Nutrient sensing in plant meristems - Springer Link

Ó Springer 2006

Plant Molecular Biology (2006) 60:981–993 DOI 10.1007/s11103-005-5749-3

Nutrient sensing in plant meristems Dennis Francis1,2,* and Nigel G. Halford1,2 1

School of Biosciences, Cardiff University, PO Box 915, Cardiff CF72 9DU, UK; 2Crop Performance and Improvement, Rothamsted Research, Harpenden AL5 2JQ, Hertfordshire, UK (*author for correspondence; e-mail [email protected])

Received 20 May 2005; accepted in revised form 5 December 2005

Key words: cell cycle, meristems, nutrient sensing, sugar sensing

Abstract Plants need nutrient to grow and plant cells need nutrient to divide. The meristems are the factories and cells that are left behind will expand and differentiate. However, meristems are not simple homogenous entities; cells in different parts of the meristem do different things. Positional cues operate that can fate cells into different tissue domains. However, founder/stem cells persist in specific locations within the meristem e.g. the quiescent centre of root apical meristem (RAM) and the lower half of the central zone of the shoot apical meristem (SAM). Given the complexity of meristems, do their cells simply respond to a diffusing gradient of photosynthate? This in turn begs the question, why do stem cell populations tend to have longer cell cycles than their immediate descendants given that like all other cells they are directly in the path of diffusing nutrient? In this review, we have examined the extent to which nutrient sensing might be operating in meristems. The scene is set for sugar sensing, the plant cell cycle, SAMs and RAMs. Special emphasis is given to the metabolic regulator, SnRK1 (SNF1-related protein kinase 1), hexokinase and the trehalose pathway in relation to sugar sensing. The unique plant cell cycle gene, cylin-dependent kinase B1;1 may have evolved to be particularly responsive to sugar signalling pathways. Also, the homeobox gene, STIMPY, emerges strongly as a link between sugar sensing, plant cell proliferation and development. Flowering can be influenced by sucrose and glucose levels and both meristem identity and organ identity genes could well be differentially sensitive to sucrose and glucose signals. We also describe how meristems deal with extra photosynthate as a result of exposure to elevated CO2. What we review are numerous instances of how developmental processes can be affected by sugars/nutrients. However, given the scarcity of knowledge we are unable to provide uncontested links between nutrient sensing and specific activities in meristems.

Introduction Higher plant meristems produce new cells that either remain in the proliferative pool or become non-proliferative as the meristem grows away from them. It is cells left by the meristems that undergo massive cell elongation, the main driver of growth. When elongation stops, most cells differentiate (e.g. tracheids and vessels). In the 1960s, Jack Van’t Hof conducted a series of experiments demonstrating that meristematic

cells were dependent on carbon supply. When pea root tips were cultured in medium depleted of sucrose, cells arrested in G1 or G2 and the roots stopped growing (Van’t Hof, 1966). They also arrested if supplied with uncouplers of oxidative phosphorylation even if those roots were supplied with a carbon source (Van’t Hof and Webster, 1973). Similarly, plant cells in culture will arrest if deprived of phosphate (T. Nagata, pers comm.). Of course much of this now seems entirely predictable but the fine sensing of nutrient delivery

982 to potential meristematic cells is not well understood. In root apical meristems (RAM) the rate of cell division drops to zero in the quiescent centre, a group of sub-terminal stem cells/founder cells in the RAM (Clowes, 1958). If there is a general diffusion gradient of nutrient into the meristems, why do cells of the quiescent centre remain inactive? Similarly, the shoot apical meristem (SAM), and youngest leaf primordia are sinks for nutrient. However, the SAM often comprises a basipetal gradient of cell division rates and cell size from the apex to the flanks. How could nutrient influence this gradient? How do meristems deal with elevated CO2? In this review, we seek answers to these questions by exploring cross-talk between cell cycle genes, homeobox genes and sugar sensing genes. The review will be neither an exhaustive cell cycle nor a detailed sugar sensing review. Both topics have been covered recently (DeWitte and Murray, 2003; Halford and Paul, 2003; Francis, 2003). Neither will it represent detailed reviews of either RAMs or SAMs (See Byrne et al., 2003; Vernoux and Benfey, 2005; Veit, 2004 and chapters this volume). Our aim is to assess putative links between nutrient sensing with the plant cell cycle and with development. We will examine the extent to which cell cycle genes or developmental genes have an obvious or cryptic need for sucrose sensing/signalling. Then we consider meristems in a developmental sense and ask whether sugars could play a role in regulating flowering, in regulating the expression of homeotic genes in SAMs and in regulating gene expression that is necessary for RAM function. What follows is setting the scene of meristems, development and the cell cycle. Against this background, sugar signalling is discussed and then we look for links between sensing and meristems.

