JXB Advance Access published online on August 23, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm169
RESEARCH PAPER |
Distinct modulations of the hexokinase1-mediated glucose response and hexokinase1-independent processes by HYS1/CPR5 in Arabidopsis
1Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan
2JST, CREST, Japan
To whom correspondence should be addressed at: Yayoi, 1-1-1 Bunkyo-ku, Tokyo 113–8657, Japan. E-mail: asyanagi{at}mail.ecc.u-tokyo.ac.jp
Received 21 March 2007; Revised 21 June 2007 Accepted 26 June 2007
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Abstract |
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The Arabidopsis mutant hypersenescence 1 (hys1), that is allelic to constitutive expresser of pathogenesis-related genes 5 (cpr5), displays phenotypes related to glucose signalling and defence responses. In the present study, it is shown that the hys1 mutation boosts the inhibitory effects of glucose upon the greening of seedlings and reduces the antagonistic activities of ethylene and cytokinin toward this inhibition. Neither the glucose content nor the sensitivities to ethylene, cytokinin, and abscisic acid were found to differ between wild-type and hys1 seedlings. However, disruption of the gene encoding hexokinase1 (HXK1), which acts as a glucose sensor, partially suppressed the glucose hypersensitive phenotype of the hys1 mutant. These results thus suggest that the hys1 mutation promotes a process associated with the HXK1-mediated glucose response during greening. By contrast, additional hys1 phenotypes, including an increase in salicylic acid (SA), production of abnormal trichomes, and early senescence, were not suppressed by the loss of HXK1. Surprisingly, the hxk1 and hys1 mutations acted synergistically towards an increased SA accumulation. Hence, HYS1/CPR5 appears to be a versatile protein that modulates both the HXK1-mediated glucose response and various HXK1-indepndent processes that are involved in growth control. A possible role for HYS1/CPR5 as a component of the networks that regulate growth control is discussed.
Key words: Arabidopsis, CPR5, cytokinin, ethylene, glucose, hexokinase, HYS1, salicylic acid, senescence, sugar signalling
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Photosynthesized sugar is not only an important energy source but also acts as a nutrient signal in plants. Indeed, sugar signalling modulates a variety of processes, including germination, seedling development, photosynthesis, carbon and nitrogen metabolism, flowering, stress responses, and senescence (Rolland et al., 2002, 2006; Yoshida, 2003; Gibson, 2005; Wingler et al., 2006), indicating a central role for sugars in plant growth regulation. Sugar signalling in plants has been suggested to include both a sucrose-specific signalling pathway and also a glucose-mediated pathway that can be further divided into hexokinase (HXK)-mediated and HXK-independent pathways (Sheen et al., 1999; Xiao et al., 2000).
HXK1 appears to mediate a major portion of the sugar signalling networks in Arabidopsis, as the Arabidopsis mutants, glucose insensitive 2 (gin2), harbouring mutations in the HXK1 gene, show phenotypic abnormalities in many processes associated with sugar signalling, including gene expression, cell proliferation, root and inflorescence growth, and the expansion and senescence of leaves. Because the gin2 phenotype was previously found to be rescued by expression of a mutant HXK1 that lacked catalytic activity, HXK1 was thus shown to function as a sugar sensor independently of its role in glucose metabolism (Moore et al., 2003). Furthermore, a recent report has suggested that HXK1 might form a signalling complex together with the 19S regulatory subunit of the 26S proteasome and the vacuolar H+-ATPase B1 in nuclei (Cho et al., 2006). However, the majority of the components of HXK-mediated glucose signalling remain unidentified.
Sugar signals are involved in plant growth control in concert with a number of hormones and various environmental stimuli, including biotic and abiotic stress signals. In this regard, a number of studies have demonstrated intimate relationships between sugar and hormone signalling in plants (Gazzarrini and McCourt, 2001; León and Sheen, 2003; Moore et al., 2003; Roitsch and González, 2004). For instance, repression of seedling development by exogenously applied sugar is reversed by the application of ethylene and cytokinin. It has also been shown that mutations conferring either abscisic acid (ABA)-deficiency or -insensitivity lead to a reduced sensitivity to exogenously applied sugar. Although the molecular mechanisms underlying such interactions are still poorly understood, the fact that the application of exogenous glucose promotes the degradation of EIN3, a key transcription factor in the ethylene signalling pathway (Yanagisawa et al., 2003), indicates the presence of a substantive mechanism that directly connects glucose and hormone signalling.
An Arabidopsis mutant, hypersenescence 1 (hys1), that was initially identified as a mutant presenting an early senescence phenotype, including early decreases in both chlorophyll and protein contents and the early accumulation of transcripts from the genes associated with senescence in rosette leaves, shows a stronger response to exogenous glucose in the dark (Yoshida et al., 2002). In addition, hys1 is allelic to constitutive expresser of pathogenesis-related genes 5 (cpr5), a mutant that displays spontaneous lesion formation, an increase in salicylic acid (SA), and abnormal trichome production (Bowling et al., 1997; Boch et al., 1998; Kirik et al., 2001; Yoshida et al., 2002). Consistent with the fact that SA accumulation is tightly linked with systemic acquired resistance (Gaffney et al., 1993), cpr5 is more resistant to pathogens (Bowling et al., 1997).
