The Plant Cell, Vol. 26: 3949–3963, October 2014, www.plantcell.org ã 2014 American Society of Plant Biologists. All rights reserved.

The Gentio-Oligosaccharide Gentiobiose Functions in the Modulation of Bud Dormancy in the Herbaceous Perennial Gentiana CW

Hideyuki Takahashi,a,1 Tomohiro Imamura,a Naotake Konno,a,2 Takumi Takeda,a Kohei Fujita,a Teruko Konishi,b Masahiro Nishihara,a and Hirofumi Uchimiyac a Iwate

Biotechnology Research Center, Kitakami, Iwate 024-0003, Japan of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, Okinawa 903-0213, Japan c Institute of Environmental Science and Technology, Saitama University, Sakura-Ku, Saitama City, Saitama 338-8570, Japan b Department

Bud dormancy is an adaptive strategy that perennials use to survive unfavorable conditions. Gentians (Gentiana), popular alpine flowers and ornamentals, produce overwintering buds (OWBs) that can persist through the winter, but the mechanisms regulating dormancy are currently unclear. In this study, we conducted targeted metabolome analysis to obtain clues about the metabolic mechanisms involved in regulating OWB dormancy. Multivariate analysis of metabolite profiles revealed metabolite patterns characteristic of dormant states. The concentrations of gentiobiose [b-D-Glcp-(1→6)-D-Glc] and gentianose [b-D-Glcp(1→6)-D-Glc-(1→2)-D-Fru] significantly varied depending on the stage of OWB dormancy, and the gentiobiose concentration increased prior to budbreak. Both activation of invertase and inactivation of b-glucosidase resulted in gentiobiose accumulation in ecodormant OWBs, suggesting that gentiobiose is seldom used as an energy source but is involved in signaling pathways. Furthermore, treatment with exogenous gentiobiose induced budbreak in OWBs cultured in vitro, with increased concentrations of sulfur-containing amino acids, GSH, and ascorbate (AsA), as well as increased expression levels of the corresponding genes. Inhibition of GSH synthesis suppressed gentiobiose-induced budbreak accompanied by decreases in GSH and AsA concentrations and redox status. These results indicate that gentiobiose, a rare disaccharide, acts as a signal for dormancy release of gentian OWBs through the AsA-GSH cycle.

INTRODUCTION Gentians (Gentiana, Gentianaceae) are herbaceous perennials that can survive several years of cycling between growth and dormancy. Prior to winter, gentians build protective structures called overwintering buds (OWBs), which are crown buds that grow from underground crown tissues, to enhance cold or freezing hardiness. After budbreak of OWBs in the spring, gentians grow vegetatively for several months and bloom from summer to autumn. The freezing tolerance of dormant OWBs has been ascribed to their consistent expression of high concentrations of stress-related proteins (Takahashi et al., 2006; Hikage et al., 2007). Late embryogenesis abundant proteins, including dehydrins, are well known to be associated with environmental stress tolerances such as freezing and drought (Close, 1997). The OWBs accumulate high levels of dehydrin transcripts, whose maximum expression level occurs during the dormant period

1 Address

correspondence to [email protected]. address: Department of Applied Biological Chemistry, Utsunomiya University, 350 Mine-machi, Utsunomiya, Tochigi 321-8505, Japan. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Hideyuki Takahashi ([email protected]). C Some figures in this article are displayed in color online but in black and white in the print edition. W Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.114.131631

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when freezing temperatures are likely to occur (Imamura et al., 2013). Furthermore, overexpression of dehydrins enhances freezing and drought tolerance in gentians (Imamura et al., 2013). Therefore, OWBs with insufficient levels of dormancy cannot survive the winter. On the basis of these findings, complete dormancy is critical to the survival of gentians, but how dormancy is regulated remains unclear at this stage. Dormancy is a period of arrested growth that is mainly due to temporal metabolic inactivity. In plants, seed and bud dormancy are well-known adaptive strategies that plants use to survive stressful seasonal environments. Seed dormancy has been widely studied through transcriptomic, proteomic, and metabolomic approaches, all demonstrating that both the induction and release of dormancy are regulated by environmental cues as well as endogenous signal molecules such as abscisic acid (ABA), gibberellic acids (GAs), and reactive oxygen species (ROS) (Finkelstein et al., 2008). In comparison to seed dormancy, little is known about bud dormancy in perennial plants. Bud dormancy is often related to plant development and growth and has been observed in floral, apical, axillary, crown, and root buds (Horvath et al., 2003). Bud dormancy can be further divided into three different states: paradormancy, growth inhibition regulated by distal organs; endodormancy, growth inhibition regulated by internal signals; and ecodormancy, growth cessation due to environmental conditions (Lang et al., 1987). The molecular mechanisms of dormancy induction, maintenance, and release in buds are becoming clearer. For example, FLOWERING LOCUS T (FT), a flowering regulatory gene, is

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related to dormant organ formation and the transition of dormancy states (Horvath, 2009). Böhlenius et al. (2006) proposed a model in which the expression level of FT determines whether a vegetative bud will grow or enter dormancy. The authors reported that transgenic poplar overexpressing Populus trichocarpa FT1 did not enter dormancy, whereas downregulation of FT1 resulted in growth cessation and bud set. These findings suggest that FT acts as an inhibitor of dormancy and an inducer of vegetative growth. Furthermore, Hsu et al. (2011) reported that two FTs regulate dormancy in vegetative buds of Populus deltoides. Since these FTs are regulated by different environmental factors, seasonal changes in environmental conditions may affect bud dormancy through these FT functions. We also identified three FT homologous genes (FT1, FT2, and TFL1) related to floral initiation, but their relationship with dormancy remains unknown (Imamura et al., 2011). Many reports have demonstrated that the transition or maintenance of dormancy states can be also affected by phytohormones (Horvath et al., 2003). ABA accumulates in the buds of many plant species when endodormancy starts (Rinne et al., 1994; Li et al., 2003). ABA also affects ethylene, which is suspected to play a role in the development of endodormancy in potato (Solanum tuberosum) tubers (Suttle, 1998). Furthermore, ABA levels decrease by catabolization prior to dormancy release (Suttle and Hultstrand, 1994), suggesting that ABA is necessary to maintain endodormancy and acts as a negative regulator of dormancy release. In contrast, GAs are maintained at a low level during endodormancy, but GA synthesis and accumulation are induced when buds begin to grow or the dormancy status shifts from endodormancy to ecodormancy (Eshel and Teper-Bamnolker, 2012). GA treatment leads to earlier budbreak in potato and Japanese apricot (Prunus mume) (Hartmann et al., 2011; Zhuang et al., 2013). Furthermore, since GAs could trigger budbreak even in ecodormant buds through GA-regulated genes or GA receptormediated signaling in Populus (Rinne et al., 2011), GAs are the key phytohormones inducing budbreak. Sugars also affect the cell cycle through GA signaling in adventitious buds of leafy spurge (Horvath et al., 2002). Moreover, exogenous sugars suppressed the growth of leafy spurge buds, and this suppression was released by GA treatment (Chao et al., 2006). Therefore, sugars are also regulators of dormancy in buds through crosstalk with phytohormones. Recently, transcriptome and proteome profiling of perennial buds revealed that several metabolic pathways not related to phytohormones were modulated during bud dormancy (Horvath et al., 2008; Victor et al., 2010; Bi et al., 2011), indicating the possibility that novel regulators of dormancy are also involved in dormancy regulation. In this study, a targeted metabolome analysis was conducted to identify candidate dormancy regulators of gentian OWBs. We found that the concentration of gentiobiose [b-D-Glcp-(1→6)-D-Glc] and gentianose [b-D-Glcp-(1→6)-D-Glc-(1→2)-D-Fru], two gentiooligosaccharides, significantly varied from one dormancy state to another. Gentiobiose and gentianose were first isolated from gentian roots in 1964 and 1882, respectively (Badenhuizen et al., 1964). Although these gentio-oligosaccharides may accumulate to high levels in some members of the Gentianaceae, such as Gentiana and Swertia (Lewis, 1984), the cellular and molecular biological functions of these compounds are not entirely understood.