The meristems Vegetative shoot apical meristem From one plastochron to the next, the shoot apical meristem (SAM) increases in size by cell division (Lyndon, 1976). At its maximum size, and through cell division, the SAM forms a bulge on its side and a sub-apical region is partitioned from

its base. As the SAM returns to its minimum size, the bulge becomes the next leaf primordium which enlarges through cell division for a while prior to cell expansion that drives growth and shape of the leaf (Dale, 1986). Also, maybe at this stage, a tiny cohort of cells in the most apical part of the primordium (at the axil between the SAM and primordium) is fated to form the next axillary bud. In Arabidopsis, the homeobox gene, WUSCHEL, is expressed very early in embryogenesis and indeed before the morphological appearance of the embryonic SAM; wus mutants cannot make a functional SAM (Mayer et al., 1998). Broadly speaking, WUS is a stem cell regulator and is expressed in a small population of cells within the lower half of the central zone, underneath the stem cells but not in stem cells themselves; WUS expressing cells prevent differentiation of the overlying cells. However, WUS expression is itself restricted through the expression of the nonhomeobox CLAVATA (CLV) family that operates in a negative feed-back loop to WUS (Schoof et al., 2000). CLV3 encodes a small polypeptide which may be a ligand for the CLV1 encoded receptor-like kinase. CLV3 expression coincides with the presumed stem cell region and thus is used as a marker for stem cells. CLV 1 then participates in a mechanism that negatively regulates the size of the WUS-expressing region (Brand et al., 2000, Schoof et al., 2000). Consistent with this model is that in clv mutants, WUS expression expands both acropetally and laterally, and the mutant forms a highly expanded SAM (Clark et al., 1996). CLV2 is thought to be an accessory protein that stabilises CLV1 but it is expressed in a domain of mainly L3 (corpus) cells that completely encompasses the cells that express WUS. CLV3 expression is restricted to layers of the central zone above the WUS-expressing cells in the vegetative shoot meristem (Figure 1). SHOOTMERISTEMLESS (STM) (Barton and Poethig, 1993; Long et al., 1996) is first expressed in the rudimentary SAM of the embryo. As development continues, STM expression is necessary to maintain the integrity of the SAM; wherever it is expressed leaves do not form (Long et al., 1996). STM over-expression can result in SAM formation while ectopic STM and WUS expression can induce ectopic CLV expression and ectopic SAMs (Gallois et al., 2002).

983 STM CLV3 CLV1

WUS

CLV1

STIP CZ ANT

MGO

PZ

PRM

PZ

HMG CoA Isoprenoid Cytokinin reductase pathway Side-chain? sugar

SnRK1

Figure 1. A general schematic of a vegetative shoot apical meristem that has just initiated a leaf primordium. The apical dome is partitioned into three histological zones zones: Central Zone (CZ), Peripheral zone (PZ) and Pith Rib Meristem (PRM). It shows interaction between some of the genes and proteins known to regulate vegetative growth of the SAM (see text). Below the cartoon is a small part of the metabolic pathway that involves SnRK1 and contributes to cytokinin biosynthesis. The dotted arrows represent PUTATIVE signalling links between sucrose and cytokinins to SAM gene function. Note that the links between genes are based on published information but this is not a portrayal of all genes known to function in the vegetative SAM. The dotted arrow are hypothesised links between nutrient induced signalling and SAM genes. STM=SHOOTMERISTEMLESS, CLV=CLAVATA, WUS=WUSCHEL, STIP=STIMPY, MGO=MGOUN, ANT=AINTEGUMENTA.

Nougarede (1967) identified the transition of cell from the central to the peripheral zone as a key event prior to leaf initiation. During the transition of cells from the central to peripheral zone (PZ), the MGOUN (MGO) gene family probably promote the transition of PZ cells to leaf founder cells (Laufs et al., 1998); primordial outgrowth is then stimulated by AINTEGUMENTA expression, which is restricted to the primordium itself (Elliot et al., 1996). Note that STM and ANT have mutually exclusive expression patterns in the vegetative SAM. This rather brief survey of gene expression (which does not include all functional genes) in vegetative SAMs must require signalling systems and, as noted by Lenhard et al. (2001), WUS is the recipient of signalling which maintains a stem cell population. So is it the general diffusion of nutrient to all zones of the apex that under-pins

