Metabolomic Profiling in Selaginella Lepidophylla

Molecular Plant Volume 6 Number 2 Pages 369385 March 2013
RESEARCH ARTICLE
Metabolomic Profiling in Selaginella lepidophylla at Various Hydration States Provides New Insights into the Mechanistic Basis of Desiccation Tolerance
AbouYobia, Bernard W.M.Woneb, WenxinXuc, Danny C.Alexanderc, LiningGuoc, John A.Ryalsc, Melvin J.Oliverd and John C.Cushmana,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
Downloaded from http://mplant.oxfordjournals.org/ at Universidad de Concepcion on June 5, 2013
ABSTRACT Selaginella lepidophylla is one of only a few species of spike mosses (Selaginellaceae) that have evolved desiccation tolerance (DT) or the ability to resurrect from an air-dried state. In order to understand the metabolic basis of DT, S.lepidophylla was subjected to a five-stage, rehydration/dehydration cycle, then analyzed using non-biased, global metabolomics profiling technology based on GC/MS and UHLC/MS/MS2 platforms. Atotal of 251 metabolites including 167 named (66.5%) and 84 (33.4%) unnamed compounds were characterized. Only 42 (16.7%) and 74 (29.5%) of compounds showed significantly increased or decreased abundance, respectively, indicating that most compounds were produced constitutively, including highly abundant trehalose, sucrose, and glucose. Several glycolysis/gluconeogenesis and tricarboxylic acid (TCA) cycle intermediates showed increased abundance at 100% relative water content (RWC) and 50% RWC. Vanillate, a potent antioxidant, was also more abundant in the hydrated state. Many different sugar alcohols and sugar acids were more abundant in the hydrated state. These polyols likely decelerate the rate of water loss during the drying process as well as slow water absorption during rehydration, stabilize proteins, and scavenge reactive oxygen species (ROS). In contrast, nitrogen-rich and -glutamyl amino acids, citrulline, and nucleotide catabolism products (e.g. allantoin) were more abundant in the dry states, suggesting that these compounds might play important roles in nitrogen remobilization during rehydration or in ROS scavenging. UV-protective compounds such as 3-(3-hydroxyphenyl)propionate, apigenin, and naringenin, were more abundant in the dry states. Most lipids were produced constitutively, with the exception of choline phosphate, which was more abundant in dry states and likely plays a role in membrane hydration and stabilization. In contrast, several polyunsaturated fatty acids were more abundant in the hydrated states, suggesting that these compounds likely help maintain membrane fluidity during dehydration. Lastly, S.lepidophylla contained seven unnamed compounds that displayed twofold or greater abundance in dry or rehydrating states, suggesting that these compounds might play adaptive roles in DT. Key words: abiotic/environmental stress; mass spectrometry; metabolomics; oxidative/photooxidative stress; desiccation tolerance; 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. E-mail jcushman@unr.edu, 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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Yobi et al. Rehydration/Dehydration Metabolomics
Within the spike mosses, the single genus Selaginella contains about 700 species that now exploit a diverse array of arctic, temperate, tropical, and semi-arid habitats (Banks, 2009). Although most spike moss species are susceptible to desiccation, a few species have evolved the ability to survive vegetative tissue drying, defined as the near complete loss (80%95%) of protoplasmic water (Tuba etal., 1998), and referred to as desiccation tolerance (DT) (Oliver etal., 2000a). Such species include S. lepidophylla (Iturriaga et al., 2006), S. bryopteris (Deeba etal., 2009), and S.tamariscina (Wang etal., 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 etal., 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 protection and inducible repair mechanisms that permit dehydration to occur within minutes while maintaining their photosynthetic apparatus relatively intact (Tuba etal., 1998; Oliver et al., 2000b, 2005). In contrast, DT angiosperms require a much longer time to dehydrate and still remain viable, presumably to allow sufficient time for mobilizing adaptive responses and accumulation of key metabolites, such as sucrose, to survive in the desiccated state (Bernacchia etal., 1996; Oliver et al., 2000a). In contrast, lycophytes represent a plant lineage that lies between mosses and angiosperms (Oliver etal., 2000a) and thus might be expected to exhibit both constitutive and inducible adaptive mechanisms of DT. Many monocot DT species lose their photosynthetic apparatus, at least partially, during the dehydration process (Farrant, 2000; Proctor and Tuba, 2002), whereas many eudicot species do not (Georgieva etal., 2009). DT Selaginella species retain their chlorophyll (and presumably their photosynthetic structures) during desiccation (Pandey etal., 2010). Members of the Selaginellales are well known for their use in traditional folk medicine. For example, S.lepidophylla has been used as a diuretic, and as a treatment for urinary and kidney infections, chronic gastritis, and gastric carcinoma (Ruiz-Bustos et al., 2009; Robles-Zepeda et al., 2011). Extracts of S.tamariscina (Ahn etal., 2006; Lee etal., 2011; Ha etal., 2012), S.doederleinii (Liu etal., 2011), and S.bryopteris (Mishra et al., 2011) have been used as anti-cancer therapeutics. Total flavonoids from S. tamariscina have also been employed as an anti-diabetic treatment (Zheng et al., 2011b). Various Selaginella species have been recognized for their potential therapeutic value as anitviral, antimicrobial, or anticancer bioactivities. For example, several novel flavones and biflavones have been characterized from S.lepidophylla (Aguilar etal., 2008), S.chrysocaulos and S.bryopteris (Swamy etal., 2006), S.labordei (Tan etal., 2009), S.moellendorffii (Cao et al., 2010; Liu et al., 2010; Wang et al., 2011; Wu and Wang, 2011), S.uncinata (Zheng etal., 2011a), and
S. tamariscina (Zhang et al., 2011). Antimicrobial alkaloids have also been characterized from S. moellendorfii (Wang etal., 2009). Despite their role in traditional medicine, comprehensive metabolite studies on Selaginella species have been limited. Previous studies have characterized only a few, highly abundant compounds such as lignin (White and Towers, 1967) and trehalose (White and Towers, 1967; Adams etal., 1990; Iturriaga et al., 2000; Vzquez-Ortz and Valenzuela-Soto, 2004; Liu etal., 2008). Arecent comparative study examined the major metabolic differences between the desiccationsensitive (DS) S. moellendorffi and the DT S. lepidophylla. 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. lepidophylla at 100% and 50% relative water contents (RWC) (Yobi etal., 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. lepidophylla. A suite of secondary compounds, such the flavonols apigenin, luteolin, and naringenin, also accumulated to greater concentrations in S.lepidophylla, where they are thought to mitigate ultraviolet light-induced free radical damage as well as aid in the stabilization of membrane structures (Hoekstra etal., 2001). Lastly, polyunsaturated fatty acids were significantly more abundant in S. lepidophylla, possibly reflecting a need for greater membrane fluidity in the dry state (Yobi etal., 2012). The exact mechanisms used to withstand DT have not been well investigated in lycophytes. To better understand the metabolic basis of DT at very low RWCs within the Selaginellaceae, the metabolome of S. lepidophylla was compared over a five-stage, rehydration/dehydration cycle using an unbiased, high-throughput metabolomics platform. This temporal study revealed that S.lepidophylla 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 etal., 2008; Shulaev etal., 2008; Urano etal., 2010). Recent studies have compared two closely related species of Sporobolus (Oliver et al., 2011) and Selaginella (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.lepidophylla.
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S.lepidophylla and Dehydration