The phenotype of the hys1/cpr5 Arabidopsis mutants might suggest that HYS1/CPR5 plays a role in organizing both sugar signalling and defence responses and thereby in controlling growth and development. Alternatively, mutations in the HYS1/CPR5 gene might result in either a phenotype related to sugar signalling or a phenotype related to cell death and defence responses, which may then influence other pathways. In this report, the enhancement of the glucose-induced inhibition of greening in the hys1 mutant seedlings is described. By employing double mutants that harbour both the hys1 mutation and a disrupted HXK1 gene, it is also shown that the impact of the hys1 mutation on greening is distinguishable from the other effects of the hys1 mutation, in terms of HXK1 dependency. Accordingly, these results indicate that HYS1/CPR5 is independently involved in both HXK1-mediated glucose responses and HXK1-independent processes during growth regulation.
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Materials and methods |
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Plant materials
Seeds of hys1-1 mutant Arabidopsis plants were provided by Dr S Yoshida (RIKEN, Japan). Arabidopsis lines harbouring a T-DNA insertion within the HXK1 locus, SALK_018086 and SALK_070739 (Alonso et al., 2003), and also abi4-1, were provided by the Arabidopsis Biological Resource Center (Ohio State University, USA). Information concerning the T-DNA insertions was obtained from the SIGnAL website (http://signal.salk.edu). Lines homozygous for the T-DNA insertion in the HXK1 gene (AGI code, At4g29130) were selected by PCR genotyping using the primers: HXK1-TDNA-RV (5'-GAAACCTAATTCCCTCTGTCTACC-3’) and LBb1 (see the SIGnAL website) for the detection of T-DNA insertion allele and HXK1-FW (5'-ATGGGTAAAGTAGCTGTTGGAGCG-3’) and HXK1-TDNA-RV for the detection of the wild-type allele. To generate double mutants, hys1-1 was crossed with the homozygous SALK_018086 or SALK_070739 line, and F2 plants homozygous for both the T-DNA insertion in the HXK1 locus and the hys1-1 allele were selected.
To examine the genotype of the HYS1 allele (AGI code, At5g64930), PCR products that were amplified with the primers, 5'-ACATTGGGCGGTGATGGCAAACCCT-3’ and 5'-GTTTATAGGACCGAACATTAGACC-3’, were digested with MvaI. The digestion of the PCR products from the wild-type allele yields two DNA fragments of 93 bp and 24 bp, whereas a single fragment of 117 bp is obtained from the hys1-1 allele.
Plant growth conditions
Surface-sterilized seeds were sown on 1/2MS plates [half-strength Murashige and Skoog salts (Murashige and Skoog, 1962), 1 mg l–1 nicotinic acid, 10 mg l–1 thiamine hydrochloride, 1 mg l–1 pyridoxine hydrochloride, 100 mg l–1 myo-inositol, 0.5 g l–1 4-morpholinoethane sulphonic acid monohydrate, and 0.8% agar, pH 5.7]. After 3 or 4 d of cold treatment, the plates were incubated under continuous illumination with light intensities of 60–80 µmol m–2 s–1 at 23 °C. For reverse transcription (RT)-PCR analysis of ERF1 transcripts, seedlings were grown on 1/2MS plates containing 1% sucrose and 5 µM L-
-(2-aminoethoxyvinyl)glycine (AVG)-HCl in the dark for 4 d and then incubated for 3 h in the dark in liquid 1/2MS medium containing 1% sucrose and 5 µM AVG-HCl, with or without 20 µM 1-amino-1-cyclopropane carboxylic acid (ACC). AVG, an inhibitor of pyridoxal enzymes including ACC synthase that is a key enzyme in the ethylene biosynthetic pathway (Yang and Hoffman, 1984), was added to the medium to reduce the endogenous ethylene levels, as described previously (Guzman and Ecker, 1990; Binder et al., 2004). For RT-PCR analysis of ARR6 transcripts, seedlings grown on 1/2MS plates containing 1% sucrose in the dark for 4 d were incubated in liquid 1/2MS medium containing 1% sucrose with or without 5 µM N6-(2-isopentenyl) adenine (2-IP). For RT-PCR analysis of ATHB-7 transcripts, seedlings grown on 1/2MS plates without sucrose in light for 15 d were treated with 100 µM ABA for 3 h. For the analysis of senescence, plants were grown on peat (Jiffy 7, Jiffy Products International AS, Kristiansand, Norway) under continuous illumination at 23 °C.
