Hexokinase-mediated sugar signaling controls expression of the ...

Journal of Plant Physiology 169 (2012) 1551–1558

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Hexokinase-mediated sugar signaling controls expression of the calcineurin B-like interacting protein kinase 15 gene and is perturbed by oxidative phosphorylation inhibition Hui-kyeong Yim a , Mi-na Lim a , Sung-eun Lee a , Jun Lim a , Yew Lee b , Yong-sic Hwang a,∗ a b

Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea Division of Biological Science and Technology, College of Science and Technology, Yonsei University, Wonju 220-710, Republic of Korea

a r t i c l e

i n f o

Article history: Received 16 April 2012 Received in revised form 14 June 2012 Accepted 15 June 2012 Keywords: Anoxia CBL-interacting protein kinase Hexokinase Oxidative phosphorylation Sugar signaling

a b s t r a c t Calcineurin B-like (CBL) interacting protein kinase 15 (CIPK15) is a newly identified positive regulator which is critical to directing the O2 deficiency signal to the sugar signaling cascade as part of Amy3D (representative Amy3 gene) regulation in rice. It is located upstream and probably contributes to reserve mobilization under anoxia. In isolated starving embryos, the temporal pattern of accumulation of CIPK15 transcripts and leaky suppression of this gene suggests that factors other than CIPK15 may also be involved in the regulation of Amy3D expression. Probing of a variety of sugars and sugar analogs has shown that hexokinase mediates the sugar regulation of CIPK15. For example, hexokinase substrates, such as mannose, 2-deoxyglucose, and other metabolizable sugars, repressed CIPK15 expression, whereas 3-O-methylglucose and 6-deoxyglucose did not. By using glucosamine, a hexokinase inhibitor, to release glucose-dependent CIPK15 suppression, we confirmed that hexokinase mediates regulation of this gene. Chemical inhibitors of mitochondrial electron transfer, proton separation or ATP synthase also effectively abolished sugar-induced repression of CIPK15. This type of interference, the release from glucose-induced repression of gene expression by inhibition of oxidative phosphorylation, was previously identified for the Amy3D gene, which suggests that hexokinase-mediated sugar signaling may be coordinated with the cellular energy status. Analysis of a transgenic rice cell line harboring the GUS reporter gene under the control of the CIPK15 promoter, and transient expression assay for 3 UTR of the CIPK15 gene indicate that sugar regulation of the rice CIPK15 gene is likely mediated by 2548-bp 5 -flanking region, with no additional post-transcriptional control. © 2012 Elsevier GmbH. All rights reserved.

Introduction Rice (Oryza sativa) is unique in its ability to anaerobically mobilize starchy endosperm, which supports the energy metabolism of the non-photosynthetic embryonic axis during anaerobic germination and post-germinative growth (Guglielminetti et al., 1995a,b; Perata et al., 1997). However, for the embryonic axis of the submerged rice seed to overcome the low energetic efficiency of fermentation, it is critical that anaerobic mobilization yields sufficient quantities of sugars. Other cereals such as barley and wheat are incapable of anaerobically breaking down the endosperm

Abbreviations: CBL, calcineurin B-like; CIPK, CBL-interacting protein kinase; DCCD, N,N -dicyclohexylcarbodimide; DNP, 2,4-dinitrophenol; GUS, ␤-glucuronidase. ∗ Corresponding author at: Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea. Tel.: +82 2 2049 6085; fax: +82 2 444 6176. E-mail address: [email protected] (Y.-s. Hwang). 0176-1617/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2012.06.003

reserve and germinating under submergence. Germination and post-germination growth under anaerobic conditions are likely to reflect the integration of many physiological and biochemical processes. However, exogenous supplementation with glucose or sucrose supports the anaerobic germination of these cereals to such an extent that anaerobic reserve mobilization is implied to play an essential role in the growth of anoxia-tolerant cereals such as rice (Perata et al., 1992). Among various hydrolytic enzymes, ␣-amylase plays the most critical role in the mobilization of the endosperm reserve. The ␣-amylase enzyme initiates the breakdown of intact starch granules, releasing glucose polymers in the form of amylose and amylopectin, which are then digested into soluble sugars by other hydrolases. Rice contains unique ␣-amylase genes that belong to the Amy3 subfamily, whose expression is induced by starvation and repressed by sugars; this explains the peak expression pattern in rice scutellum tissue during aerobic rice seed germination. Interestingly, the expression of this ␣-amylase gene is enhanced and sustained under anoxia; its expected anaerobic contribution to total ␣-amylase activity suggests that it plays a critical role in anoxic reserve mobilization (Hwang et al., 1999).