In order to discern relative metabolite abundance changes in S.lepidophylla 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 1h and 100% RWC after 24 h (Figure 1A). When hydrated, S. lepidophylla plants displayed fully expanded green microphylls (Figure1B). 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 24h of drying. The rate of water loss in S.lepidophylla was similar to S.bryopteris, a related DT species, which required about 6h to lose 80%90% of its RWC (Deeba etal., 2009; Pandey etal., 2010). During dehydration, the microphylls curl to form a tight ball (Figure1B), 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.lepidophylla is homiochlorophyllous (Tuba etal., 1998).
samples were collected from plants sampled at full dehydration 1 (7% RWC, DRY-1), partial rehydration (50% RWC, REH50), full rehydration (100% RWC, HYD), partial dehydration (50% RWC, DEH-50), and full dehydration 2 (7% RWC, DRY2), and analyzed using non-biased, global metabolome technology 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 (Figure2 and Supplemental Table1). 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%), nucleotides (5%), peptides (4%), and secondary metabolites (3%). Lastly, unnamed compounds in S.lepidophylla accounted for one-third of all compounds surveyed (Figure2).
Metabolomic Differences during a Rehydration/ DehydrationCycle

Partial Least Squares-Discriminant Analysis (PLS-DA) was used to build a comparative model of the five different hydration states in S.lepidophylla. Metabolites missing three or more out of six (50%) biological replicate values were excluded, resulting in a total of 204 metabolites for comparison. The first three PLS-DA components explained 52.2% of the variation (R2=0.50; Q2=0.40) and showed distinct clustering among each of the five states, suggesting that various metabolites likely accounted for the differences observed in the model (Figure3). The metabolomes of the two dry states
Metabolome Composition of S.lepidophylla

To assess the metabolomic response of S. lepidophylla to a rehydration/dehydration cycle, six biological replicate tissue
Figure1. Rehydration/Dehydration Rates in S.lepidophylla. (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 themean. (B) Representative images of S.lepidophylla plants at initial dry state (DRY-1), 50% rehydrated (REH-50), fully rehydrated (HYD), 50% dehydrated (DEH-50), and second dry state (DRY-2). Size bars=2cm.
Figure 2. Functional Categorization of 251 Named and Unnamed Metabolites Detected in S.lepidophylla over Five Different RWCs.
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identical conditions were used for each dehydration episode, these differences between the two dry states might be due to variation arising from repeated hydration and dehydration cycles associated with the life history of different sets of plants. Amino acids, peptides, and nucleotide metabolites were notably overrepresented in the dry states (Supplemental Table 1). In contrast, various carbohydrates, particularly 4C, 5C, and 6C sugars and sugar alcohols, lipids, or lipid metabolites (with the exception of choline phosphate), and cofactors were clearly overrepresented in the hydrated state (Supplemental Table1).
Carbohydrates and Markers of Energy Metabolism