RT-PCR
Total RNA was prepared from seedlings with TRIzol reagent (Invitrogen, CA, USA), and RT was carried out using an oligo (dT)15 primer and SuperScript II (Invitrogen). PCR was performed with Ex Taq DNA polymerase (TaKaRa Bio, Shiga, Japan) and the following primers, 5'-GAAACAGAGAATGTCTACCCTGTTC-3’ and 5'-GCCAGCTTACAAATCTCTTGTAAGG-3’ for ARR6 (AGI code, At5g62920), 5'-CTCTACGGTCTAATCGAGCAGTCC-3’ and 5'-GGTCATTCTCCGTCTCATCGAGTG-3’ for ERF1 (AGI code, At3g23240), and 5'-TGATGTCAGCAGAGCCATTCTTGACC-3’ and 5'-GATCACACATAACCCCATGACCC-3’ for ATHB-7 (AGI code, At2g46680). ß-tubulin mRNA were amplified as a control, as described in Kang and Singh (2000).
Extraction and measurement of metabolites
Metabolites were extracted by homogenizing tissues in an ice-cold medium [50 µM piperazine-N,N’-bis(2-ethanesulphonic acid) as an internal standard and 50% (v/v) methanol] for 5 min. After the exclusion of denatured proteins using a 5 kDa-cutoff centrifugal filter (Millipore Corporation, MA, USA), the salicylic acid content of the extracts was analysed by capillary electrophoresis mass spectrometry as described previously in Soga et al. (2002), with minor modifications. The glucose and sucrose contents were measured by HPAE-PAD (high-performance anion-exchange pulsed amperometric detector) on an ICS 3000 system equipped with a CarboPAC PA1 column (Dionex, CA, USA). Isocratic elution was performed using 16 mM KOH with a flow rate of 1 ml min–1. Chlorophyll was extracted from cotyledons and the first and second pairs of true leaves with dimethylformamide and the content was measured according to the method of Moran (1982).
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Results |
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The responses of the hys1 mutant to glucose, ethylene and cytokinin
Stronger effects of glucose in dark-grown hys1 seedlings have been previously shown by a measured enhancement of glucose-dependent reduction in hypocotyl elongation (Yoshida et al., 2002). Because it has been also shown that exogenously applied glucose inhibits the greening of seedlings in light (Jang et al., 1997), the glucose effects on the greening of hys1 seedlings in light was evaluated. Seedlings of the hys1 mutant and wild-type Arabidopsis of the ecotype Columbia (Col-0) developed similarly on plates containing a low concentration of glucose and also on plates containing mannitol as osmotic control. However, a severe glucose-dependent growth arrest was observed for hys1 compared with Col-0 when their respective seedlings were grown on plates containing a high concentration of glucose (see Supplementary Fig. 1 at JXB online). The ratio of green seedlings to growth-arrested seedlings was consequently lower for hys1 (see Supplementary Fig. 1 at JXB online). These data thus show that the hys1 mutation also increases the sensitivity to exogenously applied glucose during seedling development in light.
The enhancement of glucose responses in hys1 was evaluated further by investigating the effects of this mutation on the antagonistic activities of hormones against glucose using 6% glucose plates supplemented with an ethylene precursor, ACC, or a type of cytokinin, 2-IP. In Col-0 wild-type seedlings, inhibition of greening by glucose was rescued by the presence of either ACC or cytokinin, as reported previously by Moore et al. (2003) (Fig. 1A). In the hys1 seedlings, however, neither ethylene nor cytokinin effects could be observed (Fig. 1A), indicating that the actions of ethylene and cytokinin in the presence of a high concentrations of glucose are impaired. In this assay, an ethylene-insensitive mutant, etr1, harbouring a mutation in an ethylene receptor gene, was used as a negative control, as this mutation confers an oversensitivity to glucose due to the ethylene insensitivity (Zhou et al., 1998; Moore et al., 2003). In the etr1 seedlings, the antagonistic action of ACC was not observed, but the effects of the cytokinin were evident. The effects of cytokinin were reduced in etr1, however, compared with Col-0, presumably because these effects are exerted not only through its signalling pathway, but also through the promotion of ethylene production (Cary et al., 1995; Vogel et al., 1998; Chae et al., 2003).
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To eliminate the possibility that the hys1 mutation directly influences ethylene and cytokinin signalling, the growth response to these hormones was investigated in the absence of exogenous sugar. Because the ethylene-inducible triple response includes shortening hypocotyls of dark-grown seedlings, the lengths of the hypocotyls were measured in seedlings that were grown on plates containing different concentrations of ACC. The lengths of hypocotyls in both Col-0 and hys1 seedlings were shortened by ACC to a similar extent, whereas the hypocotyls of the etr1 seedlings were unaffected by this compound (Fig. 1B). The response to cytokinin in the hys1 seedlings was also evaluated by measuring the inhibition of root elongation in the presence of different concentrations of 2-IP. Repression of root elongation by 2-IP was observed for the Col-0, etr1, and hys1 seedlings to a similar extent (Fig. 1C). In addition, in both Col-0 and hys1, the expression of ethylene-responsive ERF1 and cytokinin-responsive ARR6 were similarly induced by ACC and 2-IP, respectively (Fig. 1D, E). This further indicates that normal ethylene and cytokinin signalling occurs in hys1. Because the hys1 mutant was not defective in either its sensing or signalling response to ethylene and cytokinin per se, the abolished antagonistic action of these hormones to exogenous glucose in hys1 appears to be caused by either the enhancement of sensing of endogenous glucose or the promotion of a process in response to exogenously applied glucose.