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H.-k. Yim et al. / Journal of Plant Physiology 169 (2012) 1551–1558

Intensive studies on Amy3D sugar regulation have revealed a sugar responsive cis-element (SRE), MybS1 (a transcriptional activator binding to the SRE), and sucrose non-fermenting 1-related protein kinase (SnRK1A; a positive regulator for MybS1) (Hwang et al., 1998; Lu et al., 1998, 2002, 2007). Recent molecular and genetic screening analyses have also revealed that the knock-out mutant for CIPK15 cannot mobilize the starchy endosperm and germinate under submerged conditions because it cannot produce ␣-amylase anaerobically. Thus, the CIPK15 protein has been proposed as a key component of the sensing cascade necessary for successful rice germination under flood conditions by positively regulating SnRK1A and MYBS1, which control ␣-amylase abundance under conditions of O2 deprivation (Lee et al., 2009). As ubiquitous internal secondary messengers, calcium ions mediate diverse cellular responses to various stimuli in plants and regulate a wide range of physiological processes. The “Ca2+ signature,” a combination of spatial and temporal changes in cellular Ca2+ concentrations produced in response to a particular signal, can be decoded by an array of Ca2+ sensors (Boudsocq and Sheen, 2010). Calcineurin B-like (CBL) proteins are a new family of Ca2+ sensors similar to both the regulatory ␤-subunit of calcineurin and to the neuronal Ca2+ sensor (NCS) in animals (Liu and Zhu, 1998; Kudla et al., 1999). CBLs specifically target a novel family of protein kinases, CIPKs, to relay the Ca2+ signal at the molecular level (Luan, 2009). Ca2+ has been proposed as an endogenous messenger of low O2 signaling in plants because the cytosolic Ca2+ concentration shows a transient increase in response to flooding in maize roots or to anoxic or hypoxic conditions in Arabidopsis (Subbaiah et al., 1994; Sedbrook et al., 1996). In addition, glycolytic enzymes and alcohol dehydrogenases in maize and Arabidopsis are activated by Ca2+ signaling under low O2 conditions (Subbaiah et al., 1994; Chung and Ferl, 1999). Therefore, in plants, CIPK15 may participate in the propagation of Ca2+ signaling for O2 deficiency through the CBL-CIPK network. Previous studies have shown that sugar regulation of Amy3D is under the control of hexokinase (Umemura et al., 1998) and dependent on SnRK1 activity (Lu et al., 2007). Previously, we demonstrated that inhibition of oxidative phosphorylation, which perturbs cellular energy status, interferes with hexokinase (HXK)mediated sugar regulation of Amy3D expression. In our current study, sugar regulation of CIPK15 expression is also mediated by hexokinase, and is interfered by the disturbance of mitochondrial ATP synthesis. These findings imply that HXK-mediated sugar signaling may be influenced by cellular energy status, which is presumably sensed by the SnRK1-dependent pathway. Materials and methods Plant materials and treatment of rice embryos Manually dissected rice embryos from whole rice seed (Oryza sativa L. cv. Dongjin) were surface sterilized as described in Hwang et al. (2005). Approximately 150–200 surface-sterilized embryos were placed on a single layer of 3 MM Whatman paper soaked with 10 mM calcium chloride (CaCl2 ), containing specific compounds at the indicated concentrations, in a growth chamber at 28 ◦ C. Glucosamine, 2,4-dinitrophenol (DNP), oligomycin A, N,N -dicyclohexylcarbodiimide (DCCD), and dicoumarol (3,3 -methylene-bis [4-hydroxycoumarin]) were purchased from Sigma Aldrich (St. Louis, MO, USA). Rice suspension cell cultures Suspension-cultured rice cells (O. sativa L. cv. Dongjin) were established as described by Huang et al. (1993). These cells were

