Multiple paths of sugar-sensing and a sugar/oxygen overlap for genes ...

Journal of Experimental Botany, Vol. 51, GMP Special Issue, pp. 417–427, February 2000

Multiple paths of sugar-sensing and a sugar/oxygen overlap for genes of sucrose and ethanol metabolism Karen E. Koch1, Zeng Ying, Yong Wu and Wayne T. Avigne Plant Molecular and Cellular Biology Program, Horticultural Sciences Department, University of Florida, Gainesville, Fl-32611, USA Received 2 June 1999; Accepted 26 October 1999

Abstract The two-fold purpose of this work is, first, to review current hypotheses for multiple paths of sugar-sensing in an oxygen-responsive context, and second, to present evidence for the extent of sugar/oxygen overlap regulating genes for sucrose and ethanol metabolism. Current data indicate that sugar signals in plants may be initiated by (a) hexokinases, (b) membrane sensors, (c) acetate and/or respiratory metabolites, and (d) other signals and/or crosstalk. Responses may also involve concurrent input along transduction paths by effectors such as energy charge, P status, and phytohormones. Prime candidates for initiation and/or integration of such signal integration include SNF1- and SCF-like, multi-enzyme complexes. In addition, different paths of sugar signal transduction may be linked to contrasting roles of responsive genes during feast, famine or pathogen attack. Oxygen can potentially alter sugar signals at several points, so its influence on feast and famine responses was initially tested with genes for sucrose metabolism in maize root tips. The Sus1 and Sh1 sucrose synthases in maize (typically up-regulated by carbohydrate abundance and deprivation, respectively) showed parallel responses to hypoxia (3% O [0.03l l−1 O ]) and anoxia (0% O [0l l−1 O ]) 2 2 2 2 that were consistent with involvement of similar signals. In contrast, the differential sugar-responses of the Ivr1 and Ivr2 invertases were not evident under low oxygen, and both genes were rapidly repressed. A third response was evident in the marked, sugarregulation of an oxygen-responsive Adh1 gene for alcohol dehydrogenase, which was sensitive to sugar availability from deficit to abundance, regardless of oxygen status (anaerobic to fully aerobic [40% O (0.04l l−1 2 O )]. A clear interface is thus evident between sugar 2

and oxygen signals, but this varies markedly with the genes involved and probable differences in respective transduction paths. Key words: Invertase, sucrose synthase, alcohol dehydrogenase, sugar-sensing.

Multiple paths of sugar sensing A dual role in sugar-sensing as well as glycolysis has been implicated for hexokinases The classical respiratory role of these enzymes has been that of catalyzing the first step in glycolysis, during which glucose, fructose or other hexoses are phosphorylated (Fig. 1A). More recently, data have indicated that hexokinases may also initiate sugar-sensing signals. Interestingly, this signalling function appears not to involve the hexose-P products of the reaction per se. Instead, studies in organisms from microbes to humans have indicated that sugar-sensing via hexokinases involves a different feature of the enzyme such as its conformation, phosphorylation or association with other proteins (Bell et al., 1996; Jang and Sheen, 1997; Koch, 1996; Smeekens and Rook, 1997; Smeekens, 1998). Possibilities include interactions with kinases, phosphatases, and/or membrane proteins. The latter has been reported for human cells (Newgaard and McGarry, 1995) and a similar possibility has been proposed for other species (Jang and Sheen, 1997). The mechanism of signal initiation via hexokinases remains unclear and debate persists (Meijer et al., 1998; Halford and Hardie, 1999). However, work indicates that hexokinases may initiate signals in response to C-flux through the enzyme rather than to concentrations of either hexose substrates or hexose-P products (Jang and

1 To whom correspondence should be addressed. Fax: +1 352 392 6479. [email protected] © Oxford University Press 2000

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Sheen, 1997; Koch, 1996; Smeekens and Rook, 1997; Smeekens, 1998). A key line of evidence supporting a dual role for hexokinases has been the separation of activity from signalling function. This was accomplished by transformations that up- and down-regulated native hexokinases (sensing and overall activity were altered) compared to over-expression of yeast hexokinase (activity alone was altered) (Jang and Sheen, 1997; Jang et al., 1997) Membrane-based sugar sensing may also be involved Plant membranes may contribute to sugar sensing in at least two ways (Fig. 1B). One is through association of membrane proteins (yet to be defined ) with constituents of other signal transduction pathways. Such a possibility has been suggested for hexokinases based on data from plants (Rasschaer and Malaisse, 1990) and humans (Newgaard and McGarry, 1995). Another mechanism

