e.g.

asparagine, arginine, glutamine) accumulated to significantly

greater concentrations in nearly dry or dry states (Martinelli

et al., 2007; Oliver et al., 2011). This increased accumulation of

nitrogen-rich amino acids might reflect an adaptation to the

nitrogen-limiting conditions typically encountered by S. stap-

fianus and other DT species in their natural habitat of rocky

outcrops with nitrogen-poor soils (Burke, 2002). Alternatively,

these amino acids might provide a nitrogen reservoir useful

during the early stages of rehydration before the recovery of

photosynthetic activity (Martinelli et al., 2007). In addition,

these accumulated amino acids could provide precursors for

the production of protective nitrogenous compounds, such as

the antioxidant glutathione (see Figure 7).

Citrulline, a structural analog of arginine, was the only

amino acid that was significantly (p < 0.05) more abundant

in all dry and partially dry states compared with the hydrated

state (Figure 6). This non-protein amino acid is thought to

function either as a nitrogen reserve or as a hydroxyl radi-

cal scavenger, or both, and accumulates along with arginine,

glutamate, and glutamine in response to drought stress in

a drought-tolerant wild watermelon (Kawasaki et al., 2000;

Akashi et al., 2001). Thus, citrulline might play similar roles in

S. epidophya.

The amino acid derivative 3-(3-hydroxyphenyl)propionate

was also more abundant in all dry and partially dry states

Figure 5. Differences in Sugar Alcohols and Other Sugars in S. epidophya Profiled during a Rehydration/Dehydration Cycle.

Compounds in green indicate greater relative abundance the hydrated state as shown in box plots. Dotted arrows indicate a gap in the

pathway. Box plots are as described in the Figure 4 legend. Thep- and q-values for the comparisons are presented in Supplemental Table 1.

G-6-P, glucose-6-phosphate; F-6-P, fructose-6-phosphate; Ru5P, ribulose 5-phosphate; Xu5P, xylulose 5-phosphate; E4P, erythrose 4-phosphate;

T4P, threose 4-phosphate.
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compared with the hydrated state, and significantly so in

the DRY-1 and DRY-2 states (Supplemental Table 1). Notably,the structure of 3-(3-hydroxyphenyl)propionate suggests

that it might play a protective role against UV-light dam-

age because closely related phenolic compounds, such as

3,4-dihydroxypropiophenone-3-glucoside and 4-(2-amino-

3-hydroxyphenyl)-4-oxobutanoic acid O--d-glucoside, serve

as UV-B absorbing sunscreens or filters in plants (Tegelberg

et al., 2002) and human eye lenses (Bova et al., 1999), respec-

tively. Additional research into the potential UV-protective

effects of this compound is warranted.

N-6-trimethyllysine is a biosynthetic precursor of carnitine

(Hoppel et al., 1980), a known osmoprotectant in Esherihia

oi (Cnovas et al., 2007; Verheul et al., 1998). In a recent

sister species comparison, both carnitine and acetyl carni-

tine were more abundant in DT S. epidophya than in DS

S. moeendorffii (Yobi et al., 2012). However, in the cur-

rent study, carnitine was not detected and acetyl carnitine

was more abundant in the hydrated state, except in DRY-1

(Supplemental Table 1).

Ophthalmate (glu-2-aminobutyrate-gly), a glutathione

pathway metabolite and analog of glutathione that lacks

antioxidant properties, was more abundant in all dry or

partially dry states compared with the fully hydrated state,

and significantly so in DRY-1 (Supplemental Table 1). Thiscompound was only recently reported in plants (Oliver et al.,

2011). Its accumulation pattern resembles that observed in

the DT grass S. stapfianus, in which ophthalmate abundance

increased when RWC reached 50% or less; however, the func-

tional significance of its accumulation remains unclear (Oliver

et al., 2011).