Although some findings have suggested that gentiobiose may be related to maturation processes in seeds and fruits (Dumville and Fry, 2003; Verdier et al., 2013; Aizat et al., 2014), the mechanism of gentiobiose accumulation and the induction of maturation is still obscure. Here, we report that gentiobiose is essential for budbreak of gentian OWBs. Furthermore, our results suggest a mechanism for modulating dormancy through the use of gentiobiose as a signaling molecule. RESULTS Targeted Metabolome Profiles of Gentian OWB during Dormancy In the life cycle of gentians, OWBs are produced at the end of the flowering period and grow to ;2 cm before winter begins (Figure 1A). During the winter, which extends from October to March in Japan, OWBs are dormant under the snow and sprout when warm spring weather arrives in April (Figure 1B). Once sprouted, gentians grow vegetatively (Figure 1C) and bloom in the autumn (Figure 1D). Although dormant OWBs have a similar appearance from October to March (Figure 1E), intracellular metabolic changes must be occurring to regulate dormancy and its release. To investigate metabolic regulation during dormancy, we conducted a targeted metabolome analysis using gentian OWBs harvested at the early (October), middle (January), and late (March) stages of dormancy (Figure 2). We quantified 58 metabolites and analyzed the distribution of these metabolites by principal component analysis (PCA) (Figure 2A; Supplemental Table 1). Approximately 55% of the variance in metabolites is explained by the first two principal components, PC1 (34.2%) and PC2 (20.3%). The PCA plots were clearly able to discriminate between dormant stages, implying that some of the detected metabolites were correlated with dormancy. Because PC1 distinguished the middle stage from the early and late stages, we also investigated PC1 loading values to identify metabolites that had an influence on PC1 (Figure 2B). Sucrose and gentianose had high negative values. Conversely, most amino acids, except for phenylalanine and alanine, had high positive values. Glycolytic intermediates such as phosphoenolpyruvate, 3-phosphoglycerate, glucose 6-phosphate, and fructose 1,6-bisphosphate; energy metabolites such as ATP, NADPH, and UDP-glucose (UDPG); and reduced sugars such as gentiobiose, glucose, and arabinose also had high positive values. Furthermore, the early and late stages could be distinguished by PC2. Although the highest values of PC2 loadings were observed for spermidine on the positive side and aspartate on the negative side (Supplemental Table 1), these metabolites could not be classified to a metabolic pathway. Hierarchical cluster analysis (HCA) was conducted to determine whether metabolite profiles correlated with dormancy stages. The HCA grouped the metabolites into three major clusters (Figure 2C). Cluster I could be further divided into subcluster I, which included most of the amino acids and glycolytic intermediates such as 3-phosphoglycerate, phosphoenolpyruvate, and ribulose-1,5bisphosphate, and subcluster II, which included the high-energy metabolites ATP, GTP, UDPG, and NADPH. Subcluster II also included gentiobiose, glucose, aspartate, glutamate, arginine, and glycine. Cluster II included metabolites with lower concentrations in

Gentiobiose Induces Budbreak in Gentians

Figure 1. Life Cycle of Gentian (G. triflora). (A) to (D) Gentians can survive over 5 years by cycling OWB dormancy (A), budbreak (B), vegetative growth (C), and reproductive growth (D). (E) Morphological changes in OWBs from October to April. Bars = 2 cm. [See online article for color version of this figure.]

the middle stage of dormancy, including phosphate metabolites, tricarboxylic acid (TCA) cycle metabolites, shikimate, alanine, and g-aminobutyrate. Cluster III included metabolites that had higher concentrations in the middle stage of dormancy and mainly included low-energy metabolites, TCA cycle metabolites, and polyamine-related metabolites. The Gentio-Oligosaccharide-Related Pathway Fluctuates during OWB Dormancy Multivariate analysis suggested that energy metabolites cluster with glucose and gentiobiose. Interestingly, gentiobiose concentrations peaked during the early and late stages of dormancy but were low during the middle stage, almost inverse of the gentianose concentration trend. Therefore, we speculated that gentianose is hydrolyzed to gentiobiose, which in turn is hydrolyzed to glucose that can enter glycolysis to produce energy necessary for budbreak. Although there is almost no information about the synthetic pathways of gentio-oligosaccharides in plants, we determined that gentianose could be converted to gentiobiose and

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fructose by invertase, and gentianose could be converted to sucrose and glucose by b-glucosidase (Supplemental Figure 1). On the basis of these findings, we propose a synthetic pathway for gentio-oligosaccharides (Figure 3A). During dormancy, the concentrations of glucose, gentiobiose, UDPG, and glucose 6-phosphate generally followed the same trend, gradually decreasing from October to December, and then increasing in March or April (Figure 3B). The sucrose concentration remained fairly constant from October to January but continued to decrease until April. The gentianose concentration increased from October to November but significantly decreased in March. We also measured the seasonal expression of hexokinase (HXK1 and HXK2), sucrose phosphate synthase (SPS), sucrose synthase (SUS), phosphoglucomutase (PGM), UDPG pyrophosphorylase (UGP1 and UGP2), and invertase (INV) (Figure 3C). The expression levels of HXK1, SPS, and SUS significantly increased in January, but HXK2, PGM, UGP1, and UGP2 expression tended to increase from February to April, but with no readily discernible pattern. The expression of INV almost mirrored the glucose and gentiobiose concentrations, decreasing from October to November but then strongly increasing in March and April. We also investigated the concentrations of gentiobiose and gentianose and the expression level of INV in two other gentian varieties (Supplemental Figure 2). These varieties have different flowering periods; for example, G. triflora cv Maciry blooms in July, whereas G. triflora cv SpB blooms in September. Changes in the gentiobiose and gentianose concentrations and INV expression levels showed similar trends, although there were differences in their amounts. Therefore, the observed changes seemed to be a common trend among gentian OWBs during dormancy. Because the genes encoding gentiobiose-specific glucosidases could not be identified, we measured the total gentiobiose hydrolytic activities in OWBs (Figure 4). The glucosidase activity tended to decrease from October to April. We also measured total invertase activity using gentianose or sucrose as a substrate (Figure 4). Since INV, a recombinant protein produced in the yeast Pichia pastoris, had maximum hydrolytic activity for gentianose at pH 5.0 (Supplemental Figure 3), acid invertase activity was measured. Gentianose and sucrose hydrolytic activities had almost the same pattern: low from October to March but increasing by ;6-fold in April. INV expression and invertase activity almost mirrored the gentiobiose concentration, suggesting that the gentiobiose level was mainly modulated by invertase hydrolysis of gentianose. Since the green fluorescent protein (GFP) fluorescence of the fusion protein INV-sGFP was observed in vacuoles (Supplemental Figure 4), we propose that this modulation primarily occurred in vacuoles. Gentiobiose Accumulation and INV Expression Are Correlated with the Dormant State of Gentian OWBs We found that the levels of gentiobiose and INV transcripts increased prior to budbreak of OWBs, but there was no clear evidence that these phenomena are linked to budbreak induction. Therefore, we investigated changes in gentiobiose concentration and INV levels using endodormant and ecodormant OWBs (Figure 5). First, the dormant states of field-grown OWBs were checked