may be that of direct, independent initiation of sugar signals from membrane sites (plasmamembrane, endoplasmic reticulum or tonoplast). Both sucrose and hexoses have been implicated in this process. Primary lines of evidence in plants involve analyses using nonmetabolizable and/or non-transported sugar analogues (Godt et al., 1995; Roitsch et al., 1995; Martin et al., 1997) and transgenic studies plants with specific subcellular targeting (Heineke et al., 1992; Herbers et al., 1996; Koch, 1996; Smeekins and Rook, 1997; Lalonde et al., 1999). Recent studies have begun to define membranebased sugar-sensing systems in yeast which provide testable models (Fig. 3). Acetate and/or respiratory metabolites can affect sugar-responsive genes Studies of microorganisms, humans, algae, and vascular plants indicate that acetate and certain other respiratory metabolites (Fig. 1C ) downstream of hexokinase can initiate signals of carbohydrate availability (Newgaard and McGarry, 1995; Koch, 1996, and references therein). Mechanisms for these responses have remained elusive, but appear to be distinct from those of other apparently hexokinase-linked, or membrane-based signals. Effects of pH may be involved for acetate, which moves readily across membranes. Other sensing paths and/or crosstalk may have prominent roles in sugar signalling Factors such as redox potential, adenylate balance, inorganic P, and/or phytohormones can alter, mimic, or Fig. 1. (A) Hexokinase-linked sugar sensing: Hexokinases can have dual roles as sugar sensors in addition to their classical catalysis of the first step in glycolysis. Dual roles are supported by extensive work in microbes, mammals and plants. Evidence indicates that products of the hexokinase reaction are not themselves responsible for sugar signalling, but rather some other unknown aspect of the reaction. Hypotheses include conformational changes in proteins involved in the reaction itself, or additional association with protein kinases, phosphatases or membrane constituents (see text). Other aspects of this proposed pathway are shown as currently defined for yeast in Fig. 2 (important similarities and differences are likely to emerge for plants). (B) Membrane-based sugar sensing: Plant membranes may be involved in sugar sensing either independently or in association with hexokinases. The hexokinase-independent signals may arise from glucose or sucrose sensors in plant membranes (plasmamembrane, endoplasmic retuculum, or tonoplast) via mechanisms having at least some similarities to those in yeast (see text and Fig. 3). (C ) Acetate and/or respiratory metabolites: Acetate can induce or repress genes in algae and vascular plants via a sensing mechanism other than hexokinase or membrane sugar sensors (possibly involving pH ). Evidence from microorganisms also indicates that in some instances, respiratory metabolites downstream of hexoses can affect carbohydrate-responsive genes. (D) Other signals and/or crosstalk: Adenylate balance, inorganic P, phytohormones, etc., can markedly affect sugar signals. These could act at multiple points along paths for sugar sensing, providing primary signals, and/or downstream input at central regulatory sites. Probable targets are multi-functional, multi-enzyme complexes related to the yeast SNF1 complexes (in hexokinase-linked sensing for yeast, see Fig. 2) or the SCF complexes (in membrane-based sensing for yeast, see Fig. 3). Functions of such complexes may vary considerably for plants.

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possibly initiate signals of carbohydrate availability ( Ehness et al., 1997; Halford and Hardie, 1999; Jang and Sheen, 1997; Koch, 1996; Smeekens and Rook, 1997) (Fig. 1D). Debate persists as to the relationship between these effectors and sites of signal input and initiation, since there are many points where influence could be exerted. Prominent among these are multi-functional, multi-enzyme complexes typically positioned centrally in sugar-sensing paths for yeast, and at points of crosstalk with other signal cascades. For yeast these include SNF1 kinase complexes (Fig. 2) and SCF kinase complexes (Fig. 3). Related proteins in plants, however, may or may not have similar functions. Recent work indicates widely diverse roles for SNF1-related proteins in plants, which may have roles from sugar sensing to activation of key enzymes by phosphorylation (Douglas et al., 1997; Hardie and Halford, 1999; Moorhead et al., 1999; Sugden et al., 1999).

Hexokinase-linked signalling and its potential oxygen sensitivity An overview of hexokinase-linked sugar signalling in yeast Extensive evidence from studies of yeast have provided a source of testable hypotheses and framework for comparing results from plants. In both systems, evidence suggests that signals originate from hexokinases (primarily HXK2 in yeast) which could be modulated by associations between hexokinase and other proteins. Proposed interactions include membrane sensors or transporters, as well as protein phosphatases or kinases, but the latter suggestions remain hypothetical (Fig. 2). Plant versus yeast comparisons reveal conserved features and important differences in potentials for hexokinase-linked sugar sensing. The transduction cascade pictured in Fig. 2, and related reactions in plants both appear to alter expression of sugar-responsive genes (Jang and Sheen, 1997; Koch, 1996; Smeekens and Rook, 1997; Smeekens, 1998). Several components of this path have been cloned in plants and functional analyses undertaken (reviewed in Jang and Sheen, 1997; Lalonde et al., 1999). There are also plant-specific transcription factors that respond to changes in sugar availability (Ishiguro and Nakamura, 1994; Jang and Sheen, 1997). In plants, the SNF1-like proteins occupy a prominent position in this signalling path. They have kinase activity and phosphorylate numerous substrates (Douglas et al., 1997; Johnston, 1999; Lesage et al., 1996; Sugden et al., 1999). In yeast, SNF1 also forms complexes with other proteins, and different combinations have different properties. An example is the capacity for these clusters to function as either inducers or repressors. One yeast SNF1 complex functions upstream in a transduction path leading to gene repression, whereas another (including a SIP4