Betaine (glycine betaine), a well-known osmoprotectant in

many organisms including E. oi (Miller and Ingram, 2007)

and plants (Chen and Murata, 2011), failed to display signifi-

cant changes in abundance with changes in hydration states

(Supplemental Table 1). However, this compound was more

abundant in DT S. epidophya relative to DS S. moeen-

dorffii(Yobi et al., 2012).

nucleotide Metabolism

Aside from amino acids, the relative abundances of other

nitrogen-rich metabolites within the purine and pyrimidine

nucleotide pathways were altered during the rehydration/

dehydration cycle in S. epidophya. Of the 14 nucleotides

and derivatives detected, the relative amounts of three

of them (e.g. allantoin, 1-methyladenosine, and uridine

Figure 6. Differences in Amino Acid and Nitrogen Metabolism in S. epidophya Profiled during a Rehydration/Dehydration Cycle.

Compounds in red indicate greater relative abundance in one or more dry states as shown in box plots. Box plots are as described in the Figure 4

legend. Thep- and q-values for the comparisons are presented in Supplemental Table 1.
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5-monophosphate (UMP)) were significantly (p < 0.05) more

abundant in all dry or partially dry states compared with

the fully hydrated state, and significantly so in DRY-1

(Supplemental Table 1). Inosine was also more abundant in all

of the dry or partially dry states, but this difference was not

significant. In contrast, 2-deoxyadenosine was significantly

(p < 0.05) more abundant in fully hydrated S. epidophya

compared with both dry and REH-50 states (SupplementalTable 1).

The ureides allantoin and allantoate serve as nitrogen-

rich intermediates of purine catabolism that are involved

in abiotic stress protection, apparently by quenching ROS

(Werner and Witte, 2011). Both allantoin and allantoate can

attenuate cell death and ROS accumulation in anArabidopsis

mutant defective in xanthine dehydrogenase, the key

enzyme in the catabolism of purine leading to the production

of hypoxanthine and xanthine and then to uric acid, which

is subsequently degraded to allantoin and allantoate

(Brychkova et al., 2008). Similarly, RNA interference-mediated

suppression of xanthine dehydrogenase in drought-stressed

Arabidopsis led to a marked reduction in plant growth andsignificant increases in cell death and H2O2 accumulation

(Watanabe et al., 2010). However, this stress-hypersensitive

phenotype can be reversed if the plants are pretreated

with exogenous uric acid. These results demonstrate the

importance of these ureides in nutrient recovery and

remobilization during stress. The greater relative abundance

of allantoin in all dehydrated or partially dehydrated states

examined here points to similar roles for this compound in

DT survival and recovery in S. epidophya. These results are

also similar to those observed in the DT grass S. stapfianus, in

which allantoin abundance increased when RWC fell to 40%

or less (Oliver et al., 2011).

Glutathioe ad -glutamyl Peptide Metabolism

Within glutathione metabolism and the associated

-glutamyl amino acid biosynthetic pathway, significant

differences in the relative abundance of many metaboliteswere apparent over the course of the rehydration/dehydra-

tion cycle. For example, of the nine -glutamyl amino acid

dipeptides detected, seven were more abundant in a dry

state or partially dry state relative to the hydrated state, and

four of these (e.g. -glutamylisoleucine, -glutamylleucine,

-glutamylmethionine, and -glutamylthreonine) were signif-

icantly (p < 0.05) more abundant (Figure 7 and Supplemental

Table 1). In contrast, -glutamyltryptophan was more abun-

dant in the hydrated state, except in the DEH-50 state

(Supplemental Table 1). Other pathway intermediates, includ-

ing 5-oxoproline, were up to fivefold more abundant in the

hydrated state compared with the dry or partially dry states

(Figure 7 and Supplemental Table 1). Glycine and oxidizedglutathione (GSSG) were also significantly (p < 0.05) more

abundant in the hydrated state, with the exception of the

DEH-50 state (Figure 7 and Supplemental Table 1). As men-

tioned above, ophthalmate, a glutathione pathway metabo-

lite, was more abundant in all dry or partially dry states than

in the fully hydrated state (Supplemental Table 1).