or regulates the expression of WUS, STM and the CLVs or, does it depend upon a more subtle nutrient-led signalling pathway? In other words, is nutrient simply affecting metabolism in different parts of the meristem or is it being sensed or does it initiate signalling to the network described above? In this regard, the homeobox gene STIMPY/ WOX9, is an essential gene for growth of the vegetative SAM and it positively regulates WUS expression (Wu et al., 2005). stip mutants can be rescued by sucrose, which in turn, causes more cells to divide. This work demonstrates a link between a homeobox gene with cell division, the driving force of vegetative SAM growth. Moreover the response of the stip mutant to exogenous sucrose suggest a pivotal role of STIP for cell cycle activation and coordination of developmental signals (Wu et al., 2005). There is accumulating evidence that cytokinin signalling can lead to activation of STM and WUS as the prerequisite for shoot morphogenesis (reviewed by Howell et al., 2003). However, it is unknown if cytokinin signalling is affected by the carbohydrate background in the SAM; notably, the expression of a D-type cyclins in vitro, is sensitive to both cytokinin and sugar backgrounds (Murray et al., 1998).

Floral SAMs The floral stimulus is synthesised in the leaves (Zeevart 1976). However, its precise biochemical make-up remains unknown; it may be a complex of substances that act together or synergistically to cause flowering. Upon the arrival of the floral stimulus in the SAM, it either initiates a terminal flower or switches to an inflorescence meristem. Carbohydrate has been proposed as one of those constituents (Bernier, 1998). Epidermal thin layers (ETLs) of tobacco can be manipulated to form roots, leaves or flowers according to the environment. ETLs from induced plants can form flowers but epidermal layers from vegetative plants cannot (Tran Thanh Van, 1981). Similarly, callus from flowering tobacco developed flowers when grown on a medium supplemented with glucose. However neither the presence nor absence of glucose had any ‘‘floral effect’’ on callus formed from vegetative plants (Chailakhyan et al., 1975). In Pharbitis nil, shoot apices cultured from plants one day

984 following induction formed carpels if cultured on 2% glucose but not on 2% sucrose, even if they were left for 10 weeks on sucrose (Durdan et al., 2000). Moreover, the expression of putative P. nil homologues to Arabidopsis carpel specific genes was higher in induced shoot apices cultured on glucose compared with sucrose (Parfitt et al., 2004). These switches from one developmental phase to another are just a few examples of the effects that sugars have on growth and development (Gaudin et al., 2000). Care is necessary when interpreting any nutrient effects on flowering because different species exhibit different types of floral morphogenesis. For example, while P. nil forms a terminal flower and other secondary flowers appear from floral buds, the Arabidopsis SAM stops producing leaf primordia and it becomes an inflorescence meristem that initiates secondary inflorescences that form flower primordia (Hempel and Feldman, 1994). In Arabidopss, the meristem identity genes, TERMINAL FLOWER (TFL1) and LEAFY are mutually exclusive because TFL1 and LFY are expressed strongly in the axial zone of the floral inflorescence and in the flower primordium, respectively (Schultz and Haughn, 1991; Alvarez et al., 1992; Weigel et al., 1993). tfl mutants are often limited to a solitary terminal flower whilst lfy mutants are deficient in almost all floral organs apart from occasional bracts. LFY is a transcription factor that activates another meristem identity gene, APETELLA1. Whilst ap1 mutants form the occasional flower, the double mutant lfy ap1, is unable to make flowers (Liljegren et al., 1999). Another meristem identity gene, CAULIFLOWER, is down-stream of LFY and AP1; the cal mutant does not have a distinct phenotype but double mutants cal ap1 develop into tinier versions of lfy ap1 (Liljegren et al., 1999). In the flower primordia, there is a floral meristem identity cascade: LFY on AP1 LFY +1 API on CAL. Meristem identity genes regulate organ identity genes that themselves govern the position of floral organs and floral whorls (see, for example, Weigel and Meyerowitz, 1993). Eventually, the SAM makes carpels specified by AGAMOUS or AG-like genes. Eventually, AG operates in a negative feed back loop to WUS that terminates further organ formation (Lohmann et al., 2001). Very recently, a link has been established between CDKE (HEN3) expression

and specification of stamens and carpels (Wang and Chen, 2004). This very elegant work is one of only a few reports of interfacing of cell cycle genes and development. However, the authors suggest that in this case, CDKE may be functioning outside of the cell cycle but we certainly need to know more about this interface. What could be the biochemical go-between for sugar signalling interfaces with homeotic genes? There is good evidence of a link between hexokinase, signalling and cytokinins (Wingler et al., 1998; Moore et al., 2003) but very little about signalling and floral homeotic genes.

