Accumulation of trehalose increases soluble sugar contents in rice ...

Plant Biotechnol Rep (2012) 6:89–96 DOI 10.1007/s11816-011-0210-3

ORIGINAL ARTICLE

Accumulation of trehalose increases soluble sugar contents in rice plants conferring tolerance to drought and salt stress Mark C. F. R. Redillas • Su-Hyun Park • Jang Wook Lee Youn Shic Kim • Jin Seo Jeong • Harin Jung • Seung Woon Bang • Tae-Ryong Hahn • Ju-Kon Kim

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Received: 8 December 2011 / Accepted: 13 December 2011 / Published online: 30 December 2011 Ó Korean Society for Plant Biotechnology and Springer 2011

Abstract Trehalose is a nonreducing sugar composed of two glucose units linked in an a,a-1,1-glycosidic linkage. Present in a wide variety of organisms, this sugar may serve as a source of energy and carbon and as a protective molecule against abiotic stresses. In this study, trehaloseproducing transgenic rice plants (Oryza sativa) expressing a bifunctional fusion enzyme TPSP (Ubi1:TPSP) were utilized to dissect the enigmatic role of trehalose in conferring stress tolerance to plants. Grown under normal M. C. F. R. Redillas and S.-H. Park contributed equally to this work. M. C. F. R. Redillas S.-H. Park Y. S. Kim J. S. Jeong H. Jung S. W. Bang J.-K. Kim (&) School of Biotechnology and Environmental Engineering, Myongji University, Yongin 449-728, Korea e-mail: [email protected] M. C. F. R. Redillas e-mail: [email protected] S.-H. Park e-mail: [email protected] Y. S. Kim e-mail: [email protected] J. S. Jeong e-mail: [email protected]

conditions, the Ubi1:TPSP plants produced high amounts of soluble sugars (glucose, fructose and sucrose), ranging from 1.5- to 3.5-fold over NT controls. In the time course of drought treatment, the transcripts for the drought degradable-marker genes (RbcS, FBPase, and PBZ1) persisted for two more days in Ubi1:TPSP plants before being completely degraded relative to those in NT plants, confirming the tolerance of the transgenic plants to drought. This was further supported by a delayed increase in transcript levels of the stress-inducible genes SalT, Dip1, and Wsi18 during drought stress. Similarly, Ubi1:TPSP plants showed tolerance to salt levels of up to 150 mM NaCl, as evidenced by the seedling growth and the delayed decay in RbcS and delayed increase in SalT transcript levels. The growth of NT plants was found to be slightly affected by exogenous trehalose feeding, whereas Ubi:TPSP plants remained resistant, validating the protective role of internally produced trehalose. These results suggest that the elevated production of trehalose in rice, through TPSP overexpression, increases the soluble sugar contents and enhances tolerance to both drought and salt stress. Keywords TPSP Trehalose Transgenic rice Drought stress Salt stress

H. Jung e-mail: [email protected] S. W. Bang e-mail: [email protected] J. W. Lee T.-R. Hahn Plant Metabolism Research Center, Graduate School of Biotechnology, Kyung-Hee University, Suwon 449-701, Korea e-mail: [email protected] T.-R. Hahn e-mail: [email protected]

Introduction Abiotic stress is a complex environmental constraint that limits crop production. Bioengineering pathways involved in stress signaling in order to produce stress-tolerant crops is one of the major goals of agricultural research (Penna 2003). Osmotic adjustment is an effective example of such manipulations, since the accumulation of osmoprotectants or compatible solutes is a common response observed in

