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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
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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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372 Yobi et a. Rehydration/Dehydration Metabolomics
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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Yobi et a. Rehydration/Dehydration Metabolomics 373
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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374 Yobi et a. Rehydration/Dehydration Metabolomics
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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Yobi et a. Rehydration/Dehydration Metabolomics 375
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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376 Yobi et a. Rehydration/Dehydration Metabolomics
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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Yobi et a. Rehydration/Dehydration Metabolomics 377
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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378 Yobi et a. Rehydration/Dehydration Metabolomics
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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Yobi et a. Rehydration/Dehydration Metabolomics 379
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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380 Yobi et a. Rehydration/Dehydration Metabolomics
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 -
7/28/2019 Metabolomic Profiling in Selaginella Lepidophylla
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382 Yobi et a. Rehydration/Dehydration Metabolomics
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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