InArabidopsis, and presumably in other plants, -glutamyl

amino acid cycle -glutamyltranspeptidase (also known

as -glutamyltransferase) catalyzes the interconversion of

Figure 7. Differences in -Glutamyl Amino Acid and Glutathione Metabolism in S. epidophya Profiled during a Rehydration/Dehydration Cycle.

Compounds in red or green indicate greater relative abundance in one or more dry states or hydrated state, respectively, as shown in box plots.

Dotted arrows indicate a gap in the pathway. Box plots are as described in the Figure 4 legend. Thep- and q-values for the comparisons are

presented in Supplemental Table 1. Cys-Gly, cysteinylglycine; AA, amino acid; GSH, glutathione (reduced); GSSG, glutathione disulfide (oxidized).
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glutathione (GSH, LGluCysGly) and free amino acids to

cysteinylglycine and -glutamyl amino acids (Ohkama-Ohtsu

et al., 2008). In animal systems, -glutamyl cyclo-transferase

converts -glutamyl amino acids into 5-oxoproline, releas-

ing the amino acid previously captured in the extracellular

space into the cytoplasm (Ohkama-Ohtsu et al., 2008). In

Arabidopsis, GSH degradation occurs within the cytoplasm

and transmembrane recycling of amino acids is thought tobe a minor event (Ohkama-Ohtsu et al., 2008). In S. epido-

phya, the significant increases in -glutamyl amino acids, as

well as nitrogen-rich amino acids such as glutamate, during

all stages of dehydration suggest that these peptides might

have an important function in the acquisition of DT. For

example, transmembrane amino acid recycling might occur

as it does in animal systems, and thus provide an important

mechanism for nitrogen storage during desiccation, as sug-

gested to occur in S. stapfianus (Oliver et al., 2011). Moreover,

the accumulation of -glutamyl amino acids in response to

desiccation appears to be an evolutionarily well-conserved

event. For example, -glutamylisoleucine, -glutamylleucine,

and -glutamyl-phenyalanine accumulate in S. epidophya(Figure 7), as well as in T. rurais and S. stapfianus, follow-

ing desiccation (Oliver et al., 2011). In addition, both S. epi-

dophya and S. stapfianus accumulate -glutamylglutamine

and -glutamylmethionine, whereas both S. epidophya and

T. rurais accumulate -glutamylvaline (Oliver et al., 2011). In

addition, S. epidophya accumulates -glutamylthreonine,

but not in a significant manner (Supplemental Table 1). Sister-

group comparisons between DS and DT species of Sporobous

(Oliver et al., 2011) and Seaginea (Yobi et al., 2012) dem-

onstrated that DS species fail to accumulate -glutamyl

amino acids, providing further support for an important role

for these compounds in DT. Although additional research is

needed on these compounds, storage of -glutamyl amino

acids in the dried state could provide a readily mobilized form

of nitrogen for protein synthesis following rehydration.

GSH exists in either an oxidized or a reduced form. The

oxidized form of glutathione (GSSG) showed significantly

(p < 0.05) greater abundance in S. epidophya in the

hydrated or partially hydrated states (Figure 7). Upon becom-

ing oxidized, it donates a reducing equivalent to unstable

reactive oxygen intermediates such as hydrogen peroxide

or dehydroascorbate, and then reacts instantly with a simi-

lar molecule to form glutathione disulfide (GSSG) (Mittler,

2002). Therefore, the greater abundance of GSSG observed

in hydrated S. epidophya suggests active protection againstoxidative stress at early stages of dehydration. In contrast,

S. stapfianus did not show significant accumulation of GSSG

during DT until RWC reached


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structure of lipid bilayers through inhibition of the fluid-to-gel

membrane phase transition at low hydration (Ohtake et al.,

2006; Valluru and Van den Ende, 2008). Thus, the increased

relative abundance of choline phosphate might promote

membrane hydration as well as aid in membrane stabilization

during the various stages of the rehydration/dehydration cycle.