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plant systems. Other mechanisms by which compatible solutes protect plants from stress include detoxifying radical oxygen species and stabilizing the quaternary structures of proteins to maintain their function (Penna 2003). Trehalose, [a-D-glucopyranosyl-(1?1)-a-D-glucopyranose], has been shown to efficiently stabilize dehydrated enzymes, proteins, and lipid membranes, as well as protect biological structures from damage during desiccation. Trehalose, is a nonreducing disaccharide that is found in various organisms, including bacteria, algae, fungi, yeast (Saccharomyces cerevisiae), insects, and some plants (Jang et al. 2003). In the plant kingdom, most species do not seem to accumulate detectable amounts of trehalose, with the notable exception of the highly desiccation-tolerant resurrection plants (Wingler 2002). The discovery of homologous genes for trehalose biosynthesis in Selaginella lepidophylla, Arabidopsis thaliana, and several crop plants suggests that the ability to synthesize trehalose may be widely distributed in the plant kingdom (Goddijn and van Dun 1999). This disaccharide is formed from UDP-glucose and glucose-6-phosphate, and catalyzed by the enzyme trehalose-6-phosphate synthase (TPS), producing the intermediate trehalose-6-phosphate (T-6-P). Subsequently, T-6-P is dephosphorylated into trehalose by the enzyme trehalose-6-phosphate phosphatase (TPP). Transgenic plants that express the TPS and/or TPP genes from microorganisms not only exhibit an increase in drought tolerance; they also show strong developmental alterations. Many of the transgenic plants reported are on dicot plants, which generally produce very low levels of trehalose with growth stunting (Goddijn et al. 1997; Holmstrom et al. 1996; Pilon-Smits et al. 1998; Romero et al. 1997). Interestingly, rice plants appear to be more tolerant to trehalose than dicot plants, since the exogenous application of trehalose produced no growth inhibition or visible changes in the appearance of rice (Garcia et al. 1997). A gene that encodes a bifuntional fusion enzyme TPSP, produced from the TPS and TPP in Escherichia coli, was introduced into rice (Ubi1:TPSP), producing trehalose levels of up to 0.1% of the fresh weight without any visible growth inhibition, which was coupled with increased tolerance to drought, salt, and cold stresses (Garg et al. 2002; Jang et al. 2003). In this study, we showed that trehalose accumulation, due to either internal overproduction or acquisition from an exogenous trehalose supply, was sufficient to provide the plants with tolerance to drought and salt stress.

Materials and methods Plant materials Transgenic and nontransgenic rice (Oryza sativa cv. Nakdong) plants were grown in a greenhouse for 2–4 weeks

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following five days of germination. Rice seeds were germinated in Murashige–Skoog (MS) solid medium and incubated in a growth chamber (3 dark days/2–3 light days at 28°C). They were then transplanted to soil and grown in a greenhouse (16 h light/8 h dark at 28–30°C). Each plant was grown in a pot (4 9 4 9 5 cm3; six seedlings per pot) filled with rice nursery soil (Bio-media, Kyoungju, Korea) for the indicated number days after germination. The T4 homozygous generations were used for analysis. Stress treatments For drought stress, 4-week-old nontransgenic and Ubi1:TPSP seedlings that were grown in soil were drought treated with water deprivation, while two-week-old plants were air-dried in the greenhouse under continuous light of approximately 900–1,000 lmol m-2 s-1, as described previously (Redillas et al. 2011a, b). For the salt treatments, sterilized seeds were germinated and incubated for five days in MS solid medium containing 0, 50, 100, 150, 200, and 400 mM NaCl, respectively. The growth conditions of light and temperature were the same as stated in ‘‘Plant materials.’’ Prior to each stress treatment, the plants were grown in a greenhouse under continuous light of approximately 900–1,000 lmol m-2 s-1 in a pot. To prepare the total RNA of salt-treated plants, two-week-old seedlings were transferred to a 150 mM NaCl solution in the greenhouse under identical light conditions before the leaves were sampled in liquid nitrogen. Plants prepared for drought through air-drying and salt treatments were first placed in tap water for three days for environmental adaptation. Untreated control seedlings were grown either in a greenhouse or a growth chamber under identical light conditions and harvested at time zero. Following each experimental procedure, leaf tissues were rapidly harvested into liquid nitrogen and stored at -80°C until use. RNA gel-blot analysis As described previously (Jung et al. 2011), total RNA was extracted from the leaves of transgenic and nontransgenic rice plants using Tri ReagentÒ (Molecular Research Center, Inc., Cincinnati, OH, USA). Ten micrograms of total RNA were electrophoresed on a 1.2% (w/v) agarose gel containing iodoacetamide and blotted onto a Hybond N? nylon membrane (Amersham, Little Chalfont, UK). Prepared membranes were hybridized with the gene-specific probes for the TPSP, RbcS, FBPase, PBZ1, SalT, Dip1, and Wsi18 genes. Probe DNAs were labeled with [a-32P] dCTP using a random primer labeling kit (Takara, Kyoto, Japan) according to the manufacturer’s instructions. After hybridization, membranes were washed in order with 29 SSC (0.3 M NaCl, 50 mM sodium citrate, pH 7.0),