Fig. 2. The hexokinase-linked sugar-sensing path in yeast mediates expression of ‘famine’ genes through both glucose repression and a combination of starvation induction and de-repression. In contrast, glucose-induced ‘feast’ genes in yeast are up-regulated by a different path linked to membrane sensors (Fig. 3). (1) Signal: hexokinaselinked signals for glucose repression can be initiated by one or more yeast hexokinases (mainly HXK2), but are generated by an as yet undefined means (see text). (2) Transduction: initial signals affect at least two protein kinase complexes (REG1/GLC7 and a SNF1 complex), which respectively transduce these to glucose repression versus starvation induction. REG1 (of REG1/GLC7) transfers signals for repression in two ways; it activates a MIG1 transcirptional repressor, and inactivates one or more SNF1 complexes. The SNF1 complexes are otherwise active, and in the absence of repressing sugar signals, they can be present in at least two different forms (SNF1+SNF4 or SNF1+SIP4). (3) Transcription: when a SNF1 complex includes SNF4, it can inactivate components of a prominent repressor complex that includes TUP1, SSN6 (CYC8), and MIG1. In addition, a different SNF1 complex involving SIP4 (not SNF4) can bind DNA through the SIP4 protein and function as a transcriptional activator for famine genes. Both de-repression and activation can thus contribute to upregulation of these genes under sugar deprivation. This model and its relationship to induction of feast versus famine genes is based on information on sugar-sensing in yeast (Johnston, 1999; Koch, 1997; Lesage et al., 1996) and despite some differences, shows multiple points of conservation with plants (see text, Jang and Sheen, 1997; Koch, 1996; Smeekens and Rook, 1997; Smeekens, 1998). Exceptions appear to include broader roles for hexokinase-linked signals in plants, including up-regulation of glucose-induced genes such as nitrate reductase and one of the sucrose synthases (Jang et al., 1996; Purcell et al., 1998).

protein instead of a SNF4) binds DNA and functions as an activator for starvation-induced genes (Lesage et al., 1996). Similar activity has not yet been tested in plant systems, but evidence indicates that SNF1-like proteins have a wide array of functions in plants (Douglas et al.,

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1997; Halford et al., 1999; Moorhead et al., 1999; Sugden et al., 1999). Hexokinase-linked signalling may predominate in the regulation of ‘famine genes’ although this signalling path has both inductive and repressive components (Fig. 2), in yeast these up-regulate ‘famine’ genes expressed during carbohydrate deprivation, and down-regulate them when sugars are abundant (Lesage et al., 1996; Johnston, 1999). Interestingly, glucose-induced genes are not up-regulated by this mechanism (Johnston, 1999). The situation appears to be generally similar in plants (Jang and Sheen, 1997; Jang et al., 1997), but may vary. A possible role in glucose induction was indicated for the sugar-enhanced expression of nitrate reductase in transgenic Arabidopsis plants over-expressing their own hexokinase (Jang et al., 1997). In addition, up-regulation of a sugar-inducible sucrose synthase was inhibited when antisense sequences for a SNF1-like protein were expressed in potato (Purcell et al., 1998). These results are compatible with a potentially expanded role for plant hexokinases (or related mechanisms) in the induction of ‘feast’ as well as ‘famine’ genes. The diversity of plant hexokinases and their properties may surpass those in yeast although only two hexokinases and one glucokinase (HXK1, HXK2 and GLK1) appear to mediate C-flux and signalling in yeast. Any of these can initiate sugar signals for rapid gene expression, whereas sustained glucose repression requires HXK2, and to some extent HXK1 (De Winde et al., 1996). Plants, in contrast to yeast, have more types of hexokinases, glucokinases and fructokinases with diverse properties and patterns of expression (Bouney and Saglio, 1996; Fox et al., 1998; Kanayama et al., 1998; Martinez-Barajas et al., 1997; Renz and Stitt, 1993). Balance between them can vary considerably. The significance of this situation may have some analogy to that in humans, where only one of four hexokinases (glucokinases) affects sugarsensing (Bell et al., 1996). This sensing hexokinase has the weakest affinity (highest K ) for glucose, so sugar m signals are minimal until glucose concentrations rise above the capacity for their use by the high-affinity hexokinases. In plants, the preferential utilization of glucose versus fructose substrates can be marked for a given sugar kinase (Martinez-Barajas et al., 1997; Renz and Stitt, 1993), and as noted above for yeast, this could translate into differential signals or sensing capacities (De Winde et al., 1996). In addition, biochemical analyses have revealed an unusual substrate inhibition for some of these enzymes (Martinez-Barajas et al., 1997; Renz and Stitt, 1993), that could conceivably affect a key aspect of protein configuration. Finally, plant hexokinase profiles change during development (Martinez-Barajas et al., 1997), and can be strongly altered under specific environmental conditions (Fox et al., 1998). The capacity of a given tissue

