Molecular Plant • Volume 6 • Number 2 • Pages 369–385 • March 2013 RESEARCH ARTICLE Metabolomic Profiling in Selaginella lepidophylla at Various Hydration States Provides New Insights into the Mechanistic Basis of Desiccation Tolerance Abou Yobia, Bernard W.M. Woneb, Wenxin Xuc, Danny C. Alexanderc, Lining Guoc, John A. Ryalsc, Melvin J. Oliverd and John C. Cushmana,1 a Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557–0330, USA b Department of Biological Sciences, University of Nevada, Reno, NV 89557–0314, USA c Metabolon Inc., 800 Capitola Drive, Suite 1, Durham, NC 27713, USA d US Department of Agriculture—Agricultural Research Service, Plant Genetic Research Unit, University of Missouri, Columbia, MI 65211, USA ABSTRACT Selaginella lepidophylla is one of only a few species of spike mosses (Selaginellaceae) that have evolved des- iccation 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. A total of 251 metabolites including 167 10.1093/mp/sss155 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 con- stitutively, 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 γ-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 poly- unsaturated 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 coal (Beerling, 2002; Gensel, 2008). Today, extant lycophytes comprise a single monophyletic clade of just over 1000 species. 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 1 To whom correspondence should be addressed. E-mail [email protected], million years ago during the Silurian and dominated the tel. +1 775 784 1918, fax +1 775 784 1419. Earth’s flora from the Devonian through the Carboniferous to © The Author 2013. Published by the Molecular Plant Shanghai Editorial the end of the Permian (Friedman, 2011). Dramatic decreases Office in association with Oxford University Press on behalf of CSPB and in atmospheric CO2 concentrations occurred during these IPPE, SIBS, CAS. Paleozoic epochs and the resulting decomposed flora now doi:10.1093/mp/sss155, Advance Access publication 13 December 2012 provide a major source of energy to mankind in the form of Received 9 August 2012; accepted 6 December 2012 370 Yobi et al. • Rehydration/Dehydration Metabolomics Within the spike mosses, the single genus Selaginella con- S. tamariscina (Zhang et al., 2011). Antimicrobial alkaloids tains about 700 species that now exploit a diverse array of arc- have also been characterized from S. moellendorfii (Wang tic, temperate, tropical, and semi-arid habitats (Banks, 2009). et al., 2009). Although most spike moss species are susceptible to desicca- Despite their role in traditional medicine, comprehensive tion, a few species have evolved the ability to survive vegeta- metabolite studies on Selaginella species have been limited. tive tissue drying, defined as the near complete loss (80%–95%) Previous studies have characterized only a few, highly abun- of protoplasmic water (Tuba et al., 1998), and referred to as dant compounds such as lignin (White and Towers, 1967) desiccation tolerance (DT) (Oliver et al., 2000a). Such species and trehalose (White and Towers, 1967; Adams et al., 1990; include S. lepidophylla (Iturriaga et al., 2006), S. bryopteris Iturriaga et al., 2000; Vázquez-Ortíz and Valenzuela-Soto, (Deeba et al., 2009), and S. tamariscina (Wang et al., 2010). 2004; Liu et al., 2008). A recent comparative study examined The DT trait is relatively common in algae, lichens, and mosses the major metabolic differences between the desiccation- (Alpert, 2006; Wood, 2007). However, among vascular plants, sensitive (DS) S. moellendorffi and the DT S. lepidophylla. DT is extremely rare; only about ~0.15% of species being are Several sugars (e.g. glucose, sucrose), sugar alcohols (e.g. known as resurrection plants (Oliver et al., 2000a; Porembski inositol-1-phosphate, myo-inositol, mannitol), and betaine, and Barthlott, 2000; Proctor and Pence, 2002; Proctor and which act as osmoprotectants or hydroxyl radical scavengers, Tuba, 2002). were more abundant in S. lepidophylla at 100% and 50% In terms of response to desiccation, resurrection mosses relative water contents (RWC) (Yobi et al., 2012). In addition, (e.g. bryophytes) rely on a combination of constitutive pro- a variety of γ-glutamyl amino acids, which are thought to tection and inducible repair mechanisms that permit dehy- participate in reactive oxygen species (ROS) detoxification, dration to occur within minutes while maintaining their and possible nitrogen remobilization following rehydration, photosynthetic apparatus relatively intact (Tuba et al., 1998; were markedly higher in S. lepidophylla. A suite of second- Oliver et al., 2000b, 2005). In contrast, DT angiosperms ary compounds, such the flavonols apigenin, luteolin, and require a much longer time to dehydrate and still remain via- naringenin, also accumulated to greater concentrations in ble, presumably to allow sufficient time for mobilizing adap- S. lepidophylla, where they are thought to mitigate ultravi- tive responses and accumulation of key metabolites, such as olet light-induced free radical damage as well as aid in the sucrose, to survive in the desiccated state (Bernacchia et al., stabilization of membrane structures (Hoekstra et al., 2001). 1996; Oliver et al., 2000a). In contrast, lycophytes represent Lastly, polyunsaturated fatty acids were significantly more a plant lineage that lies between mosses and angiosperms abundant in S. lepidophylla, possibly reflecting a need for (Oliver et al., 2000a) and thus might be expected to exhibit greater membrane fluidity in the dry state (Yobi et al., 2012). both constitutive and inducible adaptive mechanisms of DT. The exact mechanisms used to withstand DT have not been Many monocot DT species lose their photosynthetic appara- well investigated in lycophytes. To better understand the met- tus, at least partially, during the dehydration process (Farrant, abolic basis of DT at very low RWCs within the Selaginellaceae, 2000; Proctor and Tuba, 2002), whereas many eudicot species the metabolome of S. lepidophylla was compared over a do not (Georgieva et al., 2009). DT Selaginella species retain five-stage, rehydration/dehydration cycle using an unbiased, their chlorophyll (and presumably their photosynthetic struc- high-throughput metabolomics platform. This temporal study tures) during desiccation (Pandey et al., 2010). revealed that S. lepidophylla employs a combination of diverse Members of the Selaginellales are well known for their constitutive and inducible metabolic strategies to survive and use in traditional folk medicine. For example, S. lepidophylla recover from desiccation of vegetative tissues. has been used as a diuretic, and as a treatment for urinary and kidney infections, chronic gastritis, and gastric carci- RESULTS AND DISCUSSION noma (Ruiz-Bustos et al., 2009; Robles-Zepeda et al., 2011). Extracts of S. tamariscina (Ahn et al., 2006; Lee et al., 2011; Metabolomics approaches can provide a detailed Ha et al., 2012), S. doederleinii (Liu et al., 2011), and S. bry- understanding of an organism’s phenotype (Schauer and opteris (Mishra et al., 2011) have been used as anti-cancer Fernie, 2006; Cascante and Marin, 2008), and disposition therapeutics. Total flavonoids from S. tamariscina have also towards and response to environmental stresses (Sanchez been employed as an anti-diabetic treatment (Zheng et al., et al., 2008; Shulaev et al., 2008; Urano et al., 2010). Recent