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with 0.1% (w/v) SDS solution, 19 SSC with 0.1% (w/v) SDS solution, and then 0.59 SSC with 0.1% (w/v) SDS solution at 65°C for 15 min each. Membranes were then exposed to film on an intensifying plate and analyzed using a phosphoimage analyzer (FLA 3000, Fuji, Tokyo, Japan). Sugar feeding treatments To test trehalose accumulation in NT plants, 2-week-old seedlings were treated with 25 mM trehalose through the roots, and the leaves were collected from the plants treated for 1, 2, and 5 days with trehalose. For the sugar treatments during germination, sterilized seeds were germinated in MS solid medium containing either sorbitol, trehalose, or sucrose (100 mM each). The growth conditions (light and temperature) were the same as described in ‘‘Plant materials.’’ Metabolite measurements The starch and soluble sugar contents of the rice leaves were assayed according to the enzymatic method described by Stitt et al. (1989). Leaves (0.1–0.2 g) were ground with liquid nitrogen to a fine powder and then extracted with 10% perchloric acid. Insoluble solids in neutralized perchloric acid extracts were separated by centrifugation at 20,0009g for 5 min. The sediment was used to determine starch contents, and the aqueous phase for soluble sugars and metabolic intermediates.

Results Previously, we reported that transgenic rice plants overexpressing the bifunctional fusion gene TPSP under the maize ubiquitin promoter (Ubi1:TPSP) are drought tolerant, mainly due to trehalose accumulation (Garg et al. 2002; Jang et al. 2003). Figure 1a shows a schematic of the fusion gene TPSP constructed by connecting the TPS and TPP genes from E. coli after removing the stop codon of the TPS gene (Seo et al. 2000). The high catalytic efficiency of the bifunctional enzyme, resulting from the simultaneous catalysis of the two-step synthesis, probably reduced the accumulation of potentially deleterious T-6-P (Jang et al. 2003). To verify the drought tolerance of the Ubi1:TPSP plants, the transgenic and nontransgenic (NT) plants were subjected to drought stress, and the expression levels of TPSP and stress-responsive marker genes were probed. The latter includes the drought-degradable (RbcS, FBPase and PBZ1) and the drought-inducible (SalT, Dip1, and Wsi18) marker genes. The RNA gel blot analysis confirmed that the expression of TPSP was present only in the

Fig. 1 The Ubi1:TPSP construct and the tolerance of Ubi1:TPSP transgenic plants to dehydration. a Ubi1:TPSP consists of the maize (Zea mays) ubiquitin1 (Ubi1) promoter linked to the TPSP coding region, the 30 region of the potato proteinase inhibitor II gene (30 PinII), and a bar expression cassette containing the 35S promoter of the cauliflower mosaic virus (P 35S), the bar coding region, and the 30 region of the nopaline synthase gene (30 Nos). The TPSP coding region was made by in-frame fusion of the E. coli otsA and otsB genes, which encode trehalose-6-phosphate (T-6-P) synthase and T-6-P phosphatase, respectively (Seo et al. 2000; Jang et al. 2003). b RNA gel blot hybridization analysis using RNAs from leaves of four-weekold nontransgenic (NT) and Ubi1:TPSP transgenic plants that were stressed with drought for periods from zero to five days. Total RNA samples (10 lg each) were extracted and fractionated on a 1.2% denaturing agarose gel. RNA gel blot hybridizations were performed using the probes described in the ‘‘Materials and methods’’ section. The marker genes RbcS (ribulose-1,5-bisphosphate carboxylase/ oxygenase small subunit, AK121444), FBPase (fructose 1,6-bisphosphatase, AK062233), and PBZ1 (probenazole-inducible protein 1, AK071613) served as drought-degradable marker genes, while SalT (AK062520; Claes et al. 1990), Dip1 (dehydration stress-inducible protein 1, AY587109), and Wsi18 (water stress-inducible protein 18, AK064074) are drought-inducible marker genes. Ethidium bromide (EtBr) stained rRNAs served as a loading control

transgenic plants (Fig. 1b). In NT plants, the RbcS, FBPase, and PBZ1 transcripts were completely degraded by three days of drought stress, whereas complete degradation in Ubi1:TPSP plants occurred at a later time. In the Ubi1:TPSP plants, the transcripts of the marker genes were correlated with the expression of TPSP transcripts indicating the drought tolerance acquired following TPSP overexpression. This notion was further supported by the transcript levels of the stress-inducible genes during drought stress. Although the SalT, Dip1, and Wsi18 transcripts were induced by three days of drought stress in both NT and Ubi1:TPSP plants, the latter showed much lower