Many differences were observed in the abundance of metabolites of glycolysis/gluconeogenesis and the tricarboxylic acid (TCA) cycle. For example, glucose-6-phosphate, fructose-6-phosphate, maltose-6-phosphate, glycerate, and pyruvate showed a significantly (p<0.05) increase and peak abundance during dehydration (DEH-50) (Figure4). Several TCA cycle intermediates, such as oxaloacetate, fumarate, succinate, and alpha-ketoglutarate, also showed peak abundance during dehydration, whereas succinate was more abundant in the hydrated state (Figure4 and Supplemental Table1). Similarly, glycerate, lactate, and pyruvate were significantly (p < 0.05) more abundant in the hydrated state, with the exception of the DEH-50 state (Supplemental Table 1). The peak accumulation of many glycolysis/gluconeogenesis metabolites suggests that metabolic flux through these pathways is essential for the production of downstream products important for the acquisition ofDT. Various oligosaccharides accumulate in response to drying in the vegetative tissues of all DT tracheophytes studied to date (Alpert and Oliver, 2002). Based on the total relative ion counts, trehalose, sucrose, and glucose were the most abundant compounds within S.lepidophylla tissues and accounted for an estimated 50% of the total metabolites. These compounds tended to be more abundant in the hydrated state relative to the dry state(s) (Supplemental Table1) and their relative abundances were trehalose > sucrose > glucose. Sucrose alone, or in combination with other sugars, such as raffinose-series oligosaccharides and cyclitols, or with macromolecules, such as late embryogenesis abundant (LEA) proteins (Wolkers etal., 2001; Shimizu etal., 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 etal., 2001). S.lepidophylla synthesizes high constitutive 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 etal., 2009) and all angiosperm species examined to date, sucrose accumulation is induced following the imposition of dehydration stress (Ghasempour etal., 1998; Whittaker etal., 2001; Peters etal., 2007; Toldi etal., 2009; Oliver etal., 2011).
Figure3. Metabolites at Different Hydration States in S.lepidophylla 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 (R2 = 0.50; Q2 = 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 diamonds (moss green) represent the second dry state (DRY-2).
at 7% RWC were similar to one another. The metabolic profile 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 initial dry states, suggesting that distinct metabolic pathways operate during rehydration compared with dehydration. In comparing the S. lepidophylla metabolite profiles across 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 Table1). 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 < 0.05) greater abundance than in both dry states. An additional 46 compounds were significantly (p < 0.05) more abundant when compared with the second dry state only. Although
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Figure 4. Metabolites Associated with Energy Metabolism (Glycolysis and TCA Cycle) in S. lepidophylla Profiled during a Rehydration/ DehydrationCycle. 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. The x-axis represents the percentage of relative water content (%RWC): initial dry state (DRY-1); 50% rehydrated (REH-50); fully rehydrated (HYD); 50% dehydrated (DEH50); and second dry state (DRY-2). The y-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 Table1. G-6-P, glucose-6-phosphate; F-6-P, fructose-6-phosphate; M-6-P, mannose-6-phosphate; 3-PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate.
Trehalose, a non-reducing disaccharide, is known to accumulate in high amounts in lycophytes (Adams etal., 1990; Iturriaga etal., 2000; Liu etal., 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, several resurrection ferns and angiosperms accumulate trehalose in only very small amounts (Ghasempour etal., 1998; Farrant etal., 2009). Second, a Saccharomyces cerevisiae mutant defective in trehalose biosynthesis can maintain DT, indicating that trehalose 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 etal., 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 molecular shield function of LEA proteins in reducing desiccationinduced aggregation of proteins (Chakrabortee etal., 2012).
Interestingly, the osmoprotectant glucosylglycerol (2-O-alpha-D-glucopyranosyl-sn-glycerol) was also detected in S. lepidophylla 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 glucosylglycerol 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 etal., 1999; Chen and Murata, 2002; Rontein etal.,
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2002). A variety of polyols showed significant (p < 0.05) changes in abundance in S. lepidophylla 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 hydrated state: ribitol was 16.520.0-fold higher, sorbitol was 5.820.0fold 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 2.0fold more abundant in HYD compared with the dried states (Figure5). 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 differences were not significant (Supplemental Table1). In contrast to other DT species (Farrant etal., 2009; Oliver etal., 2011), raffinose-series sugars (e.g. galactinol, raffinose, stachyose) were not detected and probably do not participate in DT in S.lepidophylla. 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. lepidophylla 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. lepidophylla indicates several important roles for these sugars. Because polyols, especially glycerol, reduce the surface 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. pyramidilis, appears to be a result of a similar metabolic priming of the
hydrated tissue with osmolytes (Oliver etal., 2011). Slowing the rate of water loss might permit more time for the biosynthesis 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. lepidophylla and DS S. moellendorffii 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 Sporobolus stapfianus and DS S. pyramidalis 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 suggest 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. lepidophylla shows a slower rate of rehydration than does S.moellendorffii (Yobi etal., 2012). In addition to a role in water equilibrium, several polyols can also act as osmoprotectants by stabilizing protein (yeast hexokinase A) structure against thermal (Tiwari and Bhat, 2006) or acidic (Devaraneni etal., 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 abundance in S. lepidophylla compared with S. moellendorffii (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 metabolic functions such as consumption of NADH, which might enhance redox control and reduce ROS production in the cells (Shen etal., 1999). Lastly, sugar alcohols might stimulate the expression of stress-adaptive genes, as has been observed in Arabidopsis plants engineered to express mannitol (Chan etal., 2011).
Amino Acid and 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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Figure5. Differences in Sugar Alcohols and Other Sugars in S.lepidophylla Profiled during a Rehydration/DehydrationCycle. 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 Figure4 legend. The p- and q-values for the comparisons are presented in Supplemental Table1. 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.
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, trans4-hydroxyproline, and ophthalmate (Supplemental Table1). In contrast, glycine, quinate, and alanine were significantly (p<0.05) more abundant in fully hydrated S.lepidophylla compared with both dry and REH-50 states (Supplemental Table1). In comparison, quinate accumulation was induced by dehydration (at 60% RWC) in DT S.stapfianus, but not in DS S.pyramidalis (Oliver etal., 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 etal., 2007; Oliver etal., 2011). This increased accumulation of nitrogen-rich amino acids might reflect an adaptation to the
nitrogen-limiting conditions typically encountered by S.stapfianus 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 Figure7). 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 radical 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 etal., 2001). Thus, citrulline might play similar roles in S.lepidophylla. The amino acid derivative 3-(3-hydroxyphenyl)propionate was also more abundant in all dry and partially dry states
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Figure6. Differences in Amino Acid and Nitrogen Metabolism in S.lepidophylla Profiled during a Rehydration/DehydrationCycle. 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 Figure4 legend. The p- and q-values for the comparisons are presented in Supplemental Table1.
compared with the hydrated state, and significantly so in the DRY-1 and DRY-2 states (Supplemental Table1). Notably, the structure of 3-(3-hydroxyphenyl)propionate suggests that it might play a protective role against UV-light damage because closely related phenolic compounds, such as 3,4-dihydroxypropiophenone-3-glucoside and 4-(2-amino3-hydroxyphenyl)-4-oxobutanoic acid O--d-glucoside, serve as UV-B absorbing sunscreens or filters in plants (Tegelberg etal., 2002) and human eye lenses (Bova etal., 1999), respectively. Additional research into the potential UV-protective effects of this compound is warranted. N-6-trimethyllysine is a biosynthetic precursor of carnitine (Hoppel etal., 1980), a known osmoprotectant in Escherichia coli (Cnovas et al., 2007; Verheul et al., 1998). In a recent sister species comparison, both carnitine and acetyl carnitine were more abundant in DT S. lepidophylla than in DS S. moellendorffii (Yobi et al., 2012). However, in the current study, carnitine was not detected and acetyl carnitine was more abundant in the hydrated state, except in DRY-1 (Supplemental Table1). 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). This compound was only recently reported in plants (Oliver etal., 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 functional significance of its accumulation remains unclear (Oliver etal., 2011). Betaine (glycine betaine), a well-known osmoprotectant in many organisms including E. coli (Miller and Ingram, 2007) and plants (Chen and Murata, 2011), failed to display significant changes in abundance with changes in hydration states (Supplemental Table 1). However, this compound was more abundant in DT S. lepidophylla relative to DS S. moellendorffii (Yobi etal., 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. lepidophylla. Of the 14 nucleotides and derivatives detected, the relative amounts of three of them (e.g. allantoin, 1-methyladenosine, and uridine
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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 Table1). 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. lepidophylla compared with both dry and REH-50 states (Supplemental Table1). The ureides allantoin and allantoate serve as nitrogenrich 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 an Arabidopsis 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 etal., 2008). Similarly, RNA interference-mediated suppression of xanthine dehydrogenase in drought-stressed Arabidopsis led to a marked reduction in plant growth and significant 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.lepidophylla. 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 etal., 2011).
Glutathione and -glutamyl Peptide Metabolism

Within glutathione metabolism and the associated -glutamyl amino acid biosynthetic pathway, significant differences in the relative abundance of many metabolites were apparent over the course of the rehydration/dehydration 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 significantly (p < 0.05) more abundant (Figure7 and Supplemental Table 1). In contrast, -glutamyltryptophan was more abundant in the hydrated state, except in the DEH-50 state (Supplemental Table1). Other pathway intermediates, including 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 oxidized glutathione (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 mentioned above, ophthalmate, a glutathione pathway metabolite, was more abundant in all dry or partially dry states than in the fully hydrated state (Supplemental Table1). In Arabidopsis, and presumably in other plants, -glutamyl amino acid cycle -glutamyltranspeptidase (also known as -glutamyltransferase) catalyzes the interconversion of
Figure7. Differences in -Glutamyl Amino Acid and Glutathione Metabolism in S.lepidophylla Profiled during a Rehydration/DehydrationCycle. 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 Figure4 legend. The p- and q-values for the comparisons are presented in Supplemental Table1. 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, releasing 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 to be a minor event (Ohkama-Ohtsu et al., 2008). In S. lepidophylla, 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 suggested to occur in S.stapfianus (Oliver etal., 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. lepidophylla (Figure 7), as well as in T. ruralis and S. stapfianus, following desiccation (Oliver etal., 2011). In addition, both S.lepidophylla and S. stapfianus accumulate -glutamylglutamine and -glutamylmethionine, whereas both S.lepidophylla and T.ruralis accumulate -glutamylvaline (Oliver etal., 2011). In addition, S. lepidophylla accumulates -glutamylthreonine, but not in a significant manner (Supplemental Table1). Sistergroup comparisons between DS and DT species of Sporobolus (Oliver et al., 2011) and Selaginella (Yobi et al., 2012) demonstrated 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. lepidophylla in the hydrated or partially hydrated states (Figure7). Upon becoming oxidized, it donates a reducing equivalent to unstable reactive oxygen intermediates such as hydrogen peroxide or dehydroascorbate, and then reacts instantly with a similar molecule to form glutathione disulfide (GSSG) (Mittler, 2002). Therefore, the greater abundance of GSSG observed in hydrated S.lepidophylla suggests active protection against oxidative stress at early stages of dehydration. In contrast, S.stapfianus did not show significant accumulation of GSSG during DT until RWC reached <40% (Oliver etal., 2011). In terms of other antioxidant systems, S.lepidophylla failed to display the desiccation-induced accumulation of alphaand beta-tocopherols as was observed in S.stapfianus (Oliver etal., 2011). This observation suggests that ROS scavenging occurs in a constitutive manner in S. lepidophylla, which is
consistent with the constitutive production of several other classes of metabolites including sugars and sugar alcohols discussed earlier.
Secondary Metabolites
There were no significant differences in the relative abundance of secondary metabolites over the course of the rehydration/dehydration cycle with the exception of vanillate, which was more abundant in the hydrated states and significantly so compared with the DRY-2 state (Supplemental Table 1). Vanillate (and vanillic acid esters) is a well-known and potent antioxidant that exhibits protective effects against free radical-induced biomembrane damage (Tai etal., 2012). Vanillate appears to be important in DT, as this compound was found to be significantly more abundant in both sistergroup comparisons between DS and DT species of Sporobolus (Oliver etal., 2011) and Selaginella (Yobi etal., 2012). In contrast, other phenolics (e.g. caffeate), flavonols (e.g. apigenin and naringenin), and phenylpropanoids (e.g. coniferyl alcohol) were more abundant in all dry or partially dry states, but these differences were not significant (Supplemental Table 1). A flavonoid closely related to apigenin, called saponarin, is known to be induced in barley seedlings upon 68 d of UV-B exposure (Kaspar et al., 2010). Thus, apigenin is very likely to play a protective role against irradiation with UV light. In addition, apigenin likely ameliorates oxidative stress, as shown in mammalian cells (Chan et al., 2012; Suh et al., 2012). Naringenin, which is closely related in structure to apigenin, is also likely to play protective roles against both exposure to UV light and oxidative stress damage, as demonstrated in rat kidney (Renugadevi and Prabu, 2009) and hepatic (Prabu etal., 2011) cells. Apigenin, naringenin, and vanillate showed significantly (p < 0.05) greater abundance in DT S. lepidophylla compared with DS S. moellendorffii at two different hydration states (Yobi etal., 2012), reinforcing the importance of these compounds in DT.
Lipids, Phospholipids, and FattyAcids