ABA responses in the hys1 mutant
A number of mutants displaying altered responses to sugars are allelic to ABA biosynthesis or signalling mutants, suggesting that ABA is an important player in glucose signalling (Gazzarrini and McCourt, 2001; Rolland et al., 2002; León and Sheen, 2003). To investigate whether the hys1 greening phenotype is mediated through a modification in ABA signalling, the ABA-dependent inhibition of germination of hys1 seeds was analysed. Exogenously applied ABA indeed inhibited the germination of Col-0 and hys1 seeds in a similar concentration-dependent manner, whereas two control lines, the ABA-insensitive abi4 mutant (Finkelstein, 1994) and the ein2 mutant that is insensitive to ethylene and hypersensitive to ABA (Guzman and Ecker, 1990; Beaudoin et al., 2000; Ghassemian et al., 2000), displayed different responses to exogenous ABA (Fig. 2A). For further characterization of ABA signalling in hys1, ABA-responsive gene expression was monitored by semi-quantitative RT-PCR. Consistent with the results of our phenotypic analysis, the expression of ATHB-7, a well-established ABA-responsive gene (Söderman et al., 1996), was similarly induced by ABA in both the wild-type and hys1 seedlings (Fig. 2B). Furthermore, the comparable expression levels of the gene in the wild-type and hys1 seedlings, in the absence of exogenous ABA, suggested a similar accumulation of endogenous ABA in the wild-type and hys1 seedlings.
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The glucose content is unaffected by the hys1 mutation
The possibility was tested next that the hys1 mutation in Arabidopsis might induce an elevated endogenous sugar content and thereby cause an increase in the sensitivity of the plant to exogenous glucose. The glucose and sucrose contents were measured in the shoots of Col-0 and hys1 seedlings that were grown under light without any supplemented sugar for 7 d. No significant differences in these contents were subsequently found, however (Table 1), suggesting that the increased hypersensitivity to exogenously applied glucose in hys1 is caused not by an increase in the endogenous sugar content, but by a particular modification of a process for glucose perception, signalling or response.
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Disruption of the HXK1 gene partly suppresses the effects of hys1 upon greening
Both HXK-mediated and HXK-independent glucose signalling pathways have been described previously (Sheen et al., 1999; Xiao et al., 2000). To identify which of these pathways may be linked with the glucose-hypersensitive phenotype of the hys1 mutant during seedling development, double mutants were generated that harbour mutations in both the HYS1/CPR5 and HXK1 genes, and their phenotypes were analysed.
The hys1 mutation was first identified in the ecotype Col-0 background, whereas gin2 was isolated originally via the mutagenesis of seeds of the Landsberg erecta strain. To avoid any difficulties in assessing the phenotypes of our double mutant plants that may arise due to the different genetic backgrounds of the parental strains, two SALK T-DNA insertion lines were used of the Col-0 background that carry a T-DNA in HXK1 (SALK_018086 and SALK_070739), instead of the original gin2 mutant. Both lines harbour a T-DNA insertion in the first intron of the HXK1 gene (see Supplementary Fig. 2A at JXB online) and lack intact HXK1 transcripts (see Supplementary Fig. 2B at JXB online). Furthermore, these lines show a reduced sensitivity to exogenous glucose (Fig. 3), suggesting that the critical role of HXK1 in glucose signalling in Arabidopsis is independent of its ecotype.
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As shown in Fig. 3, the ratios of green seedlings for the double mutants were higher compared with hys1 and Col-0, but lower than those of the individual SALK lines when the seedlings were grown on plates containing a high concentration of glucose in light. The hys1 effect on the greening process was partially suppressed by a disruption of HXK1. If the hys1 mutation promoted uptake of exogenously applied glucose, the double mutants and the SALK lines would be expected to show an identical phenotype. Modulation of a process associated with the HXK1-mediated glucose response is therefore likely to be principally responsible for the enhanced glucose sensitivity of the hys1 mutant during seedling development.
The early senescence phenotype of hys1 is independent of HXK1-mediated glucose signalling
Senescence is a complicated phenomenon that is regulated by both developmental and environmental factors. The roles of sugars and their associated signalling pathways are also implicated in the onset of senescence (Quirino et al., 2000; Paul and Foyer, 2001; Yoshida, 2003; Roitsch and González, 2004; Buchanan-Wollaston et al., 2005; Wingler et al., 2006). The glucose-hypersensitive hys1 and glucose-insensitive gin2 mutants show an early (Yoshida et al., 2002) and late (Moore et al., 2003) onset of senescence phenotype, respectively. Thus, it was possible to speculate that the early onset of senescence in hys1 might be caused by an alteration in HXK1-mediated glucose signalling (Yoshida et al., 2002). Because the partial suppression of the hys1 greening phenotype by the disruption of the HXK1 gene suggested that HYS1 and HXK1 are not integrated into a linear cascade (Fig. 3), the possibility was also assessed that the early senescence phenotype of hys1 is caused by activation of HXK1-dependent glucose signalling. To this end, the onset of leaf senescence was compared in Col-0, SALK_018086, SALK_070739, hys1, and the hys1 hxk1 double mutants. Under our growth conditions, senescence in the cotyledons and in the first pairs of true leaves was observed in both hys1 and the double mutants grown for 20 d after germination, but not in the Col-0, SALK_018086 or SALK_070739 lines (Fig. 4A). In addition, the chlorophyll content of the 14-d-old leaves was comparable among these tested lines, but was found to have decreased more rapidly in hys1 and in the double mutants (Fig. 4B). It was concluded, therefore, that hys1 mutation-mediated early senescence is independent of HXK1 function, at least under our growth conditions. Because sugar-induced senescence is partially but not exclusively dependent on the HXK-pathway (Pourtau et al., 2006), HYS1 may therefore have a role in hexokinase-independent sugar-induced senescence or sugar-independent senescence.