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transcript levels compared to those in NT. The high levels of expression of the stress-inducible markers coincided with the time when the drought-degradable markers disappeared, reflecting the effect of drought stress on these stress-responsive genes. Collectively, these results showed that the drought-degradable and -inducible markers in Ubi1:TPSP plants were less affected by drought, confirming drought tolerance of the Ubi1:TPSP plants. Plants were also exposed to increasing salt concentrations of up to 400 mM NaCl. Figure 2a shows that when the plants were exposed to 50 mM NaCl, the Ubi1:TPSP plants showed an increase in root length, while NT plants remained relatively similar to those with 0 mM NaCl, suggesting a preference of transgenic plants for slightly elevated osmolarity. Supplied with 100–200 mM NaCl, the negative effects on growth was more pronounced in NT than in the Ubi1:TPSP plants. However, both plants suffered growth inhibition with 400 mM NaCl. Transcript levels of RbcS and SalT were observed by performing RNA gel blot analysis on the leaves of plants exposed to 150 mM NaCl—the concentration at which the two plants

showed obvious contrasts in growth (Fig. 2b). During the time course of salt treatment, the Ubi1:TPSP plants showed lower levels of SalT transcripts, while the RbcS transcripts were reduced more slowly compared to NT plants, suggesting a tolerance of the Ubi1:TPSP plants to high salinity. Collectively, these findings suggest that TPSP overexpression increased the tolerance of plants to salt as well as to drought. To determine the effects on NT plants of feeding on trehalose, 25 mM trehalose was supplied through the roots of two-week-old NT plants (NT-Tre). Figure 3a shows that, over time, accumulation of trehalose in the leaves occurred, as indicated by the arrows in the chromatogram. Since NT plants do not accumulate a significant amount of trehalose naturally, the accumulation of this molecule in the leaves clearly suggests that trehalose was transported to the shoots during exogenous feeding. To test whether the increased trehalose content affects the tolerance of plants to drought, NT-Tre plants, NT andUbi1:TPSP plants were airdried for up to 6 h. Results showed that the levels of expression of RbcS and Dip1 transcripts in NT-Tre plants

Fig. 2 Tolerance of Ubi1:TPSP transgenic plants to high salinity. a Nontransgenic (NT) and Ubi1:TPSP rice seeds were germinated and grown in the dark for three days followed by 2 days with light at 28°C on Murashige and Skoog (MS) agar medium supplemented with different concentrations of NaCl (0–400 mM). Photographs were taken at the indicated NaCl concentration. b RNA gel blot hybridization analysis was performed on the leaves using RNAs from the leaves of 2-week-old nontransgenic (NT) and Ubi1:TPSP transgenic

plants that were stressed with 150 mM NaCl for 0–24 h. Total RNA samples (10 lg each) were extracted and fractionated on a 1.2% denaturing agarose gel. RNA gel blot hybridizations were performed using the probes described in the ‘‘Materials and methods’’ section. RbcS (ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, AK121444) and SalT (AK062520) served as a stress marker gene. Ethidium bromide (EtBr) stained rRNA served as a loading control