Root apical meristems The RAM is relatively simpler than SAMs because it does not initiate organs and with its steady state growth kinetics, the meristems remains relatively constant in size as cells are left behind to elongate and differentiate in discrete tissue domains, e.g. epidermis, cortex and stele. The homozygous rootmeristemless– mutant of Arabidopsis forms a defective postembryonic RAM (Vernoux et al., 2000). The mutant phenotype has an extremely short root without any post-embryonic increase in cell numbers or cell files (Cheng et al., 1995). Notably, ROOTMERISTEMLESS is not a homeobox gene; it encodes the first enzyme, c-glutamylcysteine c-GCS of the glutathione (GSH) biosynthesis pathway. Indeed an rml- phenotype could also be visualised by treating wild type roots with an inhibitor of GSH biosynthesis but could be relieved by addition of GSH to rml1 seedlings (Cheng et al., 1995; Vernoux et al., 2000). Of interest here, ascorbate and dehydroascorbate could regulate cell division in the tobacco TBY-2 cell line. A peak in ascorbate levels but not GSH coincided with a peak in the mitotic index during an exponential growth phase of this cell line (De Pinto et al., 1999). rml mutants produce embryonic roots that either completely lack mitoses, or, they exhibit a limited number of cell divisions that lead to roots of less than 2.0 mm in length. In the same mutants, lateral root primordia begin to form through cell division but not beyond the formation of 17 cell. Remarkably, embryonic adventitious and lateral roots all arrest at about the same cell number. This would be consistent with the idea of a cell counter proposed by Lu¨ck et al. (1994), which operates on

985 transverse divisions that increase cell number within a lineage but not formative divisions that initiate two new lineages. Seventeen cell lengths is slightly puzzling, but 16 cell lengths would equate with four divisions based on the L-bootstrap theory of cell division in meristems which in turn assumes a doubling of cell length per cycle (Lu¨ck et al., 1994). Celenza and co-workers concluded that root initiation must occur in at least two stages, first the initiation/formation of a primordium, and second, the growth and development of the lateral root primordium across and out of primary tissue. Indeed, this has been confirmed genetically in Arabidopsis by analysing mutants that exhibit aberrant lateral root formation (alf). The alf1–1 mutation, an allele of rooty and sur1, overproduces IAA leading to a highly branched root phenotype. Conversely, the alf4–1 mutation will not respond to IAA resulting in a phenotype that exhibits a long primary root devoid of laterals. Lateral roots initiate in the alf3–1 mutant, but then arrest and die (DiDonato et al., 2004). Hence, the genetic analyses are entirely in line with the rml phenotype (reviewed by Malamy (2005). Clearly, the root must be able to respond to abiotic redox changes in their immediate external environment. Is this why RML is not remotely related to STM? Or will it be the case that a different ‘‘root meristemless’’ phenotype will be complemented by a ‘‘root homeobox gene’’ gene? Interestingly, in Vicia faba, although the rate of primordium formation per cm of root growth was independent of the rate of primary elongation, the number initiated per day increased in a linear fashion, with an increase in the rate at which primary roots lengthened (Thompson and MacLeod, 1981). When analysing the effects of nutrient in primary and secondary roots, this type of temporal relationship has to be accounted for.

The plant cell cycle In fission yeast the cell division cycle gene, cdc2, encodes a protein kinase that is phosphoregulated in late G2. When hitched to a partner cyclin through phosphorylation of its T161 residue, but denuded of phosphate at its Y15 by Cdc25 phosphatase, Cdc2 kinase drives cells into mitosis (Nurse, 1990). cdc2 has been cloned in virtually all major classes of eukaryotes including both higher and lower plants. Most homologues share a