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Disruption of HXK1 does not affect abnormal trichome production but produces a synergistic effect on SA accumulation in the hys1 mutant
Our current findings suggested that the HXK1-pathway is linked to the hypersensitivity of the hys1 mutant to exogenous glucose during greening (Fig. 3), but not to the early senescence phenotype in this mutant (Fig. 4). Therefore, the relationship was investigated between the HXK1-mediated glucose signalling and other known phenotypes of hys1, including the production of trichomes with an abnormal shape and the high level accumulation of SA (Bowling et al., 1997; Kirik et al., 2001; Yoshida et al., 2002). As expected, no effect was observed on hys1 mutation-dependent abnormal trichome production following the disruption to the HXK1 gene (see Supplementary Fig. 3 at JXB online), whereas an unexpected synergistic effect was found on SA accumulation in 7-d-old seedlings grown under light without sugar supplements. The accumulation of SA was higher in the hys1 seedlings than in the Col-0 seedlings (Table 2), although the absolute value that was obtained for the hys1 seedlings was lower than the values that were previously reported for 4-week-old cpr5 plants (Bowling et al., 1997; Clarke et al., 2000, 2001), presumably due to our use of young seedlings in the present analyses. A small but significant increase in SA accumulation was also observed in the hxk1 mutants. Surprisingly, the double mutants showed a remarkably higher SA content, compared with hys1 (Table 2). Because a simple additive effect resulting from the combination of the hys1 mutation and a disruption to the HXK1 gene is not sufficient to explain the large increase in SA in the double mutants, these results implied that the disruption of HXK1 did not suppress the hys1 mutation-dependent accumulation of SA, but produced a synergistic effect on SA accumulation in the hys1 background. Moreover, because the double mutants did not show the hys1 phenotype during greening (Fig. 3), the possibility that modified levels of SA enhanced HXK1-mediated glucose signalling in the hys1 seedlings was excluded.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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An Arabidopsis mutant, hys1, displays a severe glucose-induced inhibition of hypocotyl elongation in the dark, and manifests an early senescence phenotype, spontaneous lesion formation, an increase in its SA content, and abnormal trichome production (Bowling et al., 1997; Kirik et al., 2001; Yoshida et al., 2002). In the present report, it is shown that the hys1 mutation enhances glucose-dependent growth arrest in the light and reduces the antagonistic activities of ethylene and cytokinin toward glucose during greening (Figs 1, 3, 5). Based on similar sensitivities to ethylene, cytokinin, and ABA, similar levels of glucose in hys1 and Col-0, and partial suppression of the hys1 mutation by the hxk1 mutation, it is further shown that the hys1 phenotypes during greening are related to the HXK1-dependent glucose signalling pathways. On the other hand, the increase in SA, production of abnormal trichomes, and even early senescence in the hys1 mutant were found to be independent of HXK1-mediated glucose signalling, although the SA levels were shown to be synergistically modulated by HYS1 and HXK1 (Fig. 4; see Supplementary Fig. 3 at JXB online; Table 2). Hence, HYS1/CPR5 has been identified as a versatile protein that is involved in both HXK1-mediated glucose responses and various HXK1-independent processes.
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The distinct modulations of the HXK1-mediated glucose responses and HXK1-independent processes by HYS1/CPR5 may suggest a possible role for this protein in co-ordinating glucose-induced regulation and other regulatory mechanisms that are associated with growth control. At this stage, however, the possibility cannot be excluded that HYS1/CPR5 is a general factor involved in a number of additional fundamental biological processes. It has been shown that the mutations that trigger modifications in sugar signalling produce very pleiotropic effects (Nemeth et al., 1998; Yoine et al., 2006a, b). For example, a recessive mutation in the Pleiotropic regulatory locus 1 (PRL1) in Arabidopsis, encoding a conserved nuclear WD-protein, causes the transcriptional derepression of glucose responsive genes, augments the sensitivity to growth hormones including cytokinin, ethylene, abscisic acid, and auxin, stimulates the accumulation of sugars in leaves, and inhibits root elongation (Nemeth et al., 1998). The lba mutation of the Arabidopsis UPF1 RNA helicase for nonsense-mediated mRNA decay has also been shown to alter sugar signalling, accompanied by phenotypic changes that include early flowering under long-day conditions, severely stunted growth under short-day conditions, abnormal seedling growth, and bigger seeds with an abnormal shape (Yoine et al., 2006a, b). Compared with the effects of the prl1 mutation or the lba mutation, the effects of the hys1/cpr5 mutations confer a less broad range of phenotypes which appear to be specifically associated with glucose signalling and defence responses. A possible role of HYS1/CPR5 in connecting various regulatory mechanisms should therefore be further evaluated.