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Fig. 3 Accumulation of trehalose in NT plants from an exogenous supply leads to drought stress tolerance, similar to Ubi1:TPSP plants. a Chromatogram for the carbohydrates extracted from the leaves of 2-week-old NT plants that underwent 25 mM trehalose feeding for 5 days. Arrows indicate the peak for trehalose at 2.7 min. The left axis shows the range from -200 to 1,000 mV. b RNA gel blot hybridization analysis using RNAs from leaves of two-weekold NT plants, Ubi1:TPSP transgenic plants, and nontransgenic plants fed with 25 mM trehalose for 5 days (NT-Tre) that were air-dried for 0–6 h. Total RNA samples (10 lg each) were extracted and fractionated on a 1.2% denaturing agarose gel. RNA gel blot hybridizations were performed using the probes described in the ‘‘Materials and methods’’ section. RbcS and Dip1 served as stress marker genes. Ethidium bromide (EtBr) stained rRNA served as a loading control

tend to mimic those in Ubi1:TPSP plants (Fig. 3b). In NTTre plants, the RbcS transcripts were degraded more slowly and the levels of Dip1 transcripts were lower as compared to NT plants. Similarly, the Ubi1:TPSP plants showed high RbcS and lower Dip1 transcript levels throughout the 6 h drought stress period. These indicate that trehalose, either accumulated through exogenous feeding or endogenously overproduced, contributed to the tolerance of plants to drought. To examine if there were any changes in the sugar content of the leaves of Ubi1:TPSP plants, carbohydrates were measured on the leaves of two-month-old transgenic plants grown under normal conditions (Fig. 4a). Following the overexpression of TPSP, the Ubi1:TPSP leaves accumulated around 3.5-fold higher glucose and fructose contents over NT leaves. In addition, starch and sucrose also

increased by 1.5-fold in the transgenic leaves, while the phosphorylated metabolic intermediates triose-P and hexose-P did not differ between NT and Ubi1:TPSP leaves. Collectively, these results suggest that the overexpression of TPSP makes the unstressed transgenic plants behave similarly to drought-exposed nontransgenic plants by increasing their soluble sugar contents. To investigate whether exogenous feeding of sugars affects plant growth, NT and Ubi1:TPSP plants were fed with either 100 mM sorbitol, trehalose, or sucrose for five days. When the plants were fed with sorbitol, an osmotically active sugar alcohol naturally produced in plants, no significant difference between NT and Ubi1:TPSP plants was observed. On the other hand, the trehalose-fed NT plants suffered a slight reduction in height, while the Ubi1:TPSP plants remained normal. This

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Fig. 4 Sugar accumulation in Ubi1:TPSP transgenic plants, and growth of plants in response to sugar feeding. a Determination of soluble sugar and starch contents in the nontransgenic and the Ubi1:TPSP transgenic plants in 2-month-old leaves. The results are

given as mean ± SE (n = 5 separate plants). b Effects of trehalose and sucrose on the growth of rice seedlings. Seedlings were incubated in the absence (no sugar) or presence of 100 mM sorbitol, trehalose, and sucrose, respectively

suggests that the effect of the overproduction of trehalose through Ubi1:TPSP masked the negative effects of exogenous trehalose. When the plants were fed with 100 mM sucrose, a concentration similar to the endogenous concentration of sucrose in the Ubi1:TPSP plants, the transgenic plants showed a higher growth rate than the NT plants (Fig. 4b). The fact that the Ubi1:TPSP plants grew faster than NT when exogenous sucrose was supplied reflects that exogenous sucrose has an additive effect on the growth of transgenic plants, probably due to the elevated internal sucrose in the Ubi1:TPSP plants (Fig. 4a).

membranes of plants when exposed to stress by replacing hydrogen bonding through polar residues, preventing protein denaturation and fusion of membranes (Iturriaga et al. 2009). However, trehalose production in dicot plants has resulted in morphological growth defects or altered metabolism (Yeo et al. 2000; Cortina and Culiań˜ez-Macia` 2005; Miranda et al. 2007; Sua´rez et al. 2009). In our previous study, transgenic rice expressing the bifunctional enzyme TPSP derived from an in-frame fusion of TPS and TPP from E. coli resulted in the accumulation of trehalose and the tolerance of plants to abiotic stress without exerting any negative effects on plant growth (Jang et al. 2003). These effects were brought about by the increased catalytic efficiency of the TPSP fusion enzyme, which might have reduced the accumulation of potentially deleterious T-6-P while producing trehalose. In this study, the drought tolerance of Ubi1:TPSP plants was further explored, along with the changes in the

Discussion Trehalose can serve as a carbohydrate storage molecule as well as a transport sugar, similar to the function of sucrose (Mu¨ller et al. 1999). It can also stabilize proteins and