maintained in AA2 culture medium (Thompson et al., 1986) and subcultured every 10 d by transferring a 3–5 mL packed volume of the cells to 20 mL of fresh AA2 medium and actively shaking at 150 rpm and 28 ◦ C in the dark. RNA gel blot analysis Total RNA isolation, preparation of [␣-32 P]-labeled DNA probes, and analysis of the RNA gel blot were performed as described by Park et al. (2010). A probe specific for the CIPK15 gene was prepared by PCR using a primer set (Table 1). The membrane was exposed overnight to a Fuji imaging plate, and detected using a PhosphorImager system (Fujifilm FLA-7000 imaging system; Fujifilm, Tokyo, Japan). Quantitative real-time RT-PCR First-strand cDNA synthesis and real-time quantitative reverse transcription-PCR (qRT-PCR) were carried out as described by Park et al. (2010). The accumulation of fluorescent PCR products was monitored using a Thermal Cycler Dice Real Time System (Takara Shuzo, Kyoto, Japan). All procedures were performed according to the manufacturer’s instructions. The relative amplification of the rice actin gene was used as an internal control to normalize all data. Triplicates of each sample were examined to evaluate the quantitative variation in each sample, and each experiment was repeated at least 2 times. The gene-specific primers used for quantitative PCR are listed in Table 1. When we compared the results of the RNA gel blot analysis (Fig. 2A and C) with the results of real-time qPCR (Fig. 2B and D), both produced very similar results, which encouraged us to examine CIPK15 transcript levels via real-time qPCR analysis. Plasmid construction To establish a transgenic cell line that contained the CIPK15 promoter::␤-glucuronidase (GUS) construct, the 5 -flanking region of the CIPK15 gene (2548 bp) was PCR-amplified from rice genomic DNA (O. sativa L. cv. Dongjin) using a primer set (Table 1). To drive the GUS coding region, the 5 -flanking region of the CIPK15 gene was cloned into the pCAMBIA1391Z vector (GenBank accession no. AF234312) using the HindIII and BamHI restriction sites, to create pCAMBIA-CIPK15. The firefly luciferase gene (LUC) coding region was cut out of the pSP-luc+NF Fusion vector (Promega, Madison, WI, USA) using the NheI and XbaI restriction enzymes, and inserted in front of the GUS coding region of pBI221 in the XbaI site to generate the 35S::LUC:GUS:nopaline synthase (NOS) vector. The GUS coding region was removed by digestion with XbaI and SacI, and the resulting sticky ends were blunted with T4 DNA polymerase and self-religated, yielding the 35S::LUC:NOS construct. To create the 35S::LUC:CIPK15 3 UTR construct, 35S::LUC:GUS:NOS was digested with BamHI and SacI to remove the GUS coding region, and the sticky ends were blunted with T4 DNA polymerase and self-religated. The NOS terminator was replaced with the CIPK15 3 UTR fragment flanked by BamHI and SacI; this fragment was removed from the pMD20 T vector containing the PCR-amplified 3 UTR. A Renilla luciferase (RUC) fragment was removed by NheI and XbaI restriction from the pRL-TK vector (Promega) and inserted into the pBI221 vector to create 35S::RUC:GUS:NOS. The 35S::RUC:NOS vector was created by removing the GUS coding region via digestion with XbaI and SacI, blunting the sticky ends with T4 DNA polymerase, and religating. This final 35S::RUC:NOS vector was used as an internal control in the transient expression assay.


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Table 1 List of primers used in this study.

PCR

Target gene

Primer sequence

CIPK15 probe

FW RV FW RV FW RV FW RV FW RV FW RV

Amy3D probe RT qPCR

CIPK15 Actin

PCR

CIPK15 promoter

PCR

CIPK15 3 UTR

Transgenic rice cell lines The pCAMBIA1391Z vector containing CIPK15 promoter::GUS was used to transform Agrobacterium tumefaciens LBA4404 containing the pAL4404 Ti plasmid, and Agrobacterium-mediated rice transformation was performed as described by Hiei et al. (1994). The transgenic rice calli were selected on 2N6-CH solid medium (N6 salts and vitamins, 1 g/L casamino acid, 30 g/L sucrose, 2 mg/L 2,4-D, 2.0 g/L phytagel, 250 mg/L cefotaxime, and 50 mg/L hygromycin B, pH 5.8) for 3 weeks at 28 ◦ C in the dark. The hygromycin-resistant rice calli were subcultured several times and used for experiments. GUS assay For the fluorometric GUS assay, a packed-cell volume (100 ␮L) of cultured cells were homogenized in 100 ␮L of GUS extraction buffer (50 mM NaH2 PO4 , pH 7.0, 10 mM ␤-mercaptoethanol, 10 mM Na2 EDTA, 0.1% sodium lauryl sarcosine, and 0.1% Triton X-100) with a pestle. The fluorometric GUS assay was carried out as described by Hwang et al. (1998). Fluorescence was monitored using a spectrofluorometer Victor 3 (PerkinElmer, Wellesley, MA, USA) at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The fluorometric GUS activity was normalized by the amounts of protein in the cell extracts, determined by the Bradford protein assay (Bradford, 1976). A histochemical GUS assay was performed as described by Jefferson et al. (1987). Protoplast isolation and transient expression analysis The preparation and transformation of rice protoplasts were performed as described by Hwang et al. (1998). The protoplasts were resuspended in Kao and Michayluk medium (Kao and Michayluk, 1974) that contained either 0.06 M glucose/0.34 M mannitol or 0.4 M mannitol, and incubated for 24 h at 28 ◦ C in the dark without shaking. After incubation, the protoplasts were harvested by centrifugation (600 rpm for 8 min) and lysed in Reporter Lysis buffer (Promega). Firefly luciferase and Renilla luciferase activities