In contrast to the inhibition of seedling greening, the early senescence phenotype of hys1 is not suppressed by disruption of the HXK1 gene (Fig. 4), suggesting that the regulation by HYS1 might be associated with HXK1-dependent regulation in a stage- or phenomenon-specific manner. During developmental senescence, sugar accumulation is probably coupled with leaf senescence (Yoshida, 2003; Wingler et al., 2006). In this regard, antisense-HXK1 Arabidopsis plants and gin2 mutants display late onset senescence (Xiao et al., 2000; Moore et al., 2003; Pourtau et al., 2006), whereas the overexpression of hexokinase results in early senescence (Dai et al., 1999; Xiao et al., 2000). However, as evidence against the possibility that the hys1 mutation might cause early senescence through the enhancement of HXK1-mediated glucose signalling, our current data show that the early onset of leaf senescence in hys1 plants is independent of HXK1. HYS1 might thus be associated with a HXK1-independent pathway of sugar-induced senescence (Pourtau et al., 2006). Alternatively, the early senescent phenotype of hys1 might be mediated via a process unrelated to sugar-induced developmental senescence, since it was promoted by incubation in the dark (Yoshida et al., 2002), which might cause sugar-starvation. Given that SA is involved in the regulation of gene expression during developmental senescence (Morris et al., 2000; Buchanan-Wollaston et al., 2005), it is also possible that the elevated levels of SA in hys1 underlie the onset of early senescence in this mutant. It is noteworthy that the hys1 mutation was dominant over the disruption to the HXK1 gene in terms of the regulation of senescence in our present growth conditions.
The hys1 mutant produces abnormal trichome levels and shows an increased accumulation of SA (Bowling et al., 1997; Kirik et al., 2001; see Supplementary Fig. 3 at JXB online; Table 2). As expected, this hys1-dependent abnormal trichome development was found to be independent of HXK1-mediated glucose signalling, whereas an unexpected synergistic effect of the hys1 and hxk1 mutations was found in terms of SA accumulation. Gene silencing of hexokinase-encoding Hxk1 causes spontaneous lesion formation in tobacco (Kim et al., 2006). Although such severe phenotypes were not observed in the Arabidopsis HXK1 T-DNA lines under our study, it is speculated that HYS1/CPR5 might play a role in the suppression of lesion formation mediated by SA accumulation in concert with mitochondria-associated hexokinases. Future studies of these synergistic effects might provide further novel insights into the relationship between the nutrient and defence responses in plants.
The possibility that HYS1/CPR5 directly regulates HXK1 activity is unlikely as the subcellular distribution of HXK1 appears not to overlap that of HYS1/CPR5. Preliminary data have suggested that HYS1/CPR5 might be a cytoplasmic protein (data not shown) in spite of the presence of a putative nuclear localization signal and a transmembrane domain. HXK1, in contrast, is associated with the mitochondria (Rolland and Sheen, 2005) and is also present in the nucleus (Yanagisawa et al., 2003; Cho et al., 2006). Therefore, the future identification of the components under the control of HYS1/CPR5 might provide additional clues in the elucidation of the HXK-mediated glucose signalling pathways, as well as an insight into the molecular links between this network and the function of HYS1/CPR5.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Supplementary data may be found at JXB online.
Supplementary Fig. 1. The hypersensitivity to glucose of hys1.
Supplementary Fig. 2. Disruption of the HXK1 gene in the two SALK lines, SALK_070739 and SALK_018086.
Supplementary Fig. 3. Trichomes on the rosette leaves of hys1 and the hys1 hxk1 double mutants.
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Acknowledgements |
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We thank Dr Satoko Yoshida for providing the hys1 seeds and the Arabidopsis Biological Resource Center for providing the seeds for the SALK_018086, SALK_070739, and abi4-1 lines. We also thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. Funding for the SIGnAL indexed insertion mutant collection was provided by the National Science Foundation. This research was supported by CREST, JST.
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Footnotes |
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* These authors contributed equally to this work.
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Abbreviations |
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ABA, abscisic acid; ACC, 1-aminocyclopropane-1-carboxylic acid; AVG, L-
-(2-aminoethoxyvinyl) glycine; HXK, hexokinase; 2-IP, N6-(2-isopentenyl) adenine; RT, reverse transcription; SA, salicylic acid. |
References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Alonso JM, Stepanova AN, Leisse TJ, et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science (2003) 301:653–657.
Beaudoin N, Serizet C, Gosti F, Giraudat J. Interactions between abscisic acid and ethylene signaling cascades. The Plant Cell (2000) 12:1103–1115.