conserved sequence, PSTAIRE, in the T-loop of the protein (Joubes et al., 2000) and are phosphoregulated at T14 in addition to Y15 (Norbury et al., 1991). In higher eukaryotes, T14 and Y15 residues are dephosphorylated by Cdc25. This occurs when the T-loop swings towards the region at or about T161/167 and in-so-doing, reveals ATP-binding sites adjacent to T14 and Y15 (Gould et al., 1991; Morgan, 1997). The most well-documented information is for fission yeast where T167 phosphorylation is more or less coincident with cyclin binding in this region of the Cdc2 kinase (Gould et al., 1991). Cdc2 is regulated positively by Cdc25 phosphatase, and negatively by Wee1 kinase which in turn is phosphoregulated by Nim1 (new inducer of mitosis) (Russell and Nurse, 1986, 1987a, b). In Arabidopsis, there are at least five classes of CDK, A through to F, although we know little about CDK C and E (but see above for the latter). Arath;CDKAs have the conserved PSTAIRE domain, they are expressed throughout the cell cycle with peaks of kinase activity in both G1/S and G2/M (Joubes et al., 2000). However, evolutionary divergence from the conserved PSTAIRE cdc2 has occurred in some of the other plant CDKs. For example, CDKBs are unique to plants, exhibiting either PPTLARE (CDKB1) or PPTTLRE (CDKB2) in place of PSTAIRE. PPTLARE CDKs are active during S, G2 and early M-phase while PPTLRE ones only function during G2 and M phase (Joubes et al., 2000). In alfalfa, there are at least four cell cycle genes that are expressed in G2: MsCDC2: A/B, D and F. The latter is expressed at the G2/M transition and the encoded F kinase localises to substrates within the plant mitotic machinery: preprophase band, perinuclear ring, mitotic spindle and phragmoplast (Me´sza´ros et al., 2000). CDKAs but neither CDKBs nor CDKFs can complement cdc28/cdc2temperature sensitive mutants of budding/fission yeast. Note that Arath; CDKB1;1 is expressed exclusively in proliferative regions of the plant (e.g. Sorrell et al., 2002). Moroever, over expression of fission yeast cdc25 induces a small cell size in tobacco BY-2 cells through premature CDKB;1 activity (Orchard et al., 2005). Hence, this gene is unique to plants, it is only expressed in G2 and is regarded as the major plant CDK for driving cells into division. So, could it be especially responsive to a sucrose/nutrient signalling pathway?

986 Homologues to wee1 have been widely identified among eukaryotes. Notably, in Arabidopsis, Arath;WEE1 is exclusively expressed in proliferating regions of the plant (Sorrell et al., 2002) and cdc25 homologues have been long known in animals (Edgar and O’Farrell, 1990; Alphey et al.,1992). Recently, and for the first time, a full-length plant CDC25 was identified in the alga, Ostreococcus tauri; this CDC25 also complemented the temperature sensitive fission yeast mutant, cdc25– (Khadaroo et al., 2004). In Arabidopsis, there is a small CDC25 encoding only the catalytic domain although it can dephosphorylate plant CDC2 in vitro (Landrieu et al., 2004) and it can induce a small mitotic cell length phenotype in wild type fission yeast (Sorrell et al., 2005). However, unlike CDKB1;1 and Arath;WEE1, it is expressed at a low level in all tissues examined in Arabidopsis (Sorrell et al., 2005). Phosphoregulation of CDKs at G2/M is almost certainly the same in all eukaryotes. However, the Arabidopsis CDC25 is unusual in only encoding a catalytic domain and for being expressed constitutively. Also, unlike the algal one, there is yet to be convincing evidence that Arath;CDC25 can complement the cdc25)ts mutant of fission yeast. Thus, in higher plants there might be additional regulatory polypeptides that contribute to the CDK complex; perhaps the regulatory domain of CDC25 is encoded elsewhere in the genome. Clearly we know that depriving meristems of sucrose results in cell arrest in G1 or G2 (Van’t Hof, 1966) but we know very little about sugar signalling that might be required to regulate G2/ M. In turn, we do not fully understand the role of Arath;WEE1 or Arath;CDC25 in regulating cell division. However, genes known to regulate G0/ G1/S do respond to sugar availability. For example, sucrose can control expression of Cyclin D2 and D3;1 genes in the G1 phase of Arabidopsis (Riou-Khamlichi et al., 2000; Murray, this volume). Similarly, the expression of three D-cyclin genes in the apical meristems of snapdragon (Antirrhinum majus) are modulated in part by sucrose (Gaudin et al., 2000).

Sugar sensing It has been known for a decade and a half that sugar levels affect the expression of plant genes.

First evidence of this was obtained by Sheen (1990) using photosynthetic gene promoter/reporter gene fusions to show that seven maize photosynthetic genes were repressed by glucose or sucrose in a maize protoplast system. Since then, genes encoding enzymes of the glyoxylate cycle, carbohydrate metabolism, defence responses and even potato storage proteins have been shown to respond to sugars (reviewed by Halford and Paul, 2003). In a micro-array experiment, Price et al. (2004) found that 444 Arabidopsis genes were up-regulated by glucose, including those involved in biotic and abiotic responses, carbohydrate metabolism, N metabolism, lipid metabolism, inositol metabolism, secondary metabolism, nucleic acid related activities, protein synthesis and degradation, transport, signal transduction (e.g. protein kinases and phosphatases, and transcription factors), hormone synthesis and cell growth or structure. A similar number (534) were downregulated.