for sugar sensing via hexokinase-linked signalling may thus also change over time under given conditions. Sugar- and oxygen-sensitivity of hexokinases Low oxygen can markedly alter respiration and C-flux through hexokinases. Since sugar signals are hypothesized to arise from C-flux through the same reaction, this provides a point of potential overlap. Respiratory regulation of hexokinases may include adenylate effects on the reaction, and/or less direct influence via overall C-flux through glycolysis. Adenylate effects on hexokinase are also often discounted because of a general abundance of ATP, the very low K of hexokinase for ATP, and a K m eq dominated by glucose/glucose P (Roscher et al., 1998). However, recent work indicates that plant gluco- and fructokinases may be responsive to adenylates under physiological conditions ( Kanayama et al., 1998; Martinez-Barajas et al., 1997; Renz and Stitt, 1993). Further, hexokinases associated with the outer mitochondrial envelope may be preferentially supplied by mitochondrial versus cytoplasmic sources of ATP as conditions change (especially oxygen availability) (Rasschaer and Malaisse, 1990), providing a potential means of distinguishing changes in balance between glycolysis and mitochondrial respiration. In a broader context as well, adenylate balance and respiratory C-flux can show a reasonable relationship in response to oxygen and carbohydrate availability despite dominant regulation of plant glycolysis at downstream rather than upstream points in the path (Farrar and Williams, 1991; Plaxton, 1996; Roscher et al., 1998). Recent NMR analyses of maize root tips has also shown that onset of hypoxia clearly increased glycolytic rate (presumably including C-flux through hexokinase) and a modest decrease in ATP/ADP balance (Roscher et al., 1998). Sugar signalling could well be affected in each of these instances if the enhanced C-flux included a sensing hexokinase. At least two sugarinducible genes are up-regulated under hypoxia (data presented here; Zeng et al. 1998), despite expected decreases in sugar supplies under low-oxygen stress (Drew, 1997; Vartapetian and Jackson, 1997). Other aspects of low oxygen stress could also affect a hexokinase-based path of sugar sensing. In maize root tips, hypoxia can increase overall activity of glucokinase (Bouny and Saglio, 1996), and these conditions (rising ADP) are predicted to inhibit a mitochondrial, but not cytoplasmic maize hexokinase (Galina et al., 1995). In addition, changes in the patterns of hexokinase proteins present under low-oxygen have been reported (Fox et al., 1998), which could affect signalling if plant mechanisms were similar to those in yeast, where glucokinases and hexokinases can, respectively, elicit transient versus longer-term sugar responses. Finally, low oxygen could influence the extent to which gluco-, fructo- or hexokin-

Sugar-sensing and oxygen ases are associated with other proteins theoretically important to their dual roles as signal generators.

Membrane-linked sugar sensing and its potential role under low oxygen Membrane-linked sugar signalling paths are distinct from those of hexokinase sensing in yeast (Fig. 3). Present models suggest that some sugar signals in yeast are initiated by membrane sensors (RGT2 and SNF3) in response to different concentrations of exogenous sugars.

Fig. 3. The membrane-sensor-linked sugar signalling path in yeast mediates expression of ‘feast’ genes through both glucose induction and starvation repression. In contrast, starvation-induced ‘famine’ genes in yeast are up-regulated by a different path linked to hexokinases (Fig. 2). (1) Signal: signals can originate from two transporter homologues that differ from others in having distinctive cytoplasmic sites, sugar binding features, and minimal actual transport function. Signals of high glucose levels are generated from RGT2 with its low affinity for glucose, and lower levels of glucose can be sensed by SNF3 due to its higher affinity. (2) Transduction: initial phases are unclear, and remain undefined for a portion of the high-glucose, RGT2-linked path. For other signals, however, interconversions of an SCF complex are central and direct changes in the RGT transcription factor. (3) Transcription: the DNA-binding protein RGT1 becomes an activator of glucoseinduced genes if it is modified by the SCF complex such that it can no longer recruit the TUP1/SSN6(CYC8) repressor proteins. This model and its relationship to induction of feast versus famine genes is based on sugar-sensing information from yeast (Johnston, 1999; Lesage et al., 1996) and thus far is consistent with information from plants (see text; Lalonde et al., 1999). In addition, sugar sensing in plants appears to include sucrose-specific changes in expression of a membrane-bound sucrose transporter (Chiou and Bush, 1998: Lalonde et al., 1999).