5 -TTCTCCAAGGGATTTGATCTCTCTGGC-3 5 -AGGAGCGGAAACTAAATGCTTGAGAC-3 5 -CGGAGTCCCCTGCATCTTCTACG-3 5 -CTCCCGGCGTTGATGCCGTTCCT-3 5 -TAAGCCTTCAAAATTCTTCG-3 5 -TATAAACAAAACCAGGCATC-3 5 -ATGAAGATCAAGGTGGTCGC-3 5 -GTACTCAGCCTTGGCAATCC-3 5 -TCACCGACAGCGAGACGCCCGCGA-3 5 -TCTAGATATAAATCTCAGCACTAT-3 5 -GAGCTCCATGGAGATGAATAGCAG-3 5 -GAATTCTCTTGTAGGACTGTGCAC-3

were measured using a Glomax 20/20 luminometer (Promega) with the Dual-GloTM Stop & Glo® Reagent (Promega). Results Comparison of time-dependent sugar regulation patterns between CIPK15 and Amy3D Previously, semi-quantitative PCR and real-time quantitative PCR assays have indicated that glucose or sucrose in the medium significantly reduced the transcription level of CIPK15 in rice suspension cultures or isolated rice embryos (Lee et al., 2009; Park et al., 2010). Since the CIPK15 gene is assumed to be an upstream positive regulator for sugar-regulated Amy3D expression and is itself under the control of sugar regulation, we compared the kinetics of CIPK15 and Amy3D induction by incubating rice embryos in a medium with glucose or mannitol for up to 52 h. As shown in Fig. 1, the CIPK15 transcripts were detectable in 18 h-starved rice embryos; their levels peaked at 36 h and were sustained with no decrease for up to 52 h. The mRNA transcripts of Amy3D, which were undetectable in embryos incubated for less than 18 h of starvation, reached their highest levels at 36 h and decreased thereafter. Interestingly, the CIPK15 gene shows a distinctive expression pattern that is not consistent with its assumed role as an upstream positive regulator for the Amy3D gene. One example of this inconsistency is that even though the sugar repression of CIPK15 appears to be leaky, the expression of the Amy3D gene indicates tight sugar regulation. Moreover, despite the abundant expression of the CIPK15 gene, Amy3D expression rapidly decreased in rice embryos starved for 2 d, displaying a conspicuous discrepancy between the expression patterns of the 2 genes. Hexokinase-mediated sugar regulation of CIPK15 expression Various biochemical and genetic studies have demonstrated that hexokinase triggers sugar signaling of many plant genes as a sugar sensor (Rolland et al., 2006). A previous study using a

Fig. 1. Time-course analyses for sugar-regulated expression of CIPK15 and Amy3D in isolated rice embryos. Rice embryos were incubated in 60 mM glucose or mannitol medium for 0–52 h. Total RNA was isolated and analyzed to determine the levels of Amy3D and CIPK15 transcripts by RNA gel blot hybridization. The amount of total RNA loaded (40 ␮g) was determined by staining ribosomal RNA with ethidium bromide.

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Fig. 2. Effects of various sugars and glucose analogs on CIPK15 expression. Rice embryos were incubated in media containing different types of hexoses (upper panel) or sugar analogs (lower panel), as indicated. The transcription levels of CIPK15 were detected by RNA gel blot analysis (A and C) and the real-time quantitative PCR (qPCR) method (B and D). For the latter, the ratio between CIPK15 expression and rice actin gene expression in 60 mM glucose medium was set to 1 as a control. The expression ratios under other conditions are relative to the control. The error bars represent the standard deviation of the mean (n = 3).