Components of sugar sensing/signalling pathways SnRK1 Progress towards the elucidation of the signalling pathways and networks that are involved in sugar sensing in plants have relied heavily on discoveries already made in fungal (notably budding yeast (Saccharomyces cerevisiae)) and animal systems. The over-riding mechanism controlling carbon metabolism in budding yeast is glucose repression. Glucose, above a concentration of about 0.2% (w/v), affects the synthesis of dozens of enzymes, the utilisation of alternative carbon sources, gluconeogenesis, respiration and the biogenesis of mitochondria and peroxisomes (Dickinson, 1999). At the heart of the glucose repression signalling pathway in budding yeast is the sucrose nonfermenting-1 (SNF1) protein kinase (Celenza and Carlson, 1986). Remarkably, homologues of SNF1 are present in animals (AMP-activated protein kinase (AMPK)) and plants (SNF1-related protein kinase-1 (SnRK1)) (reviewed by Halford and Hardie, 1998; Halford and Paul, 2003). AMPK inactivates regulatory enzymes of ATPconsuming pathways, such as acetyl-CoA carboxylase (fatty acid synthesis) and is involved in glucose modulation of pyruvate kinase and fatty

987 acid synthase gene expression in liver cells (reviewed by Carling, 2004). SnRK1, the plant homologue of SNF1, will phosphorylate and inactivate HMG-CoA reductase, sucrose phosphate synthase (SPS) and nitrate reductase (NR) in vitro (reviewed by Halford and Hardie, 1998). It is also involved in the regulation of activity of ADP-glucose pyrophosphorylase (AGPase) through a different mechanism, redox modulation, in response to the availability of sucrose or trehalose (but not glucose) (Tiessen et al., 2003). SnRK1 also acts through the regulation of gene expression; for example, the expression of genes encoding sucrose synthase and a-amylase require SnRK1 activity in potato and wheat, respectively (Purcell et al., 1998; Laurie et al., 2003). Clearly, SnRK1 has the potential to affect carbon metabolism in different and profound ways. Hexokinase Hexokinase (HXK) has a metabolic function in catalyzing the conversion of glucose to glucose6-phosphate (the first stage in glycolysis), but it may also have a role in sensing and signalling glucose levels. This hypothesis has sparked controversy (Halford et al., 1999) but hexokinase has become widely regarded as a ubiquitous eukaryotic glucose sensor (Rolland et al., 2001). In plants, hexokinase has been implicated in the feedback control of photosynthetic gene expression (reviewed by Jang and Sheen, 1994, 1997). In recent experiments, Moore et al. (2003) used targeted mutagenesis of one of the Arabidopsis hexokinase genes, HXK1, to produce enzymes that retain signalling functions but not catalytic activities; this is good evidence of a role for hexokinase as a signalling protein. Nevertheless, there is yet to be a convincing hypothesis about how this signalling mechanism operates. Hexokinase signalling appears to affect gene expression, cell proliferation, root and inflorescence growth, leaf expansion and senescence; some of these effects may be mediated through cross-talk with light and hormone signalling (Wingler et al., 1998; Hwang and Sheen, 2001). Also glucose signalling can interact with auxin signalling to promote or inhibit growth, depending on tissue or glucose concentration (Moore et al., 2003).

The trehalose pathway The trehalose biosynthetic pathway involves two enzymes, trehalose phosphate synthase (TPS), which catalyses the formation of trehalose-6-phosphate (T6P) from glucose-6-phosphate and UDPglucose, and trehalose phosphate phosphatase (TPP), which catalyses the dephosphorylation of T6P to trehalose; trehalose is cleaved by another enzyme, trehalase. In yeast, this pathway regulates the flux of carbon into glycolysis through the control of hexokinase activity (TPS and T6P have an inhibitory effect on hexokinase) (Thevelein and Hohnmann, 1995), thereby ensuring that cellular ATP levels are not depleted through the build-up of intermediates in glycolysis (Bonini et al., 2000; Noubhani et al., 2000). The capacity to synthesise and degrade trehalose is ubiquitous in plants even though trehalose itself does not accumulate because of high trehalas activity (Bianchi et al., 1993; Drennan et al., 1993; Goddijn and van Dun, 1999). T6P functions in plants in a mechanism analogous to that in yeast which regulates flux through glycolysis (Schluepmann et al., 2003). What is sensed? As its name suggests, AMPK is activated allosterically by AMP (Carling et al., 1987, 1989), while activation of AMPK by AMP is antagonized by high concentrations of ATP. A high AMP:ATP ratio is symptomatic of low cellular energy levels and AMPK has been likened to a cellular fuel gauge (Hardie and Carling, 1997). So could AMP be sensed in plant cells as well? SnRK1 (like SNF1) is not activated allosterically by AMP in the way that AMPK is. Nevertheless, AMP can affect the phosphorylation state of SnRK1 (Sugden et al., 1999). Possibly, it acts on the protein kinase upstream of SnRK1 but this cannot be proven until that protein kinase is identified. What are the other candidates? As described above, glucose and sucrose treatments can influence flowering, but is it these metabolites themselves that are sensed or something else? Other candidate molecules include other hexoses and hexose phosphates, trehalose and trehalose 6-phosphate. If glucose is itself a signalling molecule then one possible mechanism through which it could be sensed is via its interaction with hexokinase.