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These sensors appear to have little or no role in transport per se, but exhibit distinctly different affinities for substrates either by interacting directly with sugars or indirectly with sugar transporters (Johnston, 1999; Lalonde et al., 1999). The actual signal in yeast appears to be initiated from membrane-bound sugar sensors distinguished by cytoplasmic tails that are not present in homologous, nonsensing sugar transporters (Fig. 3). Downstream are at least two transduction paths, one of which involves a multiprotein SCF complex. This complex can direct conversion of a transcriptional repressor to an activator. It does so by targeting ubiquination or similar modification of a DNA-binding protein (RGT1), which becomes an activator when it loses its capacity to recruit repressor proteins (typically TUP1 and SSN6 [CYC8] that also repress oxygen-regulated genes [Jang and Sheen, 1997]). This dual role of RGT1 as an activator and a repressor gives this membrane-linked transduction path the capacity to both up- or down-regulate genes depending on exogenous sugar concentrations. In yeast, this mechanism appears to contrast to that of hexokinase-linked signalling by mediating the up- and down-regulation of ‘feast’ rather than ‘famine’ genes (Johnston, 1999). Several lines of evidence indicate analogous transmembrane sugar sensors may function in higher plants. First, compounds that function as substrates for glucose transporters, but not hexokinases, can induce specific genes. Some of the genes encoding sucrose synthases, extracellular invertases, and patatin storage proteins respond in this way to 3-O-methylglucose and 6-deoxyglucose, consistent with the suggested involvement of membrane-linked signals (Godt et al., 1995; Roitsch et al., 1995; Martin et al., 1997). In addition, data from transgenic plants with yeast invertase targeted to vacuole, cytoplasm and extracellular spaces, indicated that elevated hexose concentrations might affect expression of some genes only in instances where membrane transit by sugars was involved (Heineke et al., 1992; Herbers et al., 1996). Also, similarities are emerging for putative hexose sensors in the membranes of yeast and plants. These proteins in both organisms show distinct cytoplasmic domains, with tails in yeast (Johnston, 1999) and internal loops or ‘bellies’ in plants (Smeekens and Rook, 1997; Lalonde et al., 1999). In addition, a sucrose-specific sensing system has been described which appears to mediate its expression of a sucrose transporter (Chiou and Bush, 1998; Lalonde et al., 1999). The potential for oxygen modulation of membranelinked sugar signals is likely to differ between hypoxia and anoxia. Membrane functions are rapidly curtailed under low oxygen stress since they are one of the most costly aspects of cell maintenance (Hochachka et al., 1996). Plasmamembrane H+-ATPase activity and protonmotive force both drop quickly under oxygen deprivation,

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and the latter was found to be especially important to the cessation of sugar transport under anoxia ( Xia and Saglio, 1990). During the progression of this stress, however, hexose transport was unaffected until ATP concentrations dropped below 70%. Recent NMR evidence indicates that this degree of ATP reduction is unlikely to occur under modest hypoxia (Roscher et al., 1998). If so, then it is possible that membrane-linked sugar signalling could remain operative until oxygen stress becomes severe. This would be consistent with differential hypoxic versus anoxic up-regulation of sugar-sensitive sucrose synthases otherwise induced by carbohydrate abundance versus deprivation, respectively (see below). Cessation of sugar signals from membrane-linked sensors could be advantageous under anoxia since this transduction path otherwise up-regulates ‘feast’ genes (typically detrimental under low oxygen, except for those encoding glycolytic enzymes).

Other paths of sugar sensing and points of oxygen sensitivity Acetate and other respiratory metabolites can influence sugar-responsive genes and are also sensitive to oxygen availability. Acetate can repress or induce sugarresponsive genes ( Koch, 1996, and unpublished data), and may do so in part from the alteration of cytoplasmic pH (acetate crosses membranes readily). Even slight changes in pH can have marked effects on processes such as glycolytic C-flux to pyruvate, adenylate cycling and concentrations of intermediates in plants (Plaxton, 1996). Acetate and low oxygen may thus share some aspects of influence on sugar-responsive gene expression. In addition, both acetate and acetaldehyde can arise during oxidative metabolism of ethanol. The adenylate balance responds to low oxygen and could alter sugar signals at several points. One of these is the effect of adenylate regulation on C-flux through glycolysis and hence the potential for sugar signals from hexokinases. Another is the likely influence of ATP depletion on membrane-based sensing systems, but as noted above, these might be less sensitive to modest changes in energy charge. In addition, both types of sensing paths could be controlled at downstream sites by regulation of SNF1-like or SCF-related multi-protein complexes in their transduction cascades. These complexes are prime candidates for input by adenylate balance. Finally, these points of adenylate influence, especially that of the SNF1like complexes, have also been suggested to have independent roles as initiators of sugar signals (Halford and Hardie, 1999). This is supported by the alteration of sugar responsiveness in one of the sucrose synthase genes in potato when plants were transformed to reduce expression of the SNF1-like kinase (SnRK1) in these plants (Purcell et al., 1998). Concurrent functions of this kinase