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Figure 2. Multivariate Analysis of Metabolites in Gentian OWBs during Dormancy. (A) PCA score plots of metabolites extracted from each of five biological replicates of OWBs (G. triflora 3 G. scabra line 05-543) harvested in October, January, and March, respectively. Scores are plotted with the first component (PC1) as the x axis and the second component (PC2) as the y axis. The proportion of the variance explained by each principal component is shown in parentheses beside each axis legend. (B) PC1 loading values of metabolites. (C) Metabolite profiling of gentian OWBs. Heat maps of metabolites extracted from each of five biological replicates of OWBs harvested in October, January, and March are compared. Hierarchical clustering of metabolites according to a dissimilarity scale is shown on the upper right. Metabolite concentrations in each OWB (five biological replicates) are normalized by a Z score transformation and represented by the Z scale. 3PGA, 3-phosphoglycerate; PEP, phosphoenolpyruvate; Arg-suc, arginosuccinate; RuBP, ribulose 1,5-bisphosphate; GABA, g-aminobutyrate; FBP, fructose 1,6-bisphosphate; 2OG, 2-oxoglutarate; G6P, glucose 6-phosphate; DHAP, dihydroxyacetone phosphate; Ru5P, ribulose 5-phosphate; R5P, ribose 5-phosphate; 6PG, 6-phosphogluconate.

(Figures 5A and 5B). Generally, when buds are incubated under optimum growth conditions, endodormant buds show no change, but ecodormant buds start budbreak. In this experiment, 3 weeks after incubation at 22°C, budbreak was determined based on the spreading of OWB tips. OWBs harvested in October and November did not show budbreak, despite a few slightly

elongated buds. In contrast, OWBs harvested in December and January showed budbreak (Figure 5B), suggesting that OWBs in October and November were in endodormancy, but buds in December and January were in ecodormancy. The timing of paradormancy in OWBs could not be determined in this study. To investigate the relationship between budbreak and gentiobiose,

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Figure 3. Seasonal Changes in Metabolite Concentrations and Transcript Abundance of Genes Probably Associated with Gentio-Oligosaccharides in Gentian OWBs.

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increases in the gentiobiose concentration and INV expression level in OWBs during incubation at 22°C were measured. Three weeks after incubation at 22°C, increased levels of gentiobiose and INV transcript were higher in OWBs harvested in December and January than those harvested in October and November (Figure 5C). The transition of dormancy in buds is regulated by the integrated amount of time exposed to chilling temperatures (Yamane et al., 2011). To obtain further evidence, we investigated OWBs exposed to several periods of constant chilling temperature (4°C) prior to transfer to 22°C. To avoid the effect of chilling temperatures in an agricultural field, OWBs collected in September were used. The appearance of OWBs was unchanged under the chilling temperature until 7 weeks (Figure 5D). Three weeks after transfer to 22°C, budbreak was observed in OWBs exposed to over 5 weeks of chilling temperature (Figure 5E), suggesting that the OWBs were in endodormancy during weeks 0 to 3 but transitioned into ecodormancy during weeks 5 to 7. The gentiobiose concentration significantly decreased after 5 weeks of incubation at 4°C, whereas the expression level of INV decreased after one week of 4°C treatment (Figure 5F). Three weeks after transfer to 22°C, gentiobiose concentrations and INV expression increased in OWBs, showing similar trends that were significantly higher in OWBs exposed to chilling for over 5 weeks than those chilled for 0 to 3 weeks (Figure 5G). Feeding Gentiobiose to in Vitro OWBs Induces Morphological and Metabolic Changes To obtain direct evidence supporting gentiobiose function in budbreak, a gentiobiose-feeding experiment using in vitro-cultured OWBs (IOWBs) was conducted. Since IOWBs had dormancy characteristics similar to field-grown OWBs (Imamura et al., 2014) and the sugar contents of IOWBs were easily controlled (Figure 6A; Supplemental Figure 5), IOWBs can be used for studying the effects of sugars on budbreak. IOWBs were cultured on solid sugar-free Murashige and Skoog (MS) medium or MS medium containing 1% (w/v) gentiobiose for 3 weeks. A comparison of the sugar concentrations revealed that gentiobiose was observed only in IOWBs cultured in the presence of gentiobiose, whereas the glucose concentrations were nearly similar (Figure 6A). Moreover, there were no morphological changes in any of the IOWBs cultured on sugar-free medium, but budbreak was frequently observed in IOWBs cultured on medium containing 1% (w/v) gentiobiose (Figures 6B and 6C).