Several lipoxygenase (LOX) activity markers (e.g. 13-hydroxy

octadecadienoic acid (13-HODE) and 2-hydroxypalmitate)showed greater relative abundance under a majority of dry

or partially dry states, but these differences were not signifi-

cant (Supplemental Table 1). These lipid peroxidation prod-

ucts might arise during dehydration from either ROS attack

or the action of LOXs (Marin et al., 1998). In addition, com-

pounds such as 13-HODE, 2-hydroxypalmitate, and 2-hydroxy-

stearate exhibited significantly (p < 0.05) greater abundance

in DT S. epidophya compared with DS S. moeendorffiiat

two different hydration states, suggesting these compounds

participate in DT mechanisms (Yobi et al., 2012). Several unsat-

urated fatty acids (e.g. arachidate, dihomo-linoleate, dihomo-

linolenate, linoleate, and linolenate) were significantly (p 2.0-fold

greater abundance under dry or partially dry conditions

in S. epidophya represent important targets for futureresearch, as these compounds might play important roles in

the acquisition of DT and could also serve as useful targets for

metabolic engineering to improve drought tolerance in crops.

Lastly, 11 unnamed compounds showed significantly increased

abundance in the REH50 state (Figure 8), which suggests that

these compounds might play roles during the recovery from

desiccation. Because S. epidophya is positioned evolutionarily

between bryophytes, which rely mostly upon repair-based

mechanisms during rehydration, and angiosperms, which rely

mainly upon dehydration-induced protection (Oliver et al.,

2000a), S. epidophya provides a useful model to bridge

our understanding of the evolutionary progression of DT

mechanisms among distantly related resurrection species.

Coclusios

This temporal evaluation of a five-stage, rehydration/dehy-

dration cycle in S. epidophya afforded a global, non-biased,

high-resolution account of the metabolite abundance changes

that occur as part of the ability of this spike moss to resurrect

from an air-dried state. Like DT mosses, S. epidophya exhibits

an obvious predisposition for DT by expressing a majority of

metabolites, such as highly abundant trehalose, sucrose, and

glucose in a constitutive manner, whereas only 16.7% and

29.5% of all compounds show significant increases or decreases

in abundance upon change in hydration state, respectively.

Many glycolysis/gluconeogenesis and TCA cycle intermediates

and many different sugar alcohols and sugar acids were more

abundant in the hydrated state. Polyols appear to play vari-

ous roles, including the slowing of water loss during drying or

water absorption during rehydration. Polyols might also serve

as osmoprotectants in stabilizing or preventing denaturation

of proteins during dehydration, in hydroxyl radical scaveng-

ing, in redox control, in reducing ROS production, and in trig-

gering stress-adaptive gene expression. Vanillate was more

abundant in the hydrated states and is a well-known and

potent antioxidant that is likely to protect against free radi-

cal-induced biomembrane damage. Protection against photo-

and oxidative damage during dehydration is also known tobe critical for DT. Citrulline, a non-protein amino acid analog

of arginine, and allantoin, a nitrogen-rich product of purine

catabolism, which likely serve as nitrogen reserves or attenu-

ate ROS accumulation and cell death, were more abundant

in all dry and partially dry states. Several amino acid-derived

secondary metabolites also appeared to play protective roles

against UV-light damage including 3-(3-hydroxyphenyl) pro-

pionate and the flavonols apigenin and naringenin in all dry
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and partially dry states. In addition, several nitrogen-rich and

-glutamyl amino acids that were markedly more abundant in

dry or partially dry S. epidophya, likely play critical roles in

the acquisition of DT through the scavenging of ROS via glu-

tathione metabolism, or the remobilization of amino acids fol-

lowing rehydration, or both. Lastly, S. epidophya contained

seven unnamed compounds that displayed twofold or greater

abundance in dry or rehydrating states, suggesting that thesecompounds might play adaptive roles in the acquisition of DT

or in the recovery from the desiccated state.