988 Whether or how hexokinase-dependent or -independent hexose signalling pathways involve SnRK1 is not clear. SnRK1 is inhibited by glucose-6-phosphate in vitro (Toroser et al., 2000) but there is no convincing evidence to show that SnRK1 activity responds to feeding with glucose (or any other sugars). That SnRK1 is required for sucrose-induced redox activation of AGPase (Tiessen et al., 2003) and for the expression of sucrose synthase in potato tubers (Purcell et al., 1998) implicate it more convincingly in sucrose/ trehalose rather than glucose signalling (although it may be involved in both).

Sugar signalling and plant development Do we have any evidence about sugar signalling that could be linked to a developmental switch? Perturbation of SnRK1 activity has profound effects on growth and development. For example, antisense SnRK1 expression in potato tubers represses sprouting if the tubers are kept at 5 °C (Halford et al., 2003), and expression of an antisense SnRK1 sequence causes abnormal pollen development and male sterility in barley (Zhang et al., 2001). In Vicia faba, an increase in sucrose levels and a decrease in hexose levels in developing cotyledons caused a steep rise in sucrose synthase activity and the onset of starch biosynthesis (Weber et al., 1996). In other words a change in sucrose:glucose ratio initiated the switch from cell proliferation to the storage and cell expansion phase. As mentioned above there is substantial evidence that links SnRK1 to sugar signalling. Direct evidence for a role of SnRK1 in meristems was obtained by Pien et al. (2001) by undertaking a differential display analysis of tomato meristems destined to form leaves. The genes showing an asymmetric pattern of expression within the meristem included one encoding SnRK1, in addition to genes encoding sucrose synthase and AGPase. A possible role for SnRK1 in meristems could be to link sugar signalling to cell cycle control (Figure 2). There has been evidence of such a link in yeast for some time. For example, snf1 mutants fail to arrest in G1 phase of the cell cycle under conditions of nutrient deprivation (ThompsonJaeger et al., 1991) and have a shorter G1 period than the wild type (Aon and Cortassa, 1999).

Furthermore, SNF1 is related to NIK1 of Saccharomyces cerevisae and its homologue, nim1, in fission yeast. NIK1 interacts with the CDC28 complex and is a negative regulator of SWE1 (the budding yeast homologue to fission yeast wee1); SWE1 is involved in calcium-dependent control of mitosis (Tanaka and Nojima, 1996). Intriguingly, over-expression of SnRK1 in yeast cells results in a reduction in cell volume to onethird of the normal (Dickinson et al., 1999). It may be that as well as performing the functions of SNF1, SnRK1 functions like NIK1 when overexpressed, pushing the yeast cells prematurely into mitosis. Note that over-expression of the putative regulatory cell cycle genes, Arath;WEE1 and Arath;CDC25 in fission yeast, induce cell lengthening and shortening, respectively (Sorrell et al., 2002, 2005). The demonstration of a cell cycle function of SnRK1 in yeast suggests that SnRK1 could cross-talk with cell cycle signalling factors. Furthermore, expression of SnRK1 in the antisense orientation in a potato cell culture induced a large mitotic cell size (D. Francis and N.G. Halford, unpublished data). ELM1, one of the protein kinases upstream of SNF1 in yeast, has also been implicated in the regulation of SWE1. elm1 mutants have a prolonged mitotic delay, fail to regulate polar bud growth during mitosis and are defective in cytokinesis (Sreenivasan and Kellogg, 1999). In vegetative SAMs, the cell cycle is much longer than in the peripheral zone. As mentioned above, the transition of a cell from the central to peripheral zone recruits new PZ cells to be fated as leaf primordial cells (Nougarede, 1967). It also results in cell size changes, from largest in the CZ to smallest in the PZ. This is the type of transition that could be governed by a SnRK1-like signal thereby linking sugar supply to a shortened cell cycle and to development (Figure 2). Sugars may also affect growth and development through cross-talk with hormone signalling pathways. For example, there is now considerable evidence for a link between abscisic acid (ABA) and sugar signalling (reviewed by Rook and Bevan, 2003). Several Arabidopsis mutants that are impaired in their response to sugars (sugar response mutants) are also affected in their response to ABA, particularly with respect to germination and seedling establishment. Perhaps sugar signalling is directly mediated by ABA (Arenas-Huertero et al., 2000; Smeekens, 2000),