Of the 32 lipid metabolism compounds surveyed, only one (i.e. choline phosphate) showed greater relative abundance in response to dehydration at all stages of dehydration and significantly (p<0.05) so in both dry states, whereas 12 compounds were significantly more abundant in the fully hydrated state compared with one or more re- or dehydration states (Supplemental Table 1). The zwitterionic choline phosphate head group is known to possess a strong water-association capacity and appears to promote water-lipid association between the onium head group and O-C=O groups in fatty acid chains (Foglia etal., 2010). Water molecules bound at this interphase might promote interaction with biomolecules such as sugars, amino acids, and membrane proteins (Disalvo etal., 2008). Membrane interaction with simple sugars or oligosaccharides has been postulated to stabilize or protect the native
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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/dehydrationcycle. 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 significant (Supplemental Table 1). These lipid peroxidation products might arise during dehydration from either ROS attack or the action of LOXs (Marin et al., 1998). In addition, compounds such as 13-HODE, 2-hydroxypalmitate, and 2-hydroxystearate exhibited significantly (p<0.05) greater abundance in DT S. lepidophylla compared with DS S. moellendorffii at two different hydration states, suggesting these compounds participate in DT mechanisms (Yobi etal., 2012). Several unsaturated fatty acids (e.g. arachidate, dihomo-linoleate, dihomolinolenate, linoleate, and linolenate) were significantly (p < 0.05) more abundant in the hydrated state compared with the DRY-1 or DRY-2 states (Supplemental Table1). Adecrease in the relative amounts of various unsaturated lipids upon dehydration has been documented in the DT angiosperms S.stapfianus (Quartacci etal., 1997) and Ramonda serbica (Quartacci etal., 2002). These alterations in unsaturated fatty acid concentrations might contribute to maintaining or increasing membrane fluidity to allow for recovery following dehydration (Upchurch, 2008). Thus, the observed trend towards greater unsaturated fatty acid abundance in the hydrated state and during dehydration suggests a predisposition towards the maintenance of membrane fluidity in S.lepidophylla.
state (Supplemental Table1). This observation stands in stark contrast to the 14 unnamed metabolites that showed 5120fold greater abundance in S.lepidophylla from a sister-group contrast between S.lepidophylla and S.moellendorffii at 50% and 100% RWC hydration states (Yobi etal., 2012). Nonetheless, the seven unnamed metabolites that showed >2.0-fold greater abundance under dry or partially dry conditions in S. lepidophylla represent important targets for future research, 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.lepidophylla 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. lepidophylla provides a useful model to bridge our understanding of the evolutionary progression of DT mechanisms among distantly related resurrection species.
Conclusions
This temporal evaluation of a five-stage, rehydration/dehydration cycle in S.lepidophylla 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.lepidophylla 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 various 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 scavenging, in redox control, in reducing ROS production, and in triggering 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 radical-induced biomembrane damage. Protection against photoand oxidative damage during dehydration is also known to be 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 attenuate 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) propionate and the flavonols apigenin and naringenin in all dry
Unnamed Metabolites
Of the 84 unnamed compounds identified in S.lepidophylla, 15 (17.8%) and 17 (20.2%) were significantly (p<0.05) more abundant in one or more dry or partially dry states than the fully hydrated state, respectively. Unnamed compounds significantly more abundant in dry or partially dry states showed 2.17.3-fold greater abundance, respectively. The unnamed compounds significantly more abundant in the fully hydrated state displayed 2.04.0-fold greater abundance compared to dry or partially dry states, respectively. In addition, nine and five unnamed compounds were always more abundant in all dehydrated states and in the hydrated state, respectively. However, these hydration-state-specific (dry/partially dry or fully hydrated) differences were not significant (Supplemental Table1). The relatively small magnitude of significant differences among the five different hydration states in S. lepidophylla suggests that metabolites that might play key roles in DT are mainly expressed constitutively. This idea is reinforced by the observation that no unnamed compound showed more than a 1.7-fold increase in relative abundance in the DEH-50
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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.lepidophylla, likely play critical roles in the acquisition of DT through the scavenging of ROS via glutathione metabolism, or the remobilization of amino acids following rehydration, or both. Lastly, S.lepidophylla contained seven unnamed compounds that displayed twofold or greater abundance in dry or rehydrating states, suggesting that these compounds might play adaptive roles in the acquisition of DT or in the recovery from the desiccated state.
METHODS
Plant Material and Water-Deficit Stress Treatments
Selaginella lepidophylla (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 2d, then reweighed to obtain dry weights. The RWC was calculated using the following formula: RWC (%)=(FwtDwt)/(FTwt-Dwt)100, where Fwt is the fresh weight at specific time points during the rehydration/dehydration cycle,
Figure8. Differences in Nine Unnamed Compounds in S.lepidophylla 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 Figure4 legend. The p- and q-values for the comparisons are presented in Supplemental Table1.
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Dwt is the weight after incubation at 65C for 2d, and FTwt is the weight after 24 h of rehydration. Samples representing 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.
in the Kyoto Encyclopedia of Genes and Genomes (KEGG) (www.genome.jp/kegg/) and Plant Metabolic Network (PMN) (www.plantcyc.org/).
Data Imputation and Statistical Analysis