METHODS

Plat Material ad Water-Deficit Stress Treatmets

Seaginea epidophya (Hooker and Greville) Spring (flower

of stone) plants were purchased in the dried state from

Hirts Gardens (Medina, OH) and kept dry until the plants

were subjected to an initial rehydration/dehydration cycle in

order to identify and remove nonviable tissues. The rate of

RWC change during the rehydration/dehydration cycle was

established by submerging nine approximately 1-year-old

plants in distilled water in a growth chamber under constant

(175 mol m2 s1) cool-white fluorescent and incandescent

light at 26C and 37% relative humidity (RH). For hydration,the plants were removed from the water, excess water was

removed by gentle blotting, and the plants were weighed

at regular intervals over a 24-h period. For dehydration, the

plants were then placed in dry trays and weighed at regular

intervals over a 24-h period. The plants were incubated at

65C for 2 d, then reweighed to obtain dry weights. The RWC

was calculated using the following formula: RWC (%) = (Fwt-

Dwt)/(FTwt-Dwt) 100, where Fwt is the fresh weight at spe-

cific time points during the rehydration/dehydration cycle,

Figure 8. Differences in Nine Unnamed Compounds in S. epidophya Profiled during a Rehydration/Dehydration Cycle.

Compounds with greater relative abundance in one or more dry states as shown in box plots are presented. Box plots are as described in the

Figure 4 legend. Thep- and q-values for the comparisons are presented in Supplemental Table 1.
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Dwt is the weight after incubation at 65C for 2 d, and FTwt

is the weight after 24 h of rehydration. Samples represent-

ing five RWCsthe initial dry state (DRY-1), 50% rehydrated

(REH-50), 100% rehydrated (HYD), 50% dehydrated (DEH-50),

and completely dehydrated (DRY-2)were then selected to

determine differences in metabolite composition over the

course of the rehydration/dehydration cycle.

Metabolomic Profilig

Six biological replicates from each species were collected at

each time point, lyophilized, and kept at 80C under her-

metic conditions prior to extraction. Then, 20 mg of each leaf

sample was extracted in 400 l of methanol containing recov-

ery standards using an automated MicroLab STAR system

(Hamilton Company, Salt Lake City, UT).

Global unbiased metabolic profiling in S. epidophya was

performed using an integrated platform consisting of a com-

bination of three independent approaches: (1) ultrahigh per-

formance liquid chromatography/tandem mass spectrometry

(UHLC/MS/MS2

) optimized for basic species, (2) UHLC/MS/MS2

optimized for acidic species, and (3) gas chromatography/mass

spectrometry (GC/MS) as described in detail in Evans et al.

(2009).

UHLC/MS/MS2 analyses were performed using a Waters

Acquity UPLC (Waters Corporation, Milford, MA) and a

Thermo-Finnigan LTQ mass spectrometer (Thermo Fisher

Scientific, Inc., Waltham, MA) equipped with an electrospray

ionization (ESI) source and linear ion-trap (LIT) mass analyzer.

Each sample was subjected to two independent UHPLC/

MS runs using separate dedicated columns optimized for

either positive ions or for negative ions. For GC/MS, samples

were re-dried under vacuum desiccation for a minimum of

24 h, derivatized under dried nitrogen using bistrimethyl-silyl-triflouroacetamide (BSTFA), and then analyzed on a

Thermo-Finnigan Trace DSQ fast-scanning single-quadrupole

MS using electron impact ionization operated at unit mass

resolving power. Chromatographic separation, followed by

full scan mass spectra, was carried out to record retention

time, molecular weight (m/z), and MS/MS2 of all detectable

ions present in the samples (see Supplemental Table 2) in

compliance with recommendations for reporting metabolite

data (Fernie et al., 2011).

Automated comparison of the ion features in the experi-

mental samples was used to identify metabolites using a

custom reference library of chemical standards. Reference

library standard entries included the retention time, molecu-

lar weight (m/z), preferred adducts, and in-source fragments

of compounds, as well as their associated MS/MS2 spectra.

Comparison of experimental samples to process blanks (water

only) and solvent blanks allowed for the removal of artifac-

tual peaks, whereas the reference library allowed the rapid

identification of metabolites in the experimental samples

with high confidence. Mapping of named metabolites was

performed onto general biochemical pathways, as provided

in the Kyoto Encyclopedia of Genes and Genomes (KEGG)

(www.genome.jp/kegg/) and Plant Metabolic Network (PMN)

(www.plantcyc.org/).