transient expression system has shown that hexokinase also mediates Amy3D sugar regulation (Umemura et al., 1998). Therefore, we performed experiments to test if the sugar-regulated expression of CIPK15, an upstream positive regulator for Amy3D, is mediated by hexokinase as well. For this study, we first examined the effectiveness of various sugars in triggering the repression of CIPK15 expression. Fig. 2 shows CIPK15 transcript levels from rice embryos incubated in media containing different hexoses or glucose analogs. Analyses of CIPK15 expression via RNA gel blot (Fig. 2A and C) and real-time quantitative PCR (Fig. 2B and D) indicate that regardless of their ability to be metabolized, all hexoses (glucose, fructose, galactose, mannose, and 2-deoxyglucose) that can be phosphorylated by hexokinase successfully suppress the expression of CIPK15, as compared to mannitol. The effective repression by mannose and 2-deoxyglucose (but not by 3-O-methylglucose or 6-deoxyglucose) demonstrates that the phosphorylation step by hexokinase is sufficient to trigger a chain of events that leads to repressed CIPK15 expression. Glucosamine, a hexokinase inhibitor, has been demonstrated to interfere effectively with the sugar signaling mediated by hexokinase (Umemura et al., 1998; Salas et al., 1965). To further confirm the critical role of hexokinase in the sugar repression of the CIPK15 gene, we tested if glucosamine was able to interfere with sugar regulation of the CIPK15 gene as well. As shown in Fig. 3, the transcript level of CIPK15 in glucose-treated embryos decreased to ∼15% of that of the mannitol control, whereas the CIPK15 transcript level in embryos incubated with glucose reached almost 90% of that of the mannitol control upon co-treatment with glucosamine. Taken together, hexokinase is involved in the sugar regulation of CIPK15 expression, consistent with the fact that CIPK15 is an upstream positive regulator for Amy3D expression under the control of hexokinase.

Interference of sugar-regulated CIPK15 expression by inhibiting oxidative phosphorylation The inability of CIPK15 knockout rice mutants to express Amy3D under waterlogged conditions indicated that anaerobic Amy3D expression requires CIPK15 expression (Lee et al., 2009). Like Amy3D, sugar regulation of CIPK15 expression was found to be affected by anaerobic conditions, implying that sustained CIPK15 expression under anaerobic conditions is critical to Amy3D expression in rice seeds germinating under submergence (Park et al., 2010). In Fig. 4, we have more carefully examined the anaerobic

Fig. 3. Effects of hexokinase inhibitor on sugar-regulated CIPK15 expression. Rice embryos were incubated for 36 h in 60 mM glucose or mannitol medium, with or without 5 mM glucosamine, at 28 ◦ C. The levels of CIPK15 transcripts were analyzed and expressed as described in Fig. 2.


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Fig. 4. Interference of sugar-regulated CIPK15 expression by oxidative phosphorylation inhibitors. Rice embryos were incubated for 36 h in 60 mM glucose or mannitol medium, with or without (A) an inhibitor for the cytochrome pathway (1 mM KCN), (B) uncouplers (30 ␮M DNP; 5 ␮M dicoumarol), (C) ATP synthase inhibitors (75 ␮g/mL oligomycin A; 3 mM DCCD), as indicated. The levels of CIPK15 transcripts were analyzed and expressed as described in Fig. 2.

factors that affect the sugar regulation of the CIPK15 gene; we performed this investigation by using several metabolic inhibitors that interfere with various steps of oxidative phosphorylation, such as the inhibitor of electron transport in the cytochrome pathway (KCN), uncouplers (DNP and dicoumarol), and ATP synthase inhibitors (DCCD and oligomycin A). All metabolic inhibitors tested effectively abolished the glucose-imposed repression of CIPK15 expression. Since all these metabolic inhibitors prevent ATP synthesis in cells, the drop in cellular energy status is likely to negatively influence the sugar regulation of CIPK15 expression. Taken together, the cellular energy status may be involved in cross-talk with hexokinase-mediated sugar regulation.

Sugar regulation of CIPK15 promoter activity in a transgenic cell line As the first step toward elucidating the molecular mechanism involved in sugar-mediated alterations in CIPK15 transcription, we examined the activity of the isolated CIPK15 promoter in the presence and absence of sugars. We constructed the pCAMBIA-CIPK15 plasmid containing the CIPK15 promoter-driven GUS gene (Fig. 5A) and used it in Agrobacterium-mediated transformation to generate a transgenic rice cell line harboring the GUS reporter gene under the control of the CIPK15 promoter. Assays for GUS enzyme activity were performed in either sugar-starved or glucose-treated

Fig. 5. Sugar regulation of the CIPK15 promoter in transgenic rice suspension-cultured cells and the role of the CIPK15 3 UTR in sugar regulation of gene expression. (A) Diagram of the 2548-bp segment of the CIPK15 5 -flanking region. (B) Fluorometric analysis of GUS activity in transgenic rice cells in response to glucose. Transgenic rice cells harboring the CIPK15 promoter-driven GUS reporter gene were incubated in AA2 medium containing 60 mM glucose (open box) or 60 mM mannitol (closed box) for up to 24 h and used for the fluorometric GUS assay. (C) Histochemical staining of transgenic rice cells in response to glucose. (D) Effect of the 3 UTR of the CIPK15 gene on sugar-regulated gene expression. The plasmid, 35S::RUC:NOS served as the internal control and the firefly luciferase activity was normalized to Renilla luciferase activity from co-transfected protoplasts. The error bars represent the standard deviation of the mean (n = 3).