To investigate how gentiobiose could induce budbreak, the metabolomes of sugar-free or gentiobiose treated IOWBs were compared. The metabolite profiles revealed that spermine and urea-cycle intermediates, including arginine, ornithine, and citrulline, were lower in IOWBs cultured in the presence of gentiobiose (Figure 7; Supplemental Table 2). In contrast, the concentrations of sulfur-containing amino acids such as methionine, cysteine, glutamyl-cysteine, homocysteine, and S-adenosylmethionine (SAM), as well as GSH and AsA were significantly higher in the IOWBs cultured in the presence of gentiobiose. GSH and AsA were the metabolites with the greatest increases in levels, with levels ;20.5- and 25.7-fold greater than those of the control, respectively (Supplemental Table 2). To obtain further insights into the mechanism, we isolated gentian homologs encoding enzymes related to these metabolites (Figure 8A). Sulfite reductase (EC 1.8.7.1) catalyzes the reduction of sulfite to sulfide, which is then used as a substrate by O-acetylserine (thiol) lyase (EC 2.5.1.47) to produce cysteine, the precursor of most sulfur-containing metabolites such as methionine and GSH. Methionine is synthesized from cysteine in three steps catalyzed by cystathionine g-synthase (EC 2.5.1.48), cystathionine b-lyase (EC 4.4.1.8), and methionine synthase (MeS; EC 2.1.1.14). SAM synthetase (SAMS; EC 2.5.1.6) converts methionine to SAM and SAM decarboxylase (SAMDC; EC 4.1.1.50) decarboxylates SAM. After the donation of a methyl group, SAM becomes S-adenosylhomocysteine (SAH), which is immediately converted to homocysteine and adenosine by SAH hydrolase (SAHH; EC 3.3.1.1). Homocysteine can be converted back to methionine, completing the methyl cycle (Rajjou et al., 2012). SAM is also converted to 1-aminocyclopropane-1carboxylic acid (ACC), and then ethylene is synthesized from ACC, which is mediated by ACC oxidase (ACO; EC 1.14.17.4) (Bleecker and Kende, 2000). GSH is synthesized from cysteine in two reactions catalyzed by g-glutamylcysteine synthetase (g-ECS; EC 6.3.2.2) and GSH synthase (EC 6.3.2.3). The expression levels of all genes, with the exception of SAMDC and ACO, were 2 to 20 times higher in IOWBs cultured with gentiobiose than those cultured on sugar-free media (Figure 8B). These results indicate that in vitro gentiobiose treatment enhanced sulfur assimilation and sulfur-containing amino acid synthesis. Furthermore, gentiobiose treatment increased the expression levels of gentian homologs of dehydroascorbate (DHA) reductase (DHAR; EC 1.8.5.1) and GSH reductase (GR; EC 1.8.1.7), which regenerate AsA and GSH from their oxidized forms, respectively. To investigate the effect of GSH and AsA on

Figure 3. (continued). (A) Predicted metabolic pathways of gentio-oligosaccharides. (B) Comparison of the concentrations of metabolites predicted to be related to the gentio-oligosaccharide metabolic pathway during dormancy in OWBs (G. triflora 3 G. scabra line 05-543). The concentration of glucose (Glc), sucrose (Suc), gentiobiose (G2), gentianose (G2F), UDPG, and glucose 6-phosphate (G6P) in OWBs harvested from October to April were measured. Each value represents the mean 6 SD calculated from five independent experiments. (C) Transcript levels of HXK, PGM, UGP, SPS, SUS, and INV in OWB harvested from October to April. Transcript levels were estimated by quantitative RT-PCR and are expressed relative values normalized against transcription levels of G. triflora Ubiquitin (UBQ). Each value represents the mean 6 SD calculated from five independent experiments. G1P, glucose-1-phosphate; GLU, glucosidase; HXK, hexokinase; PGM, phosphoglucomutase; UGP, UDPG pyrophosphorylase; SPS, sucrose phosphate synthase; SUS, sucrose synthase.

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similar to those in IOWBs treated with only gentiobiose (Figures 9A and 9B).

DISCUSSION Amino Acid and Carbohydrate Metabolism, Altered during Dormancy Transitions, Are Associated with Stress Survival and Energy Production

Figure 4. Gentio-Oligosaccharide Hydrolytic Activities in OWBs Harvested from October to April. Gentiobiose (G2)-, gentianose (G2F)-, and sucrose (Suc)-hydrolytic activity in OWBs (G. triflora 3 G. scabra line 05-543) were measured. Activities were calculated from the amount of glucose or gentiobiose produced during the reaction. Each value represents the mean 6 SD calculated from three independent experiments. Different letters indicate a significant difference at P < 0.05 according to Tukey’s test.

budbreak, IOWBs were cultured on a solid MS medium containing 1% (w/v) gentiobiose in the presence of 0, 200, or 500 mM buthionine sulfoximine (BSO), a potent inhibitor of g-ECS, the rate-limiting enzyme of GSH synthesis (Senda and Ogawa, 2004). BSO treatment suppressed the gentiobiose-increased GSH and AsA concentrations as well as the GSH:GSSG and AsA:DHA ratios in a dose-dependent manner (Figure 9A). Furthermore, BSO suppressed budbreak partially (200 mM) or completely (500 mM), whereas gentiobiose concentrations were

In this study, we conducted metabolome analyses to obtain clues about the mechanism of dormancy in gentian OWB. Fifty-eight metabolites were measured, and multivariate analysis correlated the individual concentrations of a number of metabolites with the endodormancy (October), early stage of ecodormancy (January), and late stage of eco dormancy (March) states. This analysis indicated which of the metabolites might play a role in the maintenance and regulation of the dormancy state or in the transition from one dormancy state to the next. We found that most amino acids accumulated in late ecodormant OWBs. Because the protein concentration also increased in OWBs in the transition from endodormancy (3.5 6 0.3 mg g21 dry weight [DW]) to ecodormancy (6.3 6 0.3 mg g21 DW), part of the amino acid pool might be used for the synthesis of proteins essential for budbreak or subsequent vegetative growth. Only phenylalanine displayed a different pattern, namely, phenylalanine accumulated in early ecodormant OWBs. Two previous reports have shown that phenylpropanoids synthesized from phenylalanine accumulate in peach (Prunus persica) buds exposed to prolonged cold treatment and that the accumulation improved cold and freezing tolerances (Kaneda et al., 2008; Leida et al., 2012). On the basis of these reports, phenylalanine accumulation observed in gentian OWBs might be related to a survival strategy during the winter. We also found that the concentrations of sugars as well as glycolytic intermediates fluctuated in the transition between dormant states; accumulation of abundant sugars such as sucrose, glucose, gentiobiose, and gentianose seemed to differ between the endodormancy, early ecodormancy, and late eco dormancy stages. Among these sugars, a high concentration of sucrose was observed in OWBs. This observation is consistent with sugar accumulation in the crown buds of leafy spurge (Anderson et al., 2005). Sucrose is believed to serve as a protectant against cold and freezing stresses in many plants (Tabaei-Aghdaei et al., 2003; Rekarte-Cowie et al., 2008). Furthermore, the accumulation of raffinose, a nonreducing oligosaccharide containing fructose, improves drought and cold stress tolerance in Arabidopsis (Taji et al., 2002). Gentianose also accumulated when OWBs were exposed to drought and freezing stress. Consequently, the sucrose and gentianose accumulation observed in OWBs might contribute to stress tolerance. Furthermore, sucrose is converted to glucose and fructose by invertase. The sucrose concentration decreased in late ecodormant OWBs concomitantly with the increases in INV expression and acidic invertase activity. Glucose is well known as an energy source in most organisms. Glycolysis coverts one molecule of glucose into two molecules of pyruvate and then pyruvate enters the TCA cycle where NADH is produced for respiration. In gentian OWBs, the high-energy metabolites ATP