The samples were analyzed over the course of 2 d. This required data correction for minor variations resulting from instrument inter-day tuning differences (Evans etal., 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 dividing 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 because the variance generally increased as a function of each biochemicals 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 different 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 robustness of a PLS-DA model, where R2 is the cumulative variance explained by the components. Cross-validation of R2 estimates Q2, which is the cumulative variance predicted by the model. Thus, both of these values indicate how well the overall 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 themodel. 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.lepidophylla. 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 twosample 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 comparisons. As with microarray studies, metabolomic profiling generates large numbers of metabolites, and thus FDR is typically 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
Metabolomic Profiling
Six biological replicates from each species were collected at each time point, lyophilized, and kept at 80C under hermetic conditions prior to extraction. Then, 20mg of each leaf sample was extracted in 400l of methanol containing recovery standards using an automated MicroLab STAR system (Hamilton Company, Salt Lake City,UT). Global unbiased metabolic profiling in S.lepidophylla was performed using an integrated platform consisting of a combination of three independent approaches: (1) ultrahigh performance 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 bistrimethylsilyl-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 etal., 2011). Automated comparison of the ion features in the experimental samples was used to identify metabolites using a custom reference library of chemical standards. Reference library standard entries included the retention time, molecular 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 artifactual 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
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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.
plant Craterostigma 10431050.
plantagineum.
Plant
Physiol.
111,
SUPPLEMENTARYDATA
Supplementary Data are available at Molecular Plant Online.
Bova, L.M., Wood, A.M., Jamie, J.F., and Truscott, R.J.W. (1999). UV filter compounds in human lenses: the origin of 4-(2-amino3-hydroxyphenol)-4-oxobutanoic acid O-beta-D-glucoside. Invest. Opthamol. Vis. Sci. 40, 32373244. Brychkova, G., Alikulov, Z., Fluhr, R., and Sagi, M. (2008). A critical role for ureides in dark and senescence-induced purine remobilization is unmasked in the Atxdh1 Arabidopsis mutant. Plant J. 54, 496509. Burke, A. (2002). Properties of soil pockets on arid nama Karoo inselbergs: the effect of geology and derived landforms. J. Arid Environ. 50, 219234. Cnovas, M., Bernal, V., Sevilla, A., and Iborra, J. (2007). Salt stress effects on the central and carnitine metabolisms of Escherichia coli. Biotechnol. Bioeng. 96, 722737. Cao, Y., Tan, N., Chen, J., Zeng, G., Ma, Y., Wu, Y., Yan, H., Yang, J., Lu, L., and Wang, Q. (2010). Bioactive flavones and biflavones from Selaginella moellendorffii Hieron. Fitoterapia. 81, 253258. Cascante, M., and Marin, S. (2008). Metabolomics and fluxomics approaches. Essays Biochem. 45, 6781. Chakrabortee, S., Tripathi, R., Watson, M., Schierle, G.S., Kurniawan, D.P., Kaminski, C.F., Wise, M.J., and Tunnacliffe, A. (2012). Intrinsically disordered proteins as molecular shields. Mol. Biosyst. 8, 210219. Chan, L.P., Chou, T.H., Ding, H.Y., Chen, P.R., Chiang, F.Y., Kuo, P.L., and Liang, C.H. (2012). Apigenin induces apoptosis via tumor necrosis factor receptor- and Bcl-2-mediated pathway and enhances susceptibility of head and neck squamous cell carcinoma to 5-fluorouracil and cisplatin. Biochim. Biophys. Acta. 1820, 10811091. Chan, Z., Grumet, R., and Loescher, W. (2011). Global gene expression analysis of transgenic, mannitol-producing, and salt-tolerant Arabidopsis thaliana indicates widespread changes in abiotic and biotic stress-related genes. J. Exp. Bot. 62, 47874803. Chen, T.H.H., and Murata, N. (2002). Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr. Opin. Plant. Biol. 5, 250257. Chen, T.H.H., and Murata, N. (2011). Glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications. Plant Cell Environ. 34, 120. Deeba, F., Pandey, V., Pathre, U., and Kanojiya, S. (2009). Proteome analysis of detached fronds from a resurrection plant Selaginella bryopteris: response to dehydration and rehydration. J. Proteomics Bioinformatics. 2, 108116. Devaraneni, P.K., Mishra, N., and Bhat, R. (2012). Polyol osmolytes stabilize native-like cooperative intermediate state of yeast hexokinase Aat low pH. Biochimie. 94, 947952. Disalvo, E.A., Lairion, F., Martini, F., Tymczyszyn, E., Fras, M., Almaleck, H., and Gordillo, G.J. (2008). Structural and functional properties of hydration and confined water in membrane interfaces. Biochim. Biophys. Acta. 1778, 26552670. Elbein, A.D., Pan, Y.T., Pastuszak, I., and Carroll, D. (2003). New Insights on trehalose: a multifunctional molecule. Glycobiology. 13, 17R27R.
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.
References
Adams, R.P., Kendall, E., and Kartha, K.K. (1990). Comparison of free sugars in growing and desiccated plants of Selaginella lepidophylla. Biochem. System. Ecol. 18, 107110. Aguilar, M.I., Romero, M.G., Chvez, M.I., King-Daz, B., and LotinaHennsen, B. (2008). Biflavonoids isolated from Selaginella lepidophylla inhibit photosynthesis in spinach chloroplasts. J. Agric. Food Chem. 56, 69947000. Ahn, S.H., Mun, Y.J., Lee, S.W., Kwak, S., Choi, M.K., Baik, S.K., Kim, Y.M., and Woo, W.H. (2006). Selaginella tamariscina induces apoptosis via a caspase-3-mediated mechanism in human promyelocytic leukemia cells. J. Med. Food. 9, 138144. Akashi, K., Miyake, C., and Yokota, A. (2001). Citrulline, a novel compatible solute in drought-tolerant wild watermelon leaves, is an efficient hydroxyl radical scavenger. FEBS Lett. 508, 438442. Alpert, P. (2006). Contraints of tolerance: why are desiccation-tolerant organisms so small or rare? J. Exp. Biol. 209, 15751584. Alpert, P., and Oliver, M.J. (2002). Drying without dying. In Desiccation and Survival in Plants: Drying without Dying, Black, M., and Pritchard, H.W., eds (New York, USA: CABI Publishing), pp. 143. Banks, J.A. (2009). Selaginella and 400 million years of separation. Annu. Rev. Plant Biol. 60, 223238. Beerling, D.J. (2002). Low atmospheric CO(2) levels during the Permo-Carboniferous glaciation inferred from fossil lycopsids. Proc. Natl Acad. Sci. U S A. 99, 1256712571. Bernacchia, G., Salamini, F., and Bartels, D. (1996). Molecular characterization of the rehydration process in the resurrection