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transgenic cells to determine whether the 5 -flanking region of CIPK15 is capable of sugar regulation. Fluorometric and histochemical assays revealed GUS enzyme activity to be obviously higher in sugar-starved cells than in glucose-treated cells (Fig. 5B and C, respectively). Thus, the transcriptional activity of the CIPK15 promoter was suppressed significantly in the presence of glucose, whereas it was induced under glucose-starved conditions. Interestingly, cells cultured in media containing glucose showed minimal levels of GUS enzyme activity, indicating that CIPK15 promoter activity is not repressed completely even in the presence of sugar. In addition, extended histochemical staining confirmed very weak GUS expression in cells under non-starving conditions (data not shown). These results from GUS transgenic cells are consistent with the findings of RNA gel blot analysis, as presented in Fig. 1, which shows that sugar suppression of the CIPK15 gene is not as strong as that of Amy3D, thus explaining the relatively small difference in CIPK15 promoter activity (4–5-fold) in response to sugar. Our GUS enzyme assay has demonstrated that the 2548-bp segment in the 5 -flanking region of CIPK15 can mediate sugar regulation of promoter activity, indicating that the cis elements responsible for sugar regulation must be contained within this region. In addition to transcriptional control, post-transcriptional control may contribute to sugar regulation of gene expression. For example, the 3 -untranslated region of the Amy3D gene is important because it decreases transcript stability (Chan and Yu, 1998). However, the 3 UTR of the CIPK15 gene showed no influence on transcript stability in our transient expression analysis (Fig. 5D). Discussion Due to sugar’s bi-functional nature, its cellular status is likely to affect both sugar and energy signaling inside cells. Here, we report that the sugar regulation of CIPK15, which can be affected by perturbation of mitochondrial ATP synthesis, is actually triggered by hexokinase, a well-known plant sugar sensor, implying cross-talk between sugar and energy signaling pathways. Discrepancy in the expression patterns of the CIPK15 and Amy3D genes CIPK15 is an upstream, positive regulator, directing the O2 deficiency signal to the sugar-signaling cascade in Amy3D regulation. Our comparative study of the time-dependent sugar-regulated expression levels of CIPK15 and Amy3D in rice embryos revealed a discrepancy in their expression patterns. A rapid decrease in Amy3D expression occurred after 36 h of starvation, despite the high residual levels of CIPK15 expression in rice embryos that were starved for up to 52 h. Further, the sugar regulation of CIPK15 was not as tightly controlled as that of Amy3D, as evidenced by the expression levels detected even in embryos incubated in the presence of glucose for more than 36 h. In particular, CIPK15 gene expression levels in rice embryos kept in glucose medium for more than 36 h were as high as those of the 18 h-starved embryos which showed obvious induction of Amy3D gene expression. Taken altogether, it appears that a key regulatory factor other than CIPK15 may be involved in the sugar regulation of Amy3D. Alternatively, an additional mode of regulation beyond transcriptional control may contribute to alterations in the amount of active CIPK15 protein. Hexokinase as a sugar sensor in CIPK15-mediated sugar regulation of Amy3D expression Several sugar sensors are likely to constitute a sugar-perception system that senses sugar and triggers signaling in plants (Rolland