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expression levels of the stress-responsive marker genes. The stress-degradable and -inducible markers used were RbcS, FBPase, and PBZ1, and SalT, Dip1, and Wsi18, respectively. The RNA gel blot analysis showed that transcript levels of the degradable marker genes in Ubi1:TPSP plants were less affected by drought compared to those in NT plants. Also, the expression levels of stressinducible marker genes were weaker in the transgenic plants compared to the NT plants, together indicating that transgenic plants were more tolerant to drought than NT plants. The same was true for the salt tolerance of the Ubi1:TPSP plants. We also observed that trehalose concentrations in the leaves of NT plants were increased by exogenous feeding (NT-Tre). Under drought stress, the changes in the expression level of drought-degradable RbcS transcripts in NT-Tre plants tend to mimic those in Ubi1:TPSP plants (Fig. 3b), supporting the claim that the accumulation of trehalose enhances the drought tolerance of plants. Furthermore, since the RbcS gene codes for the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, one of the key enzymes in photosynthesis, this indicates that the lower rate of RbcS transcript reduction suggests that the accumulated trehalose mitigates the negative effects of drought on the photosynthetic components of plants. This is further supported by the higher soluble sugar contents found in the leaves of unstressed Ubi1:TPSP1 plants (Fig. 4a) compared to the NT plants. Indeed, Garg et al. (2002) reported that the increased trehalose accumulation in rice correlates with higher soluble carbohydrate levels and an elevated capacity for photosynthesis under both stressed and unstressed conditions. Among these soluble sugars, sucrose plays a central role in the allocation of assimilates from source to sink organs, in addition to its function as a molecule used for storage and protection (Avigad and Dey 1997; Mu¨ller et al. 1999). The notion that sugar signaling plays major roles in responses to environmental cues is, however, still being constantly updated and elucidated (Hanson and Smeekens 2009: Rolland et al. 2006). Over the years, the role of trehalose in the acquisition of plant tolerance to stress has been linked to sugar signaling rather than an action as an osmoprotectant alone, since the concentration is too low to provide such a function (Iturriaga et al. 2009). The intermediate T-6-P has also been reported to be involved in the signaling, through sugar-mediated responses and carbohydrate allocation and metabolism (Kolbe et al. 2005). T-6-P was found to regulate the utilization of sugars for storage starch synthesis by promoting the reductive activation of AGPase in the plastid (Kolbe et al. 2005). Recently, T-6-P has been found to function as an inhibitor of SnRK1 (SNF-related kinase), a downregulator of genes involved in biosynthetic reactions (Zhang et al. 2009) that affects the transcript abundances of approximately 1,000 genes in Arabidopsis

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which play a central role in the response to starvation (Baena-Gonzalez et al. 2007). The specific function of trehalose or T-6-P is still far from understood, due to its complex involvement in the responses of plants to environmental cues. In this study, the prevention of T-6-P accumulation through high trehalose accumulation resulted in enhanced tolerance of plants to drought and salt stresses. The Km value of the bifunctional TPSP fusion enzyme for the sequential overall reaction from UDP-glucose and glucose-6-phosphate to trehalose was lower than that of an equimolar mixture of TPS and TPP, but they had similar Kcat values. This suggests that the ability to catalyze UDP-glucose and glucose-6-phosphate for T-6-P production was greater for TPSP than for TPS/ TPP (Seo et al. 2000). This might have resulted in high levels of transient T-6-P intermediates that were catalyzed later, producing a higher trehalose content with no T-6-P accumulation. The transient increase in T-6-P concentration may have played a role in the plant’s response to drought, salt, and sugar feeding, resulting in changes in carbohydrate allocation and metabolism and thus enhancing the response of the plants to stress. Collectively, the production of large amounts of osmoprotective trehalose without T-6-P accumulation plays an important role in sugar signaling, allocation, and metabolism in a manner that ultimately favors the tolerance of plants to drought and salt stress. Acknowledgments This study was supported by the Rural Development Administration under the Cooperative Research Program for Agriculture Science & Technology Development (project no. PJ906910), the Next-Generation BioGreen 21 Program (project no. PJ007971 to J.-K.K.), and by the Ministry of Education, Science and Technology under the Mid-career Researcher Program (project no. 20100026168 to J.-K.K.).

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