383
Evans, A.M., DeHaven, C.D., Barrett, T., Mitchell, M., and Milgram, E. (2009). Integrated, nontargeted ultrahigh performance liquid chromatography/electrospray ionization tandem mass spectrometry platform for the identification and relative quantification of the small-molecule complement of biological systems. Anal. Chem. 81, 66566667. Farrant, J. (2000). A comparison of mechanisms of desiccation tolerance among three angiosperm resurrection plant species. Plant Ecol. 151, 2939. Farrant, J.M., Lehner, A., Cooper, K., and Wiswedel, S. (2009). Desiccation tolerance in the vegetative tissues of the fern Mohria caffrorum is seasonally regulated. Plant J. 57, 6579. Fernie, A.R., Aharoni, A., Willmitzer, L., Stitt, M., Tohge, T., Kopka, J., Carroll, A.J., Saito, K., Fraser, P.D., and DeLuca, V. (2011). Recommendations for reporting metabolite data. Plant Cell. 23, 24772482. Foglia, F., Lawrence, M.J., Lorenz, C.D., and McLain, S.E. (2010). On the hydration of the phosphocholine headgroup in aqueous solution. J. Chem. Phys. 133, 145103145110. Friedman, W.E. (2011). Plant genomics: homoplasy heaven in a lycophyte genome. Curr. Biol. 21, R554R556. Gensel, P.G. (2008). The earliest lant plants. Ann. Rev. Ecol. Evol. Syst. 39, 459477. Georgieva, K., Rding, A., and Bchel, C. (2009). Changes in some thylakoid membrane proteins and pigments upon desiccation of the resurrection plant Haberlea rhodopensis. J. Plant Physiol. 166, 15201528. Ghasempour, H.R., Gaff, D.F., Williams, R.P.W., and Gianello, R.D. (1998). Contents of sugars in leaves of drying desiccation tolerant flowering plants, particularly grasses. Plant Growth Regul. 24, 185191. Goyal, K., Walton, L.J., and Tunnacliffe, A. (2005). LEA proteins prevent protein aggregation due to water stress. Biochem. J. 388, 151157. Ha, L.H., Thao, D.T., Huong, H.T., Minh, C.V., and Dat, N.T. (2012). Toxicity and anticancer effects of an extract from Selaginella tamariscina on a mice model. Nat. Prod. Research. 26, 11301134. Hoekstra, F.A., Golvina, E.A., and Buitink, J. (2001). Mechanisms of plant desiccation tolerance. Trends Plant Sci. 6, 431438. Hoppel, C.L., Cox, R.A., and Novak, R.F. (1980). N6-trimethyl-lysine metabolism. 3-hydroxy-N6-trimethyl-lysine and carnitine biosynthesis. Biochem. J. 188, 509519. Iturriaga, G., Cushman, M.A.F., and Cushman, J.C. (2006). An EST catalogue from the resurrection plant Selaginella lepidophylla reveals abiotic stress-adaptive genes. Plant Biol. 170, 11731184. Iturriaga, G., Gaff, D.F., and Zentella, R. (2000). New desiccationtolerant plants, including a grass, in the central highlands of Mexico, accumulate trehalose. Aust. J.Bot. 48, 153158. Kaspar, S., Matros, A., and Mock, H.P. (2010). Proteome and flavonoid analysis reveals distinct responses of epidermal tissue and whole leaves upon UV-B radiation of barley (Hordeum vulgare L.) seedlings. J. Proteome Res. 9, 24022411. Kataoka, Y., Kitadai, N., Hisatomi, O., and Nakashima, S. (2011). Nature of hydrogen bonding of water molecules in aqueous solutions of glycerol by attenuated total reflection (ATR) infrared spectroscopy. Appl. Spectrosc. 65, 436441.
Kaushik, J.K., and Bhat, R. (1998). Thermal stability of proteins inaqueous polyol solutions: role of the surface tension of water in the stabilizing effect of polyols. J. Phys. Chem. 102, 70587066. Kawasaki, S., Miyake, C., Kohchi, T., Fujii, S., Uchida, M., and Yokota, A. (2000). Responses of wild watermelon to drought stress: accumulation of an ArgE homologue and citrulline in leaves during water deficits. Plant Cell Physiol. 41, 864873. Klhn, S., and Hagemann, M. (2011). Compatible solute biosynthesis in cyanobacteria. Environ. Microbiol. 13, 551562. Lebkuecher, J.G, and Eickmeier, W.G. (1991). Reduced photoinhibition with stem curling in the resurrection plant Selaginella lepidophylla. Oecologia. 88, 597604. Lebkuecher, J.G., and Eickmeier, W.G. (1992). Photoinhibition of photophosphorylation, adendosine triphosphate content, and glyceraldehyde-3-phosphate dehydrogenase (NADP+) following high-irradiance dessiccation of Selaginella lepidophylla. Can. J.Bot. 70, 205211. Lebkuecher, J.G., and Eickmeier, W.G. (1993). Physiological benefits of stem curling for resurrection plants in the field. Ecology. 74, 10731080. Lee, S., Kim, H., Kang, J.W., Kim, J.H., Lee, D.H., Kim, M.S., Yang, Y., Woo, E.R., Kim, Y.M., Hong, J., etal. (2011). The biflavonoid amentoflavone induces apoptosis via suppressing E7 expression, cell cycle arrest at sub-G1 phase, and mitochondria-emanated intrinsic pathways in human cervical cancer cells. J. Med. Food. 14, 808816. Liu, H., Zhao, S., Zhang, Y., Wu, J., Peng, H., Fan, J., and Liao, J. (2011). Reactive oxygen species-mediated mitochondrial dysfunction is involved in apoptosis in human nasopharyngeal carcinoma CNE cells induced by Selaginella doederleinii extract. J. Ethnopharmacol. 138, 184191. Liu, J.F., Xu, K.P., Li, F.S., Shen, J., Hu, C.P., Zou, H., Yang, F., Liu, G.R., Xiang, H.L., Zhou, Y.J., etal. (2010). A new flavonoid from Selaginella tamariscina (Beauv.) Spring. Chem. Pharm. Bull. (Tokyo). 58, 549551. Liu, M.-S., Chien, C.-T., and Lin, T.-P. (2008). Constitutive components and induced gene expression are involved in the desiccation tolerance of Selaginella tamariscina. Plant Cell Physiol. 49, 653663. Marin, K., Zuther, E., Kerstan, T., Kunert, A., and Hagemann, M. (1998). The ggpS gene from Synechocystis sp. strain PCC 6803 encoding glucosyl-glycerol-phosphate synthase is involved in osmolyte synthesis. J. Bacteriol. 180, 48434849. Martinelli, T., Whittaker, A., Bochicchio, A., Vazzana, C., Suzuki, A., and Masclaux-Daubresse, C. (2007). Amino acid pattern and glutamate metabolism during dehydration stress in the resurrection plant Sporobolus stapfianus: a comparison between desiccation-sensitive and desiccation-tolerant leaves. J. Exp. Bot. 58, 30373046. Miller, E.N., and Ingram, L.O. (2007). Combined effect of betaine and trehalose on osmotic tolerance of Escherichia coli in mineral salts medium. Biotechnol. Lett. 29, 213217. Mishra, P.K., Raghuram, G.V., Bhargava, A., Ahirwar, A., Samarth, R., Upadhyaya, R., Jain, S.K., and Pathak, N. (2011). In vitro and in vivo evaluation of the anticarcinogenic and cancer
384