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Figure 5. Effect of the Dormant State of OWBs on Gentiobiose (G2) Concentration and INV Expression Level. (A) and (B) Field-grown OWBs (G. triflora cv SpB) harvested from October to January. OWBs were incubated at 22°C for 0 week (A) or 3 weeks (B). Bars = 2 cm. (C) Increased values of G2 concentration and INV expression level 3 weeks after incubation at 22°C. (D) and (E) Field-grown OWBs harvested in September and exposed to chilling (4°C) for 0 to 7 weeks. Each OWB was then incubated at 22°C for 0 week (D) or 3 weeks (E). Arrowheads indicate sprouted OWBs. Bars = 2 cm. (F) G2 concentration and INV expression level in OWBs shown in (D). (G) Increased values of G2 concentration and INV expression level in OWBs shown in (E). Values represent the mean 6 SD calculated from five independent experiments. Different letters indicate a significant difference at P < 0.05 according to Tukey’s test. [See online article for color version of this figure.]

and GTP were maintained at low levels during endodormancy to early ecodormancy but increased in late ecodormancy, suggesting that energy production was limited during the winter season and was enhanced prior to budbreak. Our results also showed that the levels of most intermediates of glycolysis and the pentose phosphate pathway increased in late ecodormant OWBs, whereas the levels of pyruvate and TCA cycle intermediates did not increase in late ecodormant OWBs. A previous study reported that transcript levels of pyruvate dehydrogenase, the key enzyme connecting glycolysis and the TCA cycle, increased prior to budbreak (Ophir et al., 2009). Since transcript levels of the gentian homolog of pyruvate dehydrogenase also increased in the late ecodormant OWB (Supplemental Figure 6), metabolic flow from glycolysis to the TCA cycle might increase in late ecodormancy in spite of the fact that there was no change in metabolite accumulation. These observations support the proposal that energy

synthesis via glycolysis, the TCA cycle, and the pentose phosphate pathway is enhanced for budbreak (Gai et al., 2013). Conversion of Gentianose to Gentiobiose Induces Budbreak of Gentian OWBs Here, we discovered that the concentrations of gentiobiose and gentianose varied during the transition of dormancy states in gentian OWBs. Although their cellular function is currently unknown, the observation that gentiobiose treatment of tomato stimulates fruit ripening (Dumville and Fry, 2003) implies that gentiobiose has a regulatory function in plant development. In gentian OWBs, the gentiobiose level was maintained at a low level during endodormancy and early ecodormancy but increased substantially in late ecodormancy (Figure 3). Our results showed that invertase converted gentianose into gentiobiose

Gentiobiose Induces Budbreak in Gentians

Figure 6. Effect of Exogenous Gentiobiose on IOWB Sprouting. (A) Glucose (Glc) and gentiobiose (G2) concentrations in IOWBs (G. triflora cv Ihatovo) 3 weeks after culturing on MS medium with sugar-free (SF) or 1% (w/v) G2. Values represent the mean 6 SD calculated from five independent experiments. Significant differences are shown (**P < 0.01; Student’s t test). (B) Photograph of IOWBs cultured on sugar-free or 1% (w/v) G2 medium for 3 weeks. Bars = 1 cm. (C) Ratio of budbreak of IOWBs cultured on sugar-free or 1% (w/v) G2 medium for 3 weeks. Numbers shown in parentheses represent the percentage. [See online article for color version of this figure.]

(Supplemental Figure 1). The gentiobiose concentration changed in parallel with invertase activity and INV expression, suggesting that the gentiobiose level was largely regulated by invertase; however, the sum of the gentiobiose and gentianose concentrations was not constant and slightly increased toward budbreak (Supplemental Figure 6). Therefore, not only invertase, but also other unknown synthetic pathways may contribute negligibly to the gentiobiose level. Gentiobiose concentrations also roughly paralleled the concentrations of glucose and the high-energy metabolites. Thus, we hypothesize that the metabolic flow from gentianose to glucose is accelerated in late ecodormancy to produce energy for budbreak. However, gentiobiose hydrolytic activity did not increase in late ecodormant OWBs (Figure 4). These results suggest that gentiobiose is not an energy source but that there is some other reason for the accumulation of gentiobiose prior to budbreak. Generally, the dormant state is an essential factor in determining whether budbreak occurs or not, and only ecodormant

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buds possess the ability to break dormancy upon return to growth-conducive conditions. The transition from endodormancy to ecodormancy requires being exposed to a prolonged chilling temperature, and gentian OWBs needed ;5 weeks of the 4°C chilling temperature for the transition (Figure 5). The significant increase in gentiobiose concentration was observed only in sprouted ecodormant OWBs, suggesting that gentiobiose accumulation consistently accompanies budbreak of OWBs. Interestingly, the gentiobiose concentration in OWBs exposed to constant chilling temperature significantly decreased 5 weeks after chilling and was maintained at a low level, whereas the expression level of INV decreased after 1 week. This discrepancy was also observed in field-grown OWBs (Figure 3). On the other hand, the increase in gentiobiose content after transfer to the growth condition was almost consistent with that of INV expression in both field-grown and constantly chilled OWBs. These observations suggest that the gentiobiose level is regulated by some INV-independent pathways during endodormancy that switches to INV-dependent machinery after the transition to ecodormancy. Furthermore, our results also showed that the induced levels of gentiobiose accumulation and the budbreak phenotype were apparently lower in chilling-treated OWBs than in field-grown OWBs. These findings signified that the degree of budbreak was parallel with the gentiobiose concentration. There are two possible reasons for the difference between chilling-treated and field-grown OWBs: (1) gradual or fluctuating decreases in temperature were necessary for the strong induction, or (2) OWBs in September accumulated low concentrations of gentianose (26.6 6 7.2 nmol mg21 DW). Our results showed that gentiobiose accumulation was due to hydrolysis of gentianose mediated by invertase. Therefore, the gentianose content may have been insufficient to induce budbreak in the OWBs, although further studies are necessary to confirm this hypothesis. Gentiobiose-Induced Budbreak Is Modulated through Sulfur Metabolism and the Redox Status of GSH and AsA Although a close relationship was observed between gentiobiose and budbreak, it was not clear whether gentiobiose accumulated in connection with the budbreak process or was an essential factor promoting budbreak; however, the results of a gentiobiose-feeding experiment using IOWBs were consistent with a functional role for gentiobiose. In vitro gentiobiose treatment frequently induced budbreak, whereas all IOWBs cultured on sugar-free media showed no morphological change. This result strongly indicates that gentiobiose is necessary for budbreak of gentian IOWBs. Simultaneously, gentiobiose increased transcript and metabolite levels associated with sulfur assimilation, sulfur-containing amino acid synthesis, and glutathione synthesis. These increases were also observed when fieldgrown OWBs began to sprout (Figure 2; Supplemental Figure 7). Although the levels of most amino acids increased prior to budbreak in field-grown OWBs, gentiobiose treatment had almost no effect on other metabolites except for sulfur-containing metabolites. Therefore, the accumulation of non-sulfur-containing amino acids might be induced in a gentiobiose-independent manner. Among the increased sulfur-containing amino acids,

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Figure 7. Mapping of Measured Metabolites into Metabolic Pathways. The concentration of metabolites in IOWBs cultured for 3 weeks on sugar-free or 1% (w/v) gentiobiose-containing MS medium were compared (n = 5; Supplemental Table 2). The ratio of metabolite concentrations between sugar-free and 1% gentiobiose are shown as different colors. The P value was calculated by Student’s t test and is represented by the size of circle for each metabolite.