et al., 2006). Hexokinase is the first plant sugar sensor identified, and is known to mediate the sugar-regulated expression of many plant genes, as evidenced by various biochemical studies using a protoplast transient expression system and by phenotypic analyses of various transgenic Arabidopsis strains (Sheen, 1990; Jang and Sheen, 1994; Jang et al., 1997). In the current study, we provide several lines of evidence showing that sugar regulation of the CIPK15 gene, an upstream regulator for the expression of Amy3D, is also mediated by hexokinase. First, the sugars that suppress CIPK15 expression share a common feature, namely, they act as a substrate for hexokinase. As implied in its name, hexokinase is relatively nonspecific and can react with most 6-carbon sugars; however, its affinity for these sugars varies with their structures (Granot, 2008). In principle, hexokinase can catalyze the phosphorylation of fructose and galactose despite its low affinity. Concurrently, as shown in Fig. 2A and B, these sugars were able to repress CIPK15 expression. Second, further metabolism of the sugar is not necessary to cause repression of the CIPK15 gene, as indicated from the repressive effects of mannose and 2-deoxyglucose. For example, mannose is phosphorylated by hexokinase, resulting in mannose-6-phosphate, but it is not further metabolized by plant cells (Salas et al., 1965; Sheu-Hwa et al., 1975; Loughman et al., 1989). A previous study of the respiration rate recovery of starved cells demonstrated that rice cells are incapable of respiring mannose as an energy source (Park et al., 2010). In the glucose analog 2-deoxyglucose, the 2-hydroxyl group is replaced by hydrogen, and thus this compound can be phosphorylated by hexokinase, but the product does not undergo additional glycolysis. Third, glucose transport per se cannot cause repression, because repression is not triggered by 3-O-methylglucose and 6-deoxyglucose, which can be efficiently taken up by cells, but are neither metabolizable glucose analogs nor substrates for hexokinase (Fig. 2C and D). Finally, glucosamine, a hexokinase inhibitor, can diminish the effect of glucose, as shown in Fig. 3. All these lines of evidence suggest that hexokinase functions as a sugar sensor to trigger repression of CIPK15 expression. This is consistent with the proposed role of CIPK15 as an upstream positive signaling component for Amy3D regulation. Previously, Umemura et al. (1998) have demonstrated the hexokinase-mediated sugar regulation of Amy3D expression by performing similar biochemical studies of rice embryos biolistically transfected with the Amy3D promoter-driven GUS reporter gene construct. Therefore, the hexokinase-mediated sugar regulation pattern observed in Amy3D expression might be a result of CIPK15 regulation. Cross-talk of sugar signaling by cellular energy status Previous biochemical studies using a mesophyll protoplast expression system showed that sugar regulation of photosynthetic genes was not triggered by direct delivery of sugar phosphates and its downstream metabolites bypassing the HXK phosphorylation steps (Sheen, 1990). Analyses of transgenic plants over-expressing the yeast sugar sensor YHXK2 (Jang et al., 1997), and Arabidopsis glucose insensive2 (gin2) mutants having molecular lesions in HXK1 (Moore et al., 2003) suggest that sugar signaling mediated through hexokinase is uncoupled from sugar metabolism. Finally, the metabolic and signaling functions of HXK1 were successfully separated in catalytically inactive HXK1 mutants by single amino acid changes in catalytic domains (Moore et al., 2003). All these data prove that sugar regulation is not a consequence of metabolic alteration. Although it is not metabolic alteration, but rather hexokinase that triggers sugar signaling, the perturbed energy status of cells appears to interfere with the signaling elicited by hexokinase. Previously, we found that inhibition of oxidative phosphorylation could de-repress the sugar regulation of the CIPK15 and Amy3D