chemopreventive potential of a flavonoid-rich fraction from a traditional Indian herb Selaginella bryopteris. Br. J.Nutr. 106, 11541168. Mittler, R. (2002). Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7, 405410. Nishizawa, A., Yabuta, Y., and Shigeoka, S. (2008). Galactinol and raffinose contribute a novel function to protect plants from oxidative damage. Plant Physiol. 147, 12511263. Nishizawa-Yokoi, A., Yabuta, Y., and Shigeoka, S. (2008). The contribution of carbohydrates including raffinose family oligosaccharides and sugar alcohols to protection of plants from oxidative damage. Plant Signal. Behav. 3, 10161018. Nuccio, M.L., Rhodes, D., McNeil, S.D., and Hanson, A.D. (1999). Metabolic engineering of plants for osmotic stress resistance. Curr. Opin. Plant Biol. 2, 128134. Ohkama-Ohtsu, N., Oikawa, A., Zhao, P., Xiang, C., Saito, K., and Oliver, D.J. (2008). A gamma-glutamyl transpeptidase-independent pathway of glutathione catabolism to glutamate via 5-oxoproline in Arabidopsis. Plant Physiol. 148, 16031613. Ohtake, S., Schebor, C., and de Pablo, J.J. (2006). Effects of trehalose on the phase behavior of DPPC-cholesterol unilamellar vesicles. Biochim. Biophys. Acta. 1758, 6573. Oliver, M.J., Guo, L., Alexander, D.C., Ryals, J.A., Wone, B.W.M., and Cushman, J.C. (2011). A sister group metabolomic contrast using untargeted global metabolomic analysis delineates the biochemical regulation underlying desiccation tolerance in Sporobolus stapfianus. Plant Cell. 23, 12311248. Oliver, M.J., Tuba, Z., and Mishler, B.D. (2000a). The evolution of vegetative desiccation tolerance in land plants. Plant Ecol. 151, 85100. Oliver, M.J., Velten, J., and Mishler, B.D. (2005). Desiccation tolerance in bryophytes: a reflection of the primitive strategy for plant survival in dehydrating habitats? Integr. Comp. Biol. 45, 788799. Oliver, M.J., Velten, J., and Wood, A.J. (2000b). Bryophytes as experimental models for the study of environmental stress tolerance: Tortula ruralis and desiccation-tolerance in mosses. Plant Ecol. 151, 7384. Pandey, V., Ranjan, S., Deeba, F., Pandey, A.K., Singh, R., and Shirke, P.A. (2010). Desiccation-induced physiological and biochemical changes in resurrection plant, Selaginella bryopteris. J. Plant Physiol. 167, 13511359. Peters, S., Mundree, S.D., Thomson, J.A., Farrant, J.M., and Keller, F. (2007). Protection mechanisms in the resurrection plant Xerophyta viscosa (Baker): both sucrose and raffinose family oligosaccharides (RFOs) accumulate in leaves in response to water deficit. J. Exp. Bot. 58, 19471956. Porembski, S., and Barthlott, W. (2000). Granitic and gneissic outcrops (inselbergs) as center of diversity for desiccation-tolerant vascular plants. Plant Ecology. 151, 1928. Prabu, S.M., Shagirtha, K., and Renugadevi, J. (2011). Naringenin in combination with vitamins C and E potentially protects oxidative stress-mediated hepatic injury in cadmium-intoxicated rats. J. Nutr. Sci. Vitaminol. (Tokyo). 57, 177185. Proctor, M., and Pence, V. (2002). Vegetative tissues: bryophytes, vascular resurrection plants and vegetative propagules. In Desiccation and Survival in Plants: Drying without Dying, Black, M., and Pritchard, H., eds (Oxford: CABI Publishing).
Proctor, M., and Tuba, Z. (2002). Poikilohydry and homoihydry: anithesis or spectrum of possibilities? New Phytol. 156, 327349. Quartacci, M.F., Forli, M., Rascio, N., Dalla Vecchia, F., Bochicchio, A., and Navari-Izzo, F. (1997). Desiccationtolerant Sporobolus stapfianus: lipid composition and cellular ultrastructure during dehydration and rehydration. J.Exp. Bot. 48, 12691279. Quartacci, M.F., Glisi, O., Stevanovi, B., and Navari-Izzo, F. (2002). Plasma membrane lipids in the resurrection plant Ramonda serbica following dehydration and rehydration. J. Exp. Bot. 53, 21592166. Ratnakumar, S., and Tunnacliffe, A. (2006). Intracellular trehalose is neither necessary nor sufficient for desiccation tolerance in yeast. FEMS Yeast Res. 6, 903913. Renugadevi, J., and Prabu, S.M. (2009). Naringenin protects against cadmium-induced oxidative renal dysfunction in rats. Toxicology. 256, 128134. Robles-Zepeda, R.E., Velzquez-Contreras, C.A., Garibay-Escobar, A., Glvez-Ruiz, J.C., and Ruiz-Bustos, E. (2011). Antimicrobial activity of northwestern Mexican plants against Helicobacter pylori. J. Med. Food. 14, 12801283. Rontein, D., Basset, G., and Hanson, A.D. (2002). Metabolic engineering of osmoprotectant accumulation in plants. Metab. Eng. 4, 4956. Ruiz-Bustos, E., Velazquez, C., Garibay-Escobar, A., Garca, Z., Plascencia-Jatomea, M., Cortez-Rocha, M.O., HernandezMartnez, J., and Robles-Zepeda, R.E. (2009). Antibacterial and antifungal activities of some Mexican medicinal plants. J. Med. Food. 12, 13981402. Sanchez, D.H., Siahpoosh, M.R., Roessner, U., Udvardi, M., and Kopka, J. (2008). Plant metabolomics reveals conserved and divergent metabolic responses to salinity. Physiol. Plant. 132, 209219. Sawangwan, T., Goedl, C., and Nidetzky, B. (2010). Glucosylglycerol and glucosylglycerate as enzyme stabilizers. Biotechnol. J. 5, 187191. Schauer, N., and Fernie, A.R. (2006). Plant metabolomics: towards biological function and mechanism. Trends Plant Sci. 11, 508516. Shen, B., Hohmann, S., Jensen, R.G., and Bohnert, H.J. (1999). Roles of sugar alcohols in osmotic stress adaptation: replacement of glycerol by mannitol and sorbitol in yeast. Plant Physiol. 121, 4552. Shimizu, T., Kanamori, Y., Furuki, T., Kikawada, T., Okuda, T., Takahashi, T., Mihara, H., and Sakurai, M. (2010). Desiccationinduced structuralization and glass formation of group 3 late embryogenesis abundant protein model peptides. Biochemistry. 49, 10931104. Shulaev, V., Cortez, D., Miller, G., and Mittler, R. (2008). Metabolomics for plant stress response. Physiol. Plant. 132, 199208. Smirnoff, N. (1992). The carbohydrates of bryophytes in relation to desiccation-tolerance. J. Bryol. 17, 185191. Storey, J.D. (2002). A direct approach to false discovery rates. J. Royal Statist. Soc. Ser. B. 64, 479498. Suh, K.S., Oh, S., Woo, J.T., Kim, S.W., Kim, J.W., Kim, Y.S., and Chon, S. (2012). Apigenin attenuates 2-deoxy-D-ribose-induced oxidative cell damage in HIT-T15 pancreatic -cells. Biol. Pharm. Bull. 35, 121126. Downloaded from http://mplant.oxfordjournals.org/ at Universidad de Concepcion on June 5, 2013
385