SAM is involved in many important plant processes. SAM is a precursor of polyamines and ethylene, both reported to regulate bud dormancy (Kaur-Sawhney et al., 1982; Suttle, 1998). Furthermore, ACO generates ethylene from ACC with the

release of HCN, which is known to regulate seed dormancy (Bleecker and Kende, 2000). However, the spermine and ACC concentrations as well as the SAMDC and ACO expression levels did not increase in response to gentiobiose treatment

Figure 8. Sulfur-Containing Amino Acid Synthesis and AsA-GSH Cycle-Related Gene Expression. (A) Metabolic pathway including sulfur assimilation, sulfur-containing amino acid synthesis, and the AsA-GSH cycle. Bold characters indicate metabolites and bold italic characters indicate enzymes. (B) The effect of gentiobiose treatment on the expression levels of enzyme genes shown in (A) in IOWBs. IOWBs were cultured on sugar-free or 1% (w/v) gentiobiose-containing MS medium for 3 weeks, and gene expression levels were compared. Each value represents the mean 6 SD calculated from five independent experiments. Significant differences are shown (*P < 0.05 and **P < 0.01; Student’s t test).

Gentiobiose Induces Budbreak in Gentians

Figure 9. Effect of BSO on the Redox Status and Concentration of GSH and AsA and Budbreak of IOWBs. (A) Effect of BSO on the concentration of GSH, AsA, and gentiobiose (G2), and the ratio of GSH/GSSG and AsA/DHA in IOWBs. IOWBs were cultured on sugar-free MS medium or MS medium containing 1% (w/v) G2 in the presence of 0, 200, or 500 µM BSO. Each value represents the mean 6 SD calculated from five independent experiments. Different letters indicate a significant difference at P < 0.05 according to Tukey’s test. Bars = 2 cm. (B) Effect of BSO on budbreak of IOWBs used for metabolite analysis shown in (A). [See online article for color version of this figure.]

(Figures 7 and 8). We also found that endodormant OWBs treated with ACC or ethephon, which is converted to ethylene, did not sprout (Supplemental Figure 8). These results suggested that polyamines and ethylene were not involved in gentiobioseinduced budbreak. As another possible reason for a relationship between SAM and budbreak, SAM serves as a methyl donor in many methyltransferase reactions (Teperino et al., 2010). In the methyl cycle, SAM donates its methyl group to a methyl acceptor, thereby becoming SAH, and then SAH is hydrolyzed by SAHH. Since SAH is a potent inhibitor of SAM-dependent methyltransferase and decreases in SAHH activity induce global DNA demethylation, SAHH is a key enzyme in the methyl cycle and in plant methylation reactions (Rocha et al., 2005). Our results showed that the expression levels of MeS, SAMS, and SAHH increased during budbreak in gentian OWBs as well as in IOWBs treated with gentiobiose, indicating that the methyl cycle was probably activated prior to budbreak to enhance methylation reactions. This hypothesis is supported by previous reports that the expression levels of methyl cycle-related genes and several methyltransferases increased when buds sprouted (Bi et al., 2011; Liu et al., 2012).

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Furthermore, gentiobiose treatment strongly induced the accumulation of GSH and AsA in IOWBs. Since the rate-limiting factors for GSH synthesis, such as cysteine concentration and transcript abundance of ECS, increased in gentiobiose-treated IOWBs, we concluded that de novo GSH synthesis was upregulated by gentiobiose; however, it is unclear how AsA accumulation was induced. In plants, GSH seems to play multiple roles throughout the life cycle, in growth, in response to pathogens, and in cell death (Ogawa, 2005). GSH also functions in the AsA-GSH cycle as an electron donor reducing DHA to AsA by DHAR and then producing GSSG, which is subsequently rereduced by GSH reductase (Foyer and Noctor, 2011). Therefore, activation of the AsA-GSH cycle results in an increase in the AsA concentration and redox status of GSH and AsA (GSH: GSSG and AsA:DHA). Because DHAR is the only enzyme directly coupling AsA and GSH, we hypothesized that DHAR was also affected by gentiobiose. Indeed, increased expression of DHAR and GR was observed in gentiobiose-treated IOWBs, suggesting that electron donation from GSH to AsA in the AsAGSH cycle might be enhanced. Importantly, changes in the concentration and redox state of GSH and AsA after treatment with BSO, an inhibitor of GSH synthesis, clearly showed that the increased concentration and redox status of AsA were mainly attributed to GSH acting as a reducing agent. Since BSO completely repressed gentiobiose-induced budbreak, either the increased concentrations or the redox state of GSH and AsA were necessary for budbreak of gentians. We also found that the concentration of total AsA (AsA + DHA) increased in gentiobiose-treated IOWBs. This finding is in accordance with previously reported results in which overexpression of wheat DHAR in tobacco (Nicotiana tabacum) resulted in significant increases in the total concentrations of GSH and AsA as well as their redox states (Chen et al., 2003). This finding implies that activation of DHAR also enhances GSH and AsA synthesis, resulting in greater increases in the GSH:GSSG and AsA:DHA ratios. Alternatively, different sources of electrons for DHA reduction could also be involved, as previously proposed (Potters et al., 2004).

Figure 10. Schematic Model of Gentiobiose-Induced Budbreak of Gentian OWBs. G2F, gentianose; G2, gentiobiose; AsA, ascorbate; MTs, methyltransferases.