Binder BM, O'Malley RC, Wang W, Moore JM, Parks BM, Spalding EP, Bleecker AB. Arabidopsis seedling growth response and recovery to ethylene. A kinetic analysis. Plant Physiology (2004) 136:2913–2920.
Boch J, Verbsky ML, Robertson TL, Larkin JC, Kunkel BN. Analysis of resistance gene-mediated defence responses in Arabidopsis thaliana plants carrying a mutation in cpr5. Molecular Plant–Microbe Interactions (1998) 11:1196–1206.[CrossRef]
Bowling SA, Clarke JD, Liu Y, Klessig DF, Dong X. The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance. The Plant Cell (1997) 9:1573–1584.[Abstract]
Buchanan-Wollaston V, Page T, Harrison E, et al. Comparative transcriptome analysis reveals significant differences in gene expression and signalling pathways between developmental and dark/starvation-induced senescence in Arabidopsis. The Plant Journal (2005) 42:567–585.[CrossRef][Web of Science][Medline]
Cary AJ, Liu W, Howell SH. Cytokinin action is coupled to ethylene in its effects on the inhibition of root and hypocotyls elongation in Arabidopsis thaliana seedlings. Plant Physiology (1995) 107:1075–1082.[Abstract]
Chae HS, Faure F, Kieber JJ. The eto1, eto2 and eto3 mutations and cytokinin treatment increase ethylene biosynthesis in Arabidopsis by increasing the stability of ACS protein. The Plant Cell (2003) 15:545–559.
Clarke JD, Aarts N, Feys BJ, Dong X, Parker JE. Constitutive disease resistance requires EDS1 in the Arabidopsis mutants cpr1 and cpr6 and is partially EDS1-dependent in cpr5. The Plant Journal (2001) 26:409–420.[CrossRef][Web of Science][Medline]
Clarke JD, Volko SM, Ledford H, Ausubel FM, Dong X. Roles of salicylic acid, jasmonic acid, and ethylene in cpr-induced resistance in Arabidopsis. The Plant Cell (2000) 12:2175–2190.
Cho YH, Yoo S-D, Sheen J. Regulatory functions of nuclear hexokinase1 complex in glucose signaling. Cell (2006) 127:579–589.[CrossRef][Web of Science][Medline]
Dai N, Schaffer A, Petreikov M, Shahak Y, Giller Y, Ratner K, Levine A, Granot D. Overexpression of Arabidopsis hexokinase in tomato plants inhibits growth, reduces photosynthesis, and induces rapid senescence. The Plant Cell (1999) 11:1253–1266.
Finkelstein R. Mutation at two new Arabidopsis ABA response loci are similar to the abi3 mutations. The Plant Journal (1994) 5:765–771.[CrossRef][Web of Science]
Gaffney T, Leslie F, Vernooij B, Negrotto D, Nye G, Uknes S, Ward E, Kessmann H, Ryals J. Requirement of salicylic acid for the induction of systemic acquired resistance. Science (1993) 261:754–756.
Gazzarrini S, McCourt P. Genetic interactions between ABA, ethylene and sugar signaling pathways. Current Opinion in Plant Biology (2001) 4:387–391.[CrossRef][Web of Science][Medline]
Ghassemian M, Nambara E, Cutler S, Kawaide H, Kamiya Y, McCourt P. Regulation of abscisic acid signaling by the ethylene response pathway in Arabidopsis. The Plant Cell (2000) 12:1117–1126.
Gibson SI. Control of plant development and gene expression by sugar signaling. Current Opinion in Plant Biology (2005) 8:93–102.[CrossRef][Web of Science][Medline]
Guzman P, Ecker JR. Exploiting the triple response of Arabidopsis to identify ethylene-related mutants. The Plant Cell (1990) 2:513–523.
Jang J-C, León P, Zhou L, Sheen J. Hexokinase as a sugar sensor in higher plants. The Plant Cell (1997) 9:5–19.[Medline]
Kang H-G, Singh KB. Characterization of salicylic acid-responsive, Arabidopsis Dof domain proteins: overexpression of OBP3 leads to growth defects. The Plant Journal (2000) 21:329–339.[CrossRef][Web of Science][Medline]
Kim M, Lim J-H, Ahn CS, Park K, Kim GT, Kim WT, Pai H-S. Mitochondria-associated hexokinases play a role in the control of programmed cell death in Nicotiana benthamiana. The Plant Cell (2006) 18:2341–2355.
Kirik V, Bouyer D, Schöbinger U, Bechtold N, Herzog M, Bonneville JM, Hülskamp M. CPR5 is involved in cell proliferation and cell death control and encodes a novel transmembrane protein. Current Biology (2001) 11:1891–1895.[CrossRef][Web of Science][Medline]
León P, Sheen J. Sugar and hormone connections. Trends in Plant Science (2003) 8:110–116.[CrossRef][Web of Science][Medline]
Moran R. Formulae for determination of chlorophyllous pigments extracted with N,N-dimethylformamide. Plant Physiology (1982) 69:1376–1381.
Moore B, Zhou L, Rolland F, Hall Q, Cheng W-H, Liu Y-X, Hwang I, Jones T, Sheen J. Role of the Arabidopsis glucose sensor HXK1 in nutrient, light, and hormonal signaling. Science (2003) 300:332–336.