genes, resulting in high-level gene expression of Amy3D in rice embryos under anaerobic conditions (Park et al., 2010). In the current study, to further support cross-talk between sugar signaling and the energy deficit, we tested several metabolic inhibitors that block various steps of mitochondrial ATP synthesis. Potassium cyanide inhibits electron transfer through the mitochondrial electron transport chain by binding tightly to the iron within a cytochrome (Ball and Cooper, 1952). The compounds DNP and dicoumarol inhibit oxidative phosphorylation by collapsing the chemiosmotic gradient to dissipate protons across the inner mitochondrial membrane, thereby uncoupling electron transport in oxidative phosphorylation (Pinchot, 1967). Dicyclohexylcarbodiimide (DCCD) forms covalent bonds with glutamate residues in the F0 subunit, thereby blocking the proton channel and stopping rotation and ATP synthesis. Oligomycin A binds directly to the ATP synthase F0 subunit and blocks the flow of protons through the channel (Linnett and Beechey, 1979). As shown in Fig. 4, all these metabolic inhibitors were very effective in relieving the sugar repression of the CIPK15 gene. In our previous study, NaN3 or anoxic treatment rapidly lowered cellular ATP levels in suspension cultures to 30–60% of the levels seen in control cells undergoing normal respiration (Park et al., 2010). Therefore, it is likely to be the energy deficit that interferes with hexokinase-elicited sugar signaling. A possible link between the sugar and energy signaling pathways Rice SnRK1A is another positive regulator for sugar regulation of Amy3D expression (Lu et al., 2007). The highly conserved Ser/Thr protein kinase SnRK1 is the plant counterpart for sucrose nonfermenting 1 (SNF1) and AMP-dependent protein kinase (AMPK), which have been proposed to act as master regulators controlling energy and metabolic adaptation in different environments to optimize growth and development (Polge and Thomas, 2007). This protein shares central roles with the orthologous yeast SNF1 and mammalian AMPK in plant energy signaling (Baena-Gonzalez et al., 2007; Baena-Gonzalez and Sheen, 2008). Rice has two SnRK1 genes, SnRK1A and SnRK1B, and interestingly, SnRK1A is able to induce itself under sugar starvation conditions, which are associated with high AMP:ATP ratios (Lu et al., 2007). In addition, SnRK1A-regulated sugar regulation of Amy3D expression can be hindered by a decrease in cellular ATP levels as well, suggesting the possibility that this protein may sense the cellular energy status (Park et al., 2010). In general, SNF1 and AMPK are active under conditions of energy limitation (high AMP:ATP) and inactive when energy supplies are abundant. Despite sharing a highly conserved energy sensor, the mechanism of sensing cellular energy status may vary among eukaryotic systems. In contrast to AMPK, which is regulated by the cellular energy charge represented by the AMP:ATP ratio, no allosteric activation of yeast SNF1 and plant SnRK1 by AMP has been demonstrated. Instead, AMP has been shown to prevent the dephosphorylation of a critical and conserved residue in the enzyme, resulting in blocking SnRK1 inactivation (Sugden et al., 1999). SnRK1 activity can be blocked by glucose-6-phosphate (G6P), the product of hexokinase activity (Toroser et al., 2000). Very little is known about the antagonistic link between the sugar and energy signaling pathways. In yeast, it is known that sugar signaling can be coordinated with SNF1 kinase activity. In response to glucose in yeast, Hxk2 has been demonstrated to translocate into the nucleus, where it interacts with a zinc-finger DNA-binding transcription factor to form a stable co-repressor complex that induces transcriptional repression in a large number of genes involved in respiration, gluconeogenesis, and the uptake and metabolism of alternative carbon sources. In contrast, SNF1 is required for the de-repression of gene expression imposed by

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Fig. 6. A model depicting interactions of putative sugar and energy signaling components. In the presence of sugar, hexokinase triggers sugar signaling for CIPK15 regulation in the nucleus. The product of hexokinase activity, glucose-6-phosphate (G6P), can also block the SnRK1 response. The high metabolic efficiency due to the mitochondrial respiration of sugar prevents an energy-deficient state in cells. In the absence of sugar, no repression of CIPK15 expression is elicited by hexokinase, leading to activation of Amy3D expression by MybS1, a transcriptional activator for the Amy3D gene. The energy deficit state caused by sugar starvation also activates the SnRK1, which orchestrates an energy-saving program via the SnRK1 pathway. Under stresses like flooding or submergence, where the mitochondrial respiration of sugar is perturbed, cells may encounter a state of energy disorder. Upon sensing the energy deficit, the SnRK1 enzyme activates the SnRK pathway, which interferes with the sugar signaling triggered by hexokinase, allowing the CIPK15 gene to be expressed, even in non-starved conditions. Direct and indirect connections are represented by solid and broken lines, respectively.

glucose via the phosphorylation of transcription factors, and this causes it to dissociate from the repressor complex and subsequently undergo nuclear export (Rolland et al., 2006). Recently, the plant sugar sensor AtHXK1 was also shown to translocate into the nucleus (Yanagisawa et al., 2003). Additionally, the vacuolar H+ -ATPase B1 (VHA-B1) and the 19S regulatory particle of the proteasome subunit (RPT5B) were shown to interact directly with HXK1 in a glucose-dependent manner, constituting a complex that directly binds to the promoters of glucose-regulated genes (Cho et al., 2006). Therefore, a regulatory system similar to that of HXK2/SNF1 in yeast may also operate in plants. As a possible model for the cross-talk between sugar signaling and cellular energy status, SnRK1A, one of the upstream positive signaling components of Amy3D regulation, is proposed to link the sugar-signaling and energy-signaling pathways (Fig. 6). At present, it is not clear whether the cross-talk between sugar signaling and cellular energy status is a general phenomenon or unique for the sugar-mediated regulation of the Amy3D or CIPK15 genes. Therefore, we plan to conduct large-scale transcriptional profiling analysis of the sugar responses to sense or antisense SnRK1 over-expression in the future to obtain further insight into the overall cross-talk between sugar- and energy-signaling pathways upon sensing glucose. Acknowledgments This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008198), Rural Development Administration, Republic of Korea, and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2012R1A1A2006267).

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