include co-ordination of key enzyme activities via phosphorylation. As in yeast, these roles may vary depending on the identity of associated proteins in each complex. CDPKs (calcium dependent protein kinases) comprise a distinctive feature of sugar signal transduction in plants (Ohto and Nakamura, 1995; Ohto et al., 1995). These kinases are present only in plants and a limited number of mainly unicellular organisms (Roberts and Harmon, 1992). The mechanism of CDPK input into sugar signalling remains unclear, but they remain primary candidates for integrating and transducing information on changing Ca availability that occur during shifts in both sugar and oxygen availability. The involvement of 14-3-3 proteins in sugar sensing is also implicated, and is supported by circumstantial evidence such as their binding to key proteins and DNA sequences. In addition, these sites may be initially phosphorylated by SNF1-like proteins or CDPKs (Douglas et al., 1997; Sugden et al., 1999). The proteins include enzymes of plant C and N resource allocation (Douglas et al., 1997; Moorhead et al., 1999; Sugden et al., 1999), cellular signalling (C MacKintosh, personal communication), and DNA-binding proteins (Ferl, 1996) that can affect sugar- and oxygen responsive genes examined thus far (RJ Ferl, unpublished data; Y Wu and K Koch, unpublished data).

Evidence for overlap in sugar and oxygen responses of genes for sucrose and ethanol metabolism Responsiveness of sucrose synthases to sugars, sensing paths, and oxygen Sugar-modulated features of sucrose synthases are compatible with both the hexokinase-linked and membranesensor-based paths of sugar signalling depending on which of the genes is being considered. The Sus1 and Sh1 genes for sucrose synthases in maize have different responses to sugars, which was unexpected ( Koch et al., 1992). They were, respectively, up- and down-regulated by carbohydrate availability. This provides a means of enhancing sucrose use when it is an abundant resource, and prioritizing key cells for import when supplies are limited. Similar overall responses are also evident among sucrose synthase isozymes of other species ( Koch et al., 1996). The mechanisms for these contrasting effects may be complex. Involvement of a hexokinase-linked path of sugar signal transduction is possible for at least some of the responses. Effects on the Sh1 gene would be compatible with the role of hexokinase-linked regulation of starvation genes in yeast (Johnston, 1999), and consistent with respiratory responses to altered sugar availability (Farrar and Williams, 1991; Plaxton, 1996). Sugar sensing pathways that regulate sucrose synthases

Sugar-sensing and oxygen and invertases may differ with sugar-response classes. Among each of the gene families are members that can be induced by carbohydrate feast or famine ( Koch et al., 1992; Xu et al., 1996). In yeast, such genes are, respectively, regulated by hexokinase- and membrane-sensor based paths (Johnston, 1999). A similar delineation between roles for different sensors may be less defined in plants (Jang et al., 1997). However, inductive responses of an extracellular invertase and a sugar-inducible sucrose synthase were both mimicked by glucose analogues that were substrates for membrane sensors, but not hexokinases (Godt et al., 1995; Roitsch et al., 1995; Smeekens and Rook, 1997). In addition, Arabidopsis plants transformed to increase or decrease expression of their own hexokinase did not show marked changes in expression of the invertases examined (JC Jang, personal communication). However, when antisense sequences of a homologue to a SNF1 component of the hexokinase-sensing path were expressed in potato, sucrose inducibility of a sucrose synthase was lost (Purcell et al., 1998). This could be interpreted as supporting an inductive role for hexokinase-linked signalling in plants, but other explanations are also possible considering the number of substrates for SNF1-type kinases (Douglas et al., 1997; Sugden et al., 1999). The latter include post-transcriptional regulation of numerous enzymes, in addition to signalling roles. Oxygen sensitivity of sucrose synthases has been reported for the Sh1 and Sus1 genes in maize at both the mRNA (McCarty et al., 1986; Talercio and Chourey, 1989) and protein abundance (Springer et al., 1986; Bailey-Serres et al., 1988). However, the extent of these responses varied between the two genes and much of the work had involved single-time-point studies. It was therefore not clear whether contrasting aspects of the responses were related to the time point of sampling, the duration of low oxygen stress, or the degree of hypoxia versus anoxia. Each of these were examined for the two differentially sugar-responsive sucrose synthase genes (Zeng et al.,1998).