Morris K, A-H-Mackerness S, Page T, John CF, Murphy AM, Carr JP, Buchnan-Wollaston V. Salicylic acid has a role in regulating gene expression during leaf senescence. The Plant Journal (2000) 23:677–685.[CrossRef][Web of Science][Medline]
Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum (1962) 15:473–497.[CrossRef]
Nemeth K, Salchert K, Putnoky P, et al. Pleiotropic control of glucose and hormone responses by PRL1, a nuclear WD protein in Arabidopsis. Genes and Development (1998) 12:3059–3073.
Paul MJ, Foyer CH. Sink regulation of photosynthesis. Journal of Experimental Botany (2001) 52:1383–1400.
Pourtau N, Jennings R, Pelzer E, Pallas J, Wingler A. Effect of sugar-induced senescence on gene expression and implications for the regulation of senescence in Arabidopsis. Planta (2006) 224:556–568.[CrossRef][Web of Science][Medline]
Quirino BF, Noh Y-S, Himelblau E, Amasino RM. Molecular aspects of leaf senescence. Trends in Plant Science (2000) 5:278–282.[CrossRef][Web of Science][Medline]
Roitsch T, González M-C. Function and regulation of plant invertases: sweet sensations. Trends in Plant Science (2004) 9:606–613.[CrossRef][Web of Science][Medline]
Rolland F, Baena-Gonzalez E, Sheen J. Sugar sensing and signaling in plants: conserved and novel mechanisms. Annual Review of Plant Biology (2006) 57:675–709.[CrossRef][Medline]
Rolland F, Moore B, Sheen J. Sugar sensing and signaling in plants. The Plant Cell (2002) 14:S185–S205.
Rolland F, Sheen J. Sugar sensing and signaling networks in plants. Biochemical Society Transactions (2005) 33:269–271.[CrossRef][Web of Science][Medline]
Sheen J, Zhou L, Jang J-C. Sugars as signaling molecules. Current Opinion in Plant Biology (1999) 2:410–418.[CrossRef][Web of Science][Medline]
Söderman E, Mattsson J, Engström P. The Arabidopsis homeobox gene ATHB-7 is induced by water deficit and by abscisic acid. The Plant Journal (1996) 10:375–381.[CrossRef][Web of Science][Medline]
Soga T, Ueno Y, Naraoka H, Ohashi Y, Tomita M, Nishioka T. Simultaneous determination of anionic intermediates for Bacillus subtilis metabolic pathways by capillary electrophoresis electrospray ionization mass spectrometry. Analytical Chemistry (2002) 74:2233–2239.[Medline]
Vogel JP, Woeste KE, Theologis A, Kieber JJ. Recessive and dominant mutation in the ethylene biosynthetic gene ACS5 of Arabidopsis confer cytokinin insensitivity and ethylene overproduction, respectively. Proceedings of the National Academy of Sciences, USA (1998) 95:4766–4771.
Wingler A, Purdy S, MacLean JA, Pourtau N. The role of sugars in integrating environmental signals during the regulation of leaf senescence. Journal of Experimental Botany (2006) 57:391–399.
Xiao W, Sheen J, Jang J-C. The role of hexokinase in plant sugar signal transduction and growth and development. Plant Molecular Biology (2000) 44:451–461.[CrossRef][Web of Science][Medline]
Yanagisawa S, Yoo S-D, Sheen J. Differential regulation of EIN3 stability by glucose and ethylene signalling in plants. Nature (2003) 425:521–525.[CrossRef][Medline]
Yang SF, Hoffman NE. Ethylene biosynthesis and its regulation in higher plants. Annual Review of Plant Physiology (1984) 35:155–189.[CrossRef][Web of Science]
Yoine M, Nishii T, Nakamura K. Arabidopsis UPF1 RNA helicase for nonsense-mediated mRNA decay is involved in seed size control and is essential for growth. Plant and Cell Physiology (2006a) 47:572–580.
Yoine M, Ohto M, Onai K, Mita S, Nakamura K. The lba1 mutation of UPF1 RNA helicase involved in nonsense-mediated mRNA decay causes pleiotropic phenotypic changes and altered sugar signalling in Arabidopsis. The Plant Journal (2006b) 47:49–62.[CrossRef][Web of Science][Medline]
Yoshida S. Molecular regulation of leaf senescence. Current Opinion in Plant Biology (2003) 6:79–84.[CrossRef][Web of Science][Medline]
Yoshida S, Ito M, Nishida I, Watanabe A. Identification of a novel gene HYS1/CPR5 that has a repressive role in the induction of leaf senescence and pathogen-defence responses in Arabidopsis thaliana. The Plant Journal (2002) 29:427–437.[CrossRef][Web of Science][Medline]
Zhou L, Jang J-C, Jones TL, Sheen J. Glucose and ethylene signal transduction crosstalk revealed by an Arabidopsis glucose-insensitive mutant. Proceedings of the National Academy of Sciences, USA (1998) 95:10294–10299.
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