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Sucrose

Glucose

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Fructose Photosynthesis

Gene expression

Senescence

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Trehalose-6phosphate

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Sucrose

Glucose

Glucose-6phosphate ADP

Cell cycle

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Glycolysis TCA cycle Respiration

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Growth and development

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ATP

Metabolic enzyme activity Gene expression Figure 2. Network of carbohydrate biochemistry shown by solid arrows. Putative sugar sensing/signalling pathways involving SnRK1, hexokinase and the trehalose biosynthetic pathway (broken arrows). Also putative links between SnRK1 with cell cycle, growth and development and gene expression are indicated by broken arrows. The inset rectangle features disaccharide and monosaccharide movement across a double membrane.

or does ABA prime tissues to respond to sugars (Rook et al., 2001)? Alternatively, ABA and sugar signalling could be essentially separate but converge and cross-talk through specific factors (Halford and Paul, 2003). In Arabidopsis, the negative cell cycle regulator of plant CDKs, ICK1 (inhibitor of CDK activity), is up-regulated by ABA (Wang et al., 2000; Zhou et al., 2003a, b). In plants, it makes eminent sense to have a sugar/ABA/cell cycle signalling mechanism that would repress growth in unfavourable conditions and it would be a neat example of sugar signalling cross-talking with the cell cycle in meristems.

Elevated CO2 and plant cell cycles Exposure of plants to elevated levels of CO2 increases plant growth, a reasonably predictable

response. In Dactylis glomerata, exposed to elevated CO2 (700 ppm, the duration of the cell cycle shortened in both root and shoot meristems in two populations, one from northern Sweden and the other from southern Portugal. The magnitude of shortening of the cell cycle was about the same in these populations. However, on exposure to elevated CO2, the proportion of rapidly dividing cells was much higher in the Portuguese compared with the Swedish population (Kinsman et al., 1997). The data were consistent with the idea that in the Portuguese population exposed to elevated CO2, more cells entered the cell cycle from G0. An extension of this model would be that plant D-type cyclins might be critical receivers of nutrient signalling. However, we now require a clearer understanding of exactly how sugar signalling cross-talks with cell cycle control in meristems.

990 Conclusions

Acknowledgements

In this review, we have questioned the concept that nutrients diffusing to major sinks (RAMs and SAMs) randomly regulate the cell cycle and development. Both SAMs and RAMs comprise distinct populations of cells within which stem cells function. WUS expression is regulated by CLV proteins in the SAM and the same type of network may be operating in the quiescent centre of RAMS but little is known about this. But, in our view, if WUS expression is regulated by general percolation of nutrient, it cannot explain the precise spatial roles that stem cells play in plant meristems. Nor can it explain why STIP but not WUS is especially sensitive to sugar supply. Evidence has slowly accumulated that links sugars to flowering both for induction and for evocation. If involved in the big decision to go floral, can sugars induce differential effects on the expression of meristems identity genes or organ identity genes or both? We know a lot about the cell cycle, development and sugar signalling but there are still large gaps in our knowledge of the interface between each. For example, is signalling initiated by sucrose, glucose or another metabolite, such as T6P or AMP? Is it carbon flux that helps to regulate development? Furthermore, we know virtually nothing about the interfaces between cell cycle and sugar signalling networks. And what about, for example, the effects of nitrate on sugar signalling? In tomato, sucrose in a nitrate background is necessary to promote flowering (Dielen et al., 2001) and effects of elevated CO2 tend to be affected by nitrate availability. As noted by Gibson (2005) a whole host of developmental processes can be affected by sugars. Our original aim was to integrate what we know about meristems, sugar signalling, the cell cycle and development. We soon became aware of much contrivance with that approach. Instead we have tried to set a scene of cell cycle, and meristems and development as a background to understanding how nutrient sensing might work in meristems. Given the lack of definite links, much more work needs to be done.

Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom. We acknowledge the BBSRC for funding some of the work reported in this review.

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