Data Imputatio ad Statistical Aalysis

The samples were analyzed over the course of 2 d. This

required data correction for minor variations resulting frominstrument inter-day tuning differences (Evans et al., 2009). If

values for a given metabolite were missing, then these were

assigned the observed minimum detection value, based on

the assumption that the missing values were below the limits

of detection. For the convenience of data visualization, the

raw area counts for each biochemical were rescaled by divid-

ing each sample value by the median value for the specific

biochemical.

Statistical analysis of the data was performed using JMP

(SAS, www.jmp.com), a commercial software package, and

R (http://cran.r-project.org/), a freely available open-source

software package. A log transformation was applied to the

observed relative abundances for each biochemical becausethe variance generally increased as a function of each bio-

chemicals average response.

Out of the 251 metabolites that were detected, a total

of 204 metabolites with no missing values were imported

into the chemometrics software Solo (Eigenvector Research,

Inc., Wenatchee, WA) for Partial Least Squares-Discriminant

Analysis (PLS-DA) to determine classification of the differ-

ent treatments (Figure 3). PLS-DA is a regression extension

of Principal Component Analysis (PCA) used to model group

classification. R2 and Q2 are used as measures for the robust-

ness of a PLS-DA model, where R2 is the cumulative variance

explained by the components. Cross-validation of R2 esti-

mates Q2, which is the cumulative variance predicted by themodel. Thus, both of these values indicate how well the over-

all model classifies and predicts group membership in a data

set. Both of these values range from 0 to 1 and the higher the

number, the more robust the model.

In order to visualize the entire data set, a heat map was

generated to show fold change for each compound identified

from the LCMS/MS2 or GCMS analyses of the tissue samples

(see Supplemental Table 1). Fold change was calculated for

each compound defined as the means ratio of each treatment

in S. epidophya. Welchs two-sample t-tests were then used

to determine whether or not each metabolite had significantly

increased or decreased in abundance. The False Discovery Rate

(FDR) was then calculated to correct for multiple Welchs two-

sample t-test comparisons for the hundreds of compounds

detected. Metabolite expression studies are very similar to

gene array studies with a very large number of statistical com-

parisons. As with microarray studies, metabolomic profiling

generates large numbers of metabolites, and thus FDR is typi-

cally calculated with multiple comparison adjustment instead

of family-wise error rate adjustments, such as the Bonferroni

or Tukey corrections. The FDR for a given set of metabolites
http://mplant.oxfordjournals.org/lookup/suppl/doi:10.1093/mp/sss155/-/DC1http://www.genome.jp/kegg/http://www.plantcyc.org/http://www.jmp.com/http://cran.r-project.org/http://mplant.oxfordjournals.org/lookup/suppl/doi:10.1093/mp/sss155/-/DC1http://mplant.oxfordjournals.org/lookup/suppl/doi:10.1093/mp/sss155/-/DC1http://cran.r-project.org/http://www.jmp.com/http://www.plantcyc.org/http://www.genome.jp/kegg/http://mplant.oxfordjournals.org/lookup/suppl/doi:10.1093/mp/sss155/-/DC1


14/17


was estimated by the q-value (Storey, 2002). Box plots were

generated for those compounds that showed a significant

increase or decrease using both the Welch two-sample t-test

and FDR (i.e.p < 0.05 and q < 0.10) significance values.

SUPPLEMEnTARY DATA

Supplementary Data are available at Moeuar Pant Onine.

FUNDING

This work was supported, in part, by a grant from the USDA

National Institute of Food and Agriculture (200755100

18374 to M.J.O. and J.C.C.) and by Hatch funding (NAES-0341)

from the Nevada Agricultural Experiment Station.

AckNOwlEDGMENTS

The authors would also like to thank Mary Ann Cushman

for her helpful and clarifying comments on the manuscript.

Mention of a trademark or proprietary product does not

constitute a guarantee or warranty of the product by the

United States Department of Agriculture and does not imply

its approval to the exclusion of other products that may also

be suitable. No conflict of interest declared.

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