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Although we demonstrated here that the gentiobiose-induced budbreak of IOWB occurred through the synthesis of sulfurcontaining amino acids, GSH, and AsA, the induction mechanisms for these syntheses and for budbreak by GSH and AsA are still unknown; however, in the latter case, some mechanisms can be hypothesized. Several studies have shown that ROS are regulatory factors for budbreak, and levels of GSH and AsA as well as gene expression of the related antioxidant enzymes increase during seasonal or hydrogen cyanamide-induced budbreak probably to control ROS levels (Halaly et al., 2008; Walton et al., 2009; Gai et al., 2013). Furthermore, Considine and Foyer (2014) proposed that cellular redox signaling through GSH and AsA controls cell division through modulation of cell cycle progression from the G1 phase to the S phase. In many plant species, cells in dormant buds are arrested at primarily the G1 phase, and progression from the G1 phase to the S phase is promoted during budbreak in many plant species (Devitt and Stafstrom, 1995; Campbell et al., 1996; Horvath et al., 2002). Therefore, it is possible that high redox statuses of GSH and AsA are required for cell cycle progression leading to budbreak. Based on these findings, GSH and AsA act as multifunctional agents leading to dormancy release. Similar functions might be operating in gentian; this possibility should be examined in the future. Furthermore, the increased concentrations of sulfur-related metabolites observed in gentian OWBs during budbreak are characteristics common to the break of seed dormancy (Rajjou et al., 2012). However, since gentiobiose did not affect germination or sulfur metabolite concentrations in Arabidopsis thaliana seeds (Supplemental Figure 9), gentiobiose seems not to be involved in the acceleration of seed dormancy release, at least in Arabidopsis. Previous reports showed that gentiobiose accumulation was observed in maturing seeds of Medicago (Verdier et al., 2013) as well as in ripening fruits of tomato (Solanum lycopersicum) and Capsicum (Dumville and Fry, 2003; Aizat et al., 2014). More directly, Dumville and Fry (2003) revealed that fruit ripening was accelerated by gentiobiose infiltration, suggesting the possibility that gentiobiose hastens maturation processes in seeds and fruits, probably through ethylene-related pathways as the authors proposed. On the other hand, our results indicated that ethylene biosynthesis and the signaling pathway were not responsible for budbreak in IOWBs induced by gentiobiose (Figure 8). A role of gentiobiose in gentian OWBs is modulated through sulfur metabolism as mentioned above, but the sensing and the downstream signaling mechanisms are largely unknown. Although there are several lines of evidence that sugars act as signaling molecules in plants (Smeekens et al., 2010; Rabot et al., 2012), the distribution and behavior of gentiobiose is less characterized in most plant species. Therefore, more detailed studies of sugars, including gentiobiose, as signaling molecules are necessary to gain insight into the functions regulating various growth and developmental processes. In summary, we propose that gentiobiose and gentianose, two gentio-oligosaccharides that accumulate abundantly in gentian, are associated with dormancy regulation of OWBs (Figure 10). Our results revealed that budbreak of OWBs occurred consistently with gentiobiose accumulation derived from enhanced hydrolysis of gentianose by INV and that gentiobiose

was seldom used as an energy source. Since gentiobiose treatment induced budbreak in vitro and the induction was suppressed by depletion of GSH and AsA, we concluded that gentiobiose serves as a signal to break dormancy mainly through GSH- and AsA-utilizing systems. Gentiobiose treatment also activated the methyl cycle, implying that a methyltransferase-related reaction was also involved. Sugar phosphates, monosaccharides, and sucrose are thought to be important regulators of plant growth and development. Our results highlight the possibility that gentiooligosaccharides also function as a signal to modulate plant metabolism, and they provide further insight into sugar-mediated signaling. METHODS Plant Materials Three-year-old Gentiana triflora 3 Gentiana scabra (05-543) hybrid plants were used for chronologic measurements. Two-year-old G. triflora cv SpB plants were used to evaluate the effects of the dormant states. These OWBs were harvested from plants grown in an agricultural field at the Iwate Biotechnology Research Center from September 2009 to April 2010 or September 2010 to April 2011, frozen in liquid nitrogen, and lyophilized. G. triflora cv Ihatovo was used for in vitro culture, and IOWBs were induced by the method described by Imamura et al. (2014). Metabolite Analysis Tissue samples were pulverized in a Micro Smash M100 (TOMY) and homogenized with ice-cold 50% (v/v) methanol containing 50 mM 1,4piperazineethanesulfonate and methionine sulfone as internal standards for quantification with a capillary electrophoresis-quadrupole time-offlight mass spectrometry (CE-Q-TOFMS) apparatus (Agilent Technologies). Homogenates were then centrifuged at 20,000g for 5 min, the supernatant was filtered through a 3-kD-cutoff filter (Millipore), and the filtrates were used for analysis. Nucleotides and ascorbate were quantified by CE-Q-TOFMS according to the method of Takahashi et al. (2009). To measure DHA, 8 mL of the filtrate was added to 2 mL of 50 mM Tris-HCl (pH 7.0) containing 50 mM DTT and incubated at room temperature for 30 min to convert DHA to AsA, and the total AsA concentration was quantified by CE-Q-TOFMS. The DHA concentration was calculated as the difference between the total AsA concentration and the AsA concentration. Sugars were measured as described by Takahashi et al. (2010). Sucrose and gentianose were quantified with a Dionex Bio-LC system and CarboPac PA1 anion-exchange column (Dionex), and glucose and gentiobiose were quantified by liquid chromatography (LC)-QTOFMS (Agilent Technologies) with a ZORBAX Eclipse XDB-C18 column (150 3 4.6 mm; Agilent Technologies) after derivatization with 1-phenyl-3methyl-5-pyrazolone. Other metabolites were quantified by LC-Q-TOFMS with a ZORBAX Eclipse plus-C18 column (250 3 4.6 mm; Agilent Technologies). The mobile phases consisting of 0.1% (v/v) formic acid and 0.1% (v/v) formic acid in acetonitrile (85:15, v/v) or 10 mM ammonium acetate-acetonitrile (95:5, v/v) were used to separate cationic or anionic metabolites, respectively, at a flow rate of 0.2 mL/min. Sugars were also analyzed by thin-layer chromatography (TLC) on a silica gel 60 F254 plate (Merck) using ethyl acetate-acetic acid-water (3:2:1) as the mobile phase and stained thymol-sufuric acid reagent. Accuracy was verified with known concentrations of reference standard compounds. Metabolome data were normalized by a Z score transformation before processing by PCA and HCA as described previously (Takahashi et al., 2012). All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS v.10.0).

Gentiobiose Induces Budbreak in Gentians

Molecular Cloning of Genes Encoding Gentio-OligosaccharideRelated Enzymes Total RNA was extracted from leaves of G. triflora with an RNAs-ici!-P kit (Rizo) and incubated with RNase-free DNase I (Wako) to eliminate genomic DNA. First-strand cDNA was synthesized from 500 ng of total RNA using an RNA PCR kit (AMV) version 3.0 (Takara) with oligo(dT) primer according to the manufacturer’s instructions. Full-length open reading frame sequences were obtained from our G. triflora EST library obtained by the method of Nakatsuka et al. (2013) and were isolated by PCR. Primers are shown in Supplemental Table 3. The cDNAs were cloned into the pCR4 TOPO vector (Invitrogen) and sequenced with the ABI PRISM Big Dye Terminator Cycle Sequencing kit using an ABI 3100 DNA sequencer (Applied Biosystems).