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availability. Alternately, a membrane sensor could also be sensitive to oxygen input if membrane transport were involved. Oxygen effects on C-flux differ for hypoxia and anoxia. Analyses of maize root tips have indicated that glycolytic rates rise under hypoxia, but rapidly collapse under anoxia in this system (reviewed by Vartapetian and Jackson, 1997). NMR analyses of living maize root tips also show marked rises in C-flux with hypoxia, despite only about 20–30% shifts in adenylate balance (Roescher et al., 1998). Other approaches have yielded similar results (Drew 1997; Vartapetian and Jackson, 1997). Oxygen treatments employed here (Fig. 3) might thus have had comparable effects on C-flux as did those of limited versus abundant supplies of sugars. The consistent contrast in response of the Sus1 and Sh1 sucrose synthase genes under these conditions is compatible with predicted results of hexokinase-linked signalling, but membrane-linked signals may be involved for Sus1. Overlap between sugar and oxygen signals does not include invertase responses Invertases may be subject to different means of sugar and/or oxygen regulation. Although the pattern of sugar responses by the Ivr1 and Ivr2 invertases paralleled that of the two sucrose synthases shown in Fig. 4, both invertases were rapidly down-regulated under hypoxia or anoxia (data not shown). This suggests that one or more aspects of their regulation differs from that of the sucrose synthase genes. In yeast, the Suc2 gene for invertase has been a model reporter for dissecting the hexokinaselinked path of sugar sensing (Gancedo, 1998; Johnston, 1999). However, plant invertases appear to be regulated differently. As noted above, Arabidopsis invertases did

‘Feast and famine’ versus hypoxia and anoxia for Sus1 and Sh1 A striking parallel is evident in Fig. 3 between differential responses of the maize sucrose synthase genes (Sus1 and Sh1) to contrasts in sugar availability ( limited versus abundant in Fig. 3A), and to differences oxygen supply (anoxia [0% O ] versus hypoxia [3% O ] in Fig. 3B). This 2 2 comparison was undertaken to test whether variations in the oxygen responses of the sucrose synthase genes showed predicted similarities to contrasts in carbohydrate regulation based on current models for sugar signalling. A carbohydrate sensing system linked to C-flux through hexokinase could potentially respond to changes in glycolytic rate brought about by either sugar or oxygen

Fig. 4. Relative abundance of Sh1 and Sus1 sucrose synthase mRNA in maize root tips after 24 h of (A) depleted versus abundant sugar supplies (0.2% glucose [11 mM ] versus 2.0% glucose [111 mM ]) or (B) anoxia versus hypoxia (0% O versus 3% O [0.03 l l−1]) in the presence 2 2 of 2% glucose. For sugar treatments, 1 cm root tips were excised and cultured in aerated White’s nutrient medium with glucose or osmotic controls (-glucose and pentaerythritol [not shown]). For oxygen experiments, root tips were excised for analyses after whole-seedling treatments (airflow c. 1 l min−1) c. 5 d after germination. Anoxic treatments were probably preceded by short periods of hypoxia during oxygen depletion. RNA was extracted and quantified as in Xu et al. ( Xu et al., 1996). ((A) was compiled from data in Koch et al., 1992, and (B) from data in Zeng et al., 1998; in each instance, bars represent means±SE of three separate experiments.)

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not respond to transgenic manipulation of hexokinase activity (JC Jang, personal communication), nor were they altered in potato plants with antisense sequences for a SNF1 homologue (Purcell et al., 1998). Contrasting responses of sucrose synthases and invertases in the latter could indicate potentially different modes of regulation. It is not surprising, therefore, that oxygen responses of invertases may also differ despite initially observed similarities in patterns of sugar modulation among invertase and sucrose synthase family members. Anaerobic regulatory mechanisms may also override sugar sensing for many genes. Under low oxygen, a strong limitation is imposed on synthesis of all but a very few proteins in plants (Sachs et al., 1996; Vartapetian and Jackson, 1997). In many instances, this involves still more stringent regulation at the translational than transcriptional levels (Fennoy et al., 1995, 1998). Resultant decreases in protein biosynthesis can provide one of the most prominent reductions in ATP costs under lowoxygen conditions (Hochachka et al., 1996; Vartapetian and Jackson, 1997). This limited expression also indicates that readily testable instances of overlap between sugar and oxygen signalling may be restricted to relatively few genes. Especially prominent among these are likely to be genes that facilitate use of sucrose while limiting ATP consumption. Advantages of invertase repression under low oxygen may extend beyond costs of protein biosynthesis alone. NMR data from maize root tips, together with earlier studies, indicate that invertases can contribute prominently to ‘futile’ cycling of sugars, and that this uses considerable ATP (Dieuaide-Noubhani et al, 1995). Sugar cycling also occurs in other species (often as glucose resynthesis), where it constitutes a significant energy cost that is rapidly repressed under low oxygen to conserve ATP (Hochachka et al., 1996). Invertases may also be disadvantageous under low oxygen due to their production of twice as many moles of hexoses (gluc+fruc) from sucrose than does sucrose synthase (fruc+UDPgluc). Possible differences in metabolic influence of these different products are debated (Drew, 1997), but sucrose is clearly preferred over hexoses as a carbon source during anoxia or low-oxygen (Germain et al., 1997). In addition, the invertase reaction produces double the substrates for up-regulation of other hexose- responsive genes. Although some of these would be advantageous under low oxygen (e.g. for enhanced glycolysis), others could enhance unwanted C-costs of biosynthetic processes (e.g. starch and protein storage). Sugar-responsiveness of Adh1 Alcohol dehydrogenase1 responds to sugars in fullyoxygenated maize root tips. Figure 5 shows that Adh1