Swamy, R.C., Kunert, O., Schhly, W., Bucar, F., Ferreira, D., Rani, V.S., Kumar, B.R., and Appa Rao, A.V. (2006). Structurally unique biflavonoids from Selaginella chrysocaulos and Selaginella bryopteris. Chem Biodivers. 3, 405413. Tai, A., Sawano, T., and Ito, H. (2012). Antioxidative properties of vanillic acid esters in multiple antioxidant assays. Biosci. Biotechnol. Biochem. 76, 314318. Tan, W.J., Xu, J.C., Li, L., and Chen, K.L. (2009). Bioactive compounds of inhibiting xanthine oxidase from Selaginella labordei. Nat. Prod. Res. 23, 393398. Tegelberg, R., Aphalo, P.J., and Julkunen-Tiitto, R. (2002). Effects of long-term, elevated ultraviolet-B radiation on phytochemicals in the bark of silver birch (Betula pendula). Tree Physiol. 22, 12571263. Tiwari, A., and Bhat, R. (2006). Stabilization of yeast hexokinase A by polyol osmolytes: correlation with the physicochemical properties of aqueous solutions. Biophys. Chem. 124, 9099. Toldi, O., Tuba, Z., and Scott, P. (2009). Vegetative desiccation tolerance: is it a goldmine for bioengineering crops? Plant Sci. 176, 187199. Tuba, Z., Proctor, M.C.F., and Csintalan, Z. (1998). Ecological responses of homoiochlorophyllous and poikilochlorophyllous desiccation tolerance plants: a comparison and an ecological perspective. Plant Growth Regulation. 24, 211217. Upchurch, R.G. (2008). Fatty acid unsaturation, mobilization, and regulation in the response of plants to stress. Biotechnol. Lett. 30, 967977. Urano, K., Kurihara, Y., Seki, M., and Shinozaki, K. (2010). Omics analyses of regulatory networks in plant biotic stress responses. Curr. Opin. Plant. Biol. 13, 132138. Valluru, R., and Van den Ende, W. (2008). Plant fructans in stress environments: emerging concepts and future prospects. J. Exp. Bot. 59, 29052916. Vzquez-Ortz, F.A., and Valenzuela-Soto, E.M. (2004). HPLC determination of trehalose in Selaginella lepidophylla plants. J. Liquid Chromat. Related Technol. 27, 19371946. Verheul, A., Wouters, J.A., Rombouts, F.M., and Abee, T. (1998). A possible role of ProP, ProU and CaiT in osmoprotection of Escherichia coli by carnitine. J. Appl. Microbiol. 85, 10361046. Wang, H.S., Sun, L., Wang, Y.H., Shi, Y.N., Tang, G.H., Zhao, F.W., Niu, H.M., Long, C.L., and Li, L. (2011). Carboxymethyl flavonoids and a monoterpene glucoside from Selaginella moellendorffii. Arch. Pharm. Res. 34, 12831288. Wang, X., Chen, S., Zhang, H., Shi, L., Cao, F., Guo, L., Xie, Y., Wang, T., Yan, X., and Dai, S. (2010). Desiccation tolerance mechanism in resurrection fern-ally Selaginella tamariscina revealed by physiological and proteomic analysis. J. Proteomics. Res. 9, 65616577. Wang, Y., Long, C., Yang, F., Wang, X., Sun, Q., Wang, H., Shi, Y., and Tang, G. (2009). Pyrrolidinoindoline alkaloids from Selaginella moellendorfii. J. Nat. Prod. 72, 11511154.
Watanabe, S., Nakagawa, A., Izumi, S., Shimada, H., and Sakamoto, A. (2010). RNA interference-mediated suppression of xanthine dehydrogenase reveals the role of purine metabolism in drought tolerance in Arabidopsis. FEBS Lett. 584, 11811186. Weng, L., Chen, C., Zuo, J., and Li, W. (2011). Molecular dynamics study of effects of temperature and concentration on hydrogen-bond abilities of ethylene glycol and glycerol: implications for cryopreservation. J. Phys. Chem. A. 115, 47294737. Werner, A.K., and Witte, C.P. (2011). The biochemistry of nitrogen mobilization: purine ring catabolism. Trends. Plant. Sci. 16, 381387. White, E., and Towers, G.H.N. (1967). Comparative biochemistry of the lycopods. Phytochemistry. 6, 663667. Whittaker, A., Bochicchio, A., Vazzana, C., Lindsey, G., and Farrant, J. (2001). Changes in leaf hexokinase activity and metabolite levels in response to drying in the desiccation-tolerant species Sporobolus stapfianus and Xerophyta viscosa. J. Exp. Bot. 52, 961969. Wolkers, W.F., McCready, S., Brandt, W.F., Lindsey, G.G., and Hoekstra, F.A. (2001). Isolation and characterization of a D-7 LEA protein from pollen that stabilizes glasses in vitro. Biochim. Biophys. Acta. 1544, 196206. Wood, A.J. (2007). New frontiers in bryology and lichenology: the nature and distribution of vegetative desiccation-tolerance in hornworts, liverworts and mosses. The Bryologist. 110, 163177. Wu, B., and Wang, J. (2011). Phenolic compounds from Selaginella moellendorfii. Chem. Biodivers. 8, 17351747. Xie, G., and Timasheff, S.N. (1997). Mechanism of the stabilization of ribonuclease Aby sorbitol: preferential hydration is greater for the denatured than for the native protein. Protein Sci. 6, 211221. Yobi, A., Wone, B.W.M., Xu, W., Alexander, D.C., Guo, L., Ryals, J.A., Oliver, M.J., and Cushman, J.C. (2012). Comparative metabolic profiling between desiccation-sensitive and desiccation-tolerant species of Selaginella reveals insights into the resurrection trait. Plant J. 72, 983999. Zhang, Y.X., Li, Q.Y., Yan, L.L., and Shi, Y. (2011). Structural characterization and identification of biflavones in Selaginella tamariscina by liquid chromatography-diode-array detection/ electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 25, 21732186. Zheng, J.X., Zheng, Y., Zhi, H., Dai, Y., Wang, N.L., Fang, Y.X., Du, Z.Y., Zhang, K., Li, M.M., Wu, L.Y., et al.. (2011a). New 3,8-linked biflavonoids from Selaginella uncinata displaying protective effect against anoxia. Molecules. 16, 62066214. Zheng, X.K., Zhang, L., Wang, W.W., Wu, Y.Y., Zhang, Q.B., and Feng, W.S. (2011b). Anti-diabetic activity and potential mechanism of total flavonoids of Selaginella tamariscina (Beauv.) Spring in rats induced by high fat diet and low dose STZ. J. Ethnopharmacol. 137, 662668. Downloaded from http://mplant.oxfordjournals.org/ at Universidad de Concepcion on June 5, 2013

Metabolomic Profiling in Selaginella Lepidophylla

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Metabolomic Profiling in Selaginella Lepidophylla

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Molecular Plant Volume 6 Number 2 Pages 369385 March 2013

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Yobi et al. Rehydration/Dehydration Metabolomics

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RESULTS AND DISCUSSION

Yobi et al. Rehydration/Dehydration Metabolomics

S.lepidophylla and Dehydration

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Metabolomic Differences during a Rehydration/ DehydrationCycle

Metabolome Composition of S.lepidophylla

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Carbohydrates and Markers of Energy Metabolism

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Sugar Alcohol Metabolism

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Amino Acid and Peptide Metabolism

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Yobi et al. Rehydration/Dehydration Metabolomics

Glutathione and -glutamyl Peptide Metabolism

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Lipids, Phospholipids, and FattyAcids

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Yobi et al. Rehydration/Dehydration Metabolomics

Data Imputation and Statistical Analysis

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plant Craterostigma 10431050.

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Yobi et al. Rehydration/Dehydration Metabolomics

Yobi et al. Rehydration/Dehydration Metabolomics

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