Quantitative RT-PCR Analysis Quantification of gene transcript levels by quantitative RT-PCR analysis was conducted according to the method of Imamura et al. (2014). For synthesizing first-strand cDNA, 500 ng of total RNA was reverse transcribed using a High Capacity cDNA reverse transcription kit (Applied Biosystems) with random primers according to the manufacturer’s instructions. Quantitative PCR was performed using StepOnePlus kit (Applied Biosystems) with a Kapa SYBR Fast ABI Prism qPCR kit (Kapa Biosystems). Gt-UBQ was used as an internal control in each experiment. Primers used for quantitative PCR are shown in Supplemental Table 3. Enzyme Activities Associated with Gentio-Oligosaccharides For enzymatic analyses, 20 mg of freeze-dried OWB was homogenized in 1 mL of extraction buffer (50 mM HEPES, pH 7.5, 1 mM EDTA, 20% glycerol, and 1 mM DTT). After centrifugation at 20,000g for 10 min, the supernatant was washed by at least a 1:100,000 dilution with extraction buffer using a 3-kD-cutoff filter (Millipore) to remove mono- and polysaccharides and was subsequently used as crude extract for further enzymatic analyses. Gentiobiose-hydrolytic activity was measured in a reaction mixture containing 50 mM sodium phosphate (pH 7.0), 3 mM gentiobiose, and 200 mg of total protein from the crude extract. After incubation at 30°C for 1 h, hydrolyzed glucose was measured by LC-TOFMS (Takahashi et al., 2010). To measure gentianose- and sucrose-hydrolytic activity, a reaction mixture containing 50 mM sodium citrate (pH 5.0), 10 mM gentianose or 10 mM sucrose, and 200 mg of total protein from the crude extract was incubated at 37°C for 1 h. Hydrolyzed gentiobiose and glucose were measured as described by Takahashi et al. (2010). Expression and Purification of Recombinant INV Protein An EasySelect Pichia Expression Kit (Invitrogen) was used for transformation of recombinant INV. The transformant was cultivated in growth medium and then in induction medium as described by Konno et al. (2009). Recombinant INV protein was purified with a HiPrep phenyl column (1.6 3 10 cm; GE Healthcare) and a DEAE- TOYOPEARL PAK 650S anion exchange column (0.83 7.5 cm; Tosoh Co.) as described previously (Konno and Sakamoto, 2011) and analyzed by SDS-PAGE on 12% polyacrylamide gels. Purified INV protein was detected either with Coomassie Brilliant Blue (Nacalai) or by protein gel blotting using the antiMyc antibody (1:1000; Invitrogen) followed by peroxidase-conjugated anti-mouse secondary antibody (1:50,000; Life Technologies). Subcellular Localization Open reading frames of INV and At-GRP5 were cloned into the pGWB5 vector containing the cauliflower mosaic virus 35S promoter and the sGFP gene (Nakagawa et al., 2007). Leaves of 3- to 5-week-old Nicotiana benthamiana plants were coinfiltrated with Agrobacterium tumefaciens

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(GV3101) containing the pGWB5 constructs and a viral silencing suppressor gene (Voinnet et al., 2003). The subcellular localization of the GFP fusion protein was examined by confocal microscopy (FLOVIEW FV1000; Olympus) 3 d after incubation under constitutive dark conditions. Gentiobiose-Feeding Experiment To assess budbreak using IOWBs, we used solid MS medium containing 0.2% (w/v) gellan gum. After chilling for 7 weeks, bud tips of IOWBs were cut into ;2-cm segments, placed on solid MS medium containing 1% (w/v) gentiobiose, and cultured for 3 weeks at 22°C with a 16/8-h light/dark photoperiod at a light intensity of 40 mmol m22 s21. Control bud tips were handled similarly except gentiobiose was omitted from the medium. To assess the uptake of gentiobiose, IOWBs were cultured for 24 h on solid MS medium containing 0.5% 2-aminopyridine-tagged gentiobiose (G2-AP), which synthesized and purified according to the method of Hase et al. (1978). After metabolite extraction, G2-AP was separated by TLC and the fluorescence of G2-AP was detected by a LAS-4000 image analyzer (Fujifilm). Arabidopsis thaliana seeds (Columbia-0) were surface sterilized with 10% sodium hypochlorite, placed on sugar-free or 1% gentiobiose-containing MS agar medium, and incubated at 4°C for 4 d before transferring to a growth chamber at 22°C under continuous light (60 mmol m22 s21). Seeds were harvested at each time point after the transfer, washed with distilled water five times, and freeze-dried. Accession Numbers Sequence data from this article can be found in the GenBank/EMBL/ DDBJ databases under the following accession numbers: INV (AB916692), HXK (AB916693), PGM (AB916694), UGP (AB916695), SPS (AB916696), SUS (AB916697), SiR (AB916698), OASTL1 (AB916699), OASTL2 (AB916700), OASTL3 (AB916701), ECS (AB916702), GSHS (AB916703), CBL (AB916704), CGS (AB916705), MeS1 (AB916706), MeS2 (AB916707), SAMS1 (AB916708), SAMS2 (AB916709), SAMDC (AB916710), ACO (AB980799), SAHH1 (AB916711), SAHH2 (AB916712), DHAR1 (AB916713), DHAR2 (AB916714), GR1 (AB916715), GR2 (AB916716), and PDH (LC003013). Supplemental Data The following materials are available in the online version of this article. Supplemental Figure 1. TLC Analysis of Sugars Hydrolyzed from Gentianose. Supplemental Figure 2. Variety Comparison of Changes in GentioOligosaccharide Concentrations and INV Expression in OWBs. Supplemental Figure 3. Characterization of Recombinant INV Protein. Supplemental Figure 4. Subcellular Localization of INV-sGFP. Supplemental Figure 5. Uptake of G2-AP into IOWBs. Supplemental Figure 6 Changes in PDH Expression and the Sum Concentrations of Gentiobiose and Gentianose in Field-Grown OWBs. Supplemental Figure 7. Changes in Gentiobiose-Affected Gene Expression in Field-Grown OWBs before and during Budbreak. Supplemental Figure 8. Effect of Treatment of Ethylene Precursors on Budbreak of Gentian OWBs. Supplemental Figure 9. Effect of Gentiobiose Treatment on Germination of Arabidopsis Seeds. Supplemental Table 1. Metabolite Profiles of OWBs [G. triflora 3 G. scabra (05-543)] Harvested in October, January, and March. Supplemental Table 2. Metabolite Concentrations in IOWBs (G. triflora cv Ihatovo) Cultured on Sugar-Free or 1% (w/v) Gentiobiose-Containing MS Medium.

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Supplemental Table 3. List of Primers Used in This Study. Supplemental References.

ACKNOWLEDGMENTS This research was supported by a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science, and Technology (23780029 and 25850023 to H.T.; 24780030 to T.I.). We thank Kazumichi Fujiwara and Takashi Nakazato for providing helpful advice and Atsumi Higuchi for technical assistance.

AUTHOR CONTRIBUTIONS H.T., T.T., M.N., and H.U. designed the research. H.T., I.T., N.K., T.T., K.F., and T.K. performed the research. H.T. analyzed the data. H.T. and M.N. wrote the article.

Received September 4, 2014; revised September 4, 2014; accepted September 30, 2014; published October 17, 2014.

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Gentiobiose Induces Budbreak in Gentians

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The Gentio-Oligosaccharide Gentiobiose Functions in the Modulation of Bud Dormancy in the Herbaceous Perennial Gentiana Hideyuki Takahashi, Tomohiro Imamura, Naotake Konno, Takumi Takeda, Kohei Fujita, Teruko Konishi, Masahiro Nishihara and Hirofumi Uchimiya Plant Cell 2014;26;3949-3963; originally published online October 17, 2014; DOI 10.1105/tpc.114.131631 This information is current as of January 9, 2015 Supplemental Data

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