Fig. 5. Relative abundance of Adh1 alcohol dehydrogenase mRNA in maize root tips after 24 h exposure to different sugar concentrations (%, w/v) under fully oxygenated (40% O [0.4 l l−1]) or anaerobic 2 conditions (0–3% O [0.03 l l−1]). Root tips (1.0 cm) were excised and 2 cultured as described for Fig. 4, except that air flow through culture vessels was maintained at c. 50 ml min−1. -glucose was used as a nonmetabolizable osmotic control. Glucose concentrations of 0.2, 0.5, 2.0, and 4.0% (w/v) corresponded to 11, 27, 111, and 222 mM, respectively, with 2.0% most closely approximating endogenous concentrations in intact controls. Loading controls were provided by actin mRNA abundance (Nairn et al., 1988).

can be induced by sugars at physiological concentrations (between 0 and 222 mM glucose) even when root tips were aerated with 40% O . The possibility remains that 2 sugars can induce endogenous hypoxia, although internally low oxygen is common in vivo (Ober and Sharp, 1996; Drew, 1997; Thomson and Greenway, 1991), and is likely minimized here by the high concentration of exogenous oxygen utilized. Other stresses can also affect Adh1 expression (Dolferus et al., 1994), or the rate of O 2 use, but use of -glucose as a non-metabolizable glucose control rules out osmotic effects in the present study (results of other osmotic controls were similar but are not shown). Clear effects of sugar abundance were observed at each glucose concentration tested (Fig. 5), and maximal sugar-induction of Adh1 in aerated root tips was equivalent to that observed under low oxygen. At least some contribution from a sugar-sensing system appears to be likely. Sugar-responsiveness of Adh1 persists under hypoxia and anoxia. Figure 6 indicates that oxygen influence on amounts of Adh1 mRNA is affected by sugar availability. Message abundance increased with glucose supplies under both hypoxia and anoxia, and required less sugar to do so under hypoxia. This is consistent with expected effects of hypoxia versus anoxia on overall carbohydrate balance

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Concluding comments A review of existing evidence for mechanisms of sugar sensing in yeast and plants indicates that there are several points of potential sensitivity to oxygen. Analysis of maize Sus1 and Sh1 sucrose synthases, invertases, and Adh1 alcohol dehydrogenase shows that responses to sugar and oxygen overlap for key genes of sucrose and ethanol metabolism, but in different ways. Results are consistent with the presence of multiple paths for sugar sensing, each of which may regulate genes having similar overall roles (e.g. during feast, famine, or pathogen attack).

References

Fig. 6. Relative abundance of Adh1 alcohol dehydrogenase mRNA in maize root tips after 24 h exposure to different sugar concentrations under (A) hypoxia (3% O ) and (B) anoxia (0% O ). Root tips (1.0 cm) 2 2 were excised and cultured as described for Fig. 3, except that airflow through the culture vessels was maintained at c. 50 ml min−1. -glucose was used as a non-metabolizable osmotic control. Glucose concentrations of 0.2, 0.5, 2.0, and 4.0% (w/v) corresponded to 11, 27, 111, and 222 mM, respectively, with 2.0% most closely approximating endogenous concentrations in intact, aerobic controls. Loading controls were provided by actin mRNA abundance (Nairn et al., 1988).

(Bouny and Saglio, 1996; Drew 1998; Vartapetian and Jackson, 1997), and may or may not involve additional effects on signalling paths. Either way, Adh1 induction is a sugar-modulated process at all oxygen concentrations tested. This facet of its regulation could have considerable significance to cells exposed to endogenous hypoxia or to flooding stresses.

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