Cyclic nucleotides in higher plants- the enduring paradox

New Phytol. (1999), 143, 427–455

Tansley Review No. 106 Cyclic nucleotides in higher plants : the enduring paradox RUSSELL P. NEWTON"*, LUC ROEF#, ERWIN WITTERS#  HARRY VAN ONCKELEN# " Biochemistry Group, School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK # Laboratorium voor Plantenbiochemie en -fysiologie, Department of Biology, Universiteit Antwerpen (UIA), Universiteitsplein 1, B-2610, Antwerp, Belgium Received 23 October 1998 ; accepted 17 May 1999  Summary I.  II.       c   III.       c   IV.   V. 

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VI. c-  , , c-   -  VII.    c VIII. c     IX.   References

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 For three decades, hypotheses relating to the occurrence and function of cyclic nucleotides in higher plants have been highly controversial. Although cyclic nucleotides had been shown to have key regulatory roles in animals and bacteria, investigations with higher plants in the 1970s and early 1980s were criticized on the basis of (i) a lack of specificity of effects apparently elicited by cyclic nucleotides, (ii) the equivocal identification of putative endogenous cyclic nucleotides and (iii) ambiguity in the identification of enzymes connected with cyclic nucleotide. More recent evidence based on more rigorous identification procedures has demonstrated conclusively the presence of cyclic nucleotides, nucleotidyl cyclases and cyclic nucleotide phosphodiesterases in higher plants, and has identified plant processes subject to regulation by cyclic nucleotides. Here we review the history of the debate, the recent evidence establishing the presence of these compounds and their role ; future research objectives are discussed.

I.  The hypothesis that adenosine 3h,5h-cyclic monophosphate (cAMP) performs a regulatory and\or signal transduction role in higher plants has been variously described over the past three decades as ‘ non-existent ’, ‘ unequivocally established ’, ‘ un-

likely ’, ‘ probable ’ and ‘ controversial ’. Few with active interests in the relevant areas of plant biochemistry and physiology have retained a neutral or indifferent viewpoint on the concept. A recent critique (Trewavas, 1997) described believers, of which company we have long been members, ‘ who reported meaningful micromolar concentrations,

*Author for correspondence (fax j44 1792 295447 ; e-mail r.p.newton!swansea.ac.uk). Abbreviations : ATF, activating transcription factor ; cAMP, adenosine 3h,5h-cyclic monophosphate ; CBP, CREB-binding protein ; cdTMP, 2h-deoxythymidine 3h,5h-cyclic monophosphate ; cCMP, cytidine 3h,5h-cyclic monophosphate ; cGMP, guanosine 3h,5h-cyclic monophosphate ; CID, collision-induced dissociation ; cIMP, inosine 3h,5h-cyclic monophosphate ; cPKA and cPKB, cAMP-dependent protein kinases ; cUMP, uridine 3h,5h-cyclic monophosphate ; CRE, cAMP-response element ; CREB, cAMP-response-elementbinding protein ; ESI, electrospray ionization ; FAB, fast atom bombardment ; G-protein, GTP-binding protein ; Ins(1,4,5)P , inositol $ 1,4,5-trisphosphate ; MIKE, mass-analysed kinetic energy ; MSMS, tandem MS ; PAL, phenylalanine ammonia-lyase ; VBP1, Vicia faba DNA-binding protein.

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Cell membrane

Rs

Inhibitory hormone/ neurotransmitter

Gs

Gi

Adenylyl cyclase

ATP

Ion channel

Ri

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Protein R–R

R–R Cytoplasm A-PK

C–C C

C

Protein – PO4

Nucleus

CREB CREM

C

CREB – PO4 CREM – PO4

CRE

Activation of cAMP-induced genes

Fig. 1. Role of cyclic AMP in mammals. Diagram of mechanisms of action of cAMP in the mammalian cell. Molecular conversions are represented by solid arrows, regulatory effects by dotted arrows. Rs/i, receptor ; Gs/i, G-protein ; PDE, phosphodiesterase ; A-PK, cAMP-dependent protein kinase ; R-R and C-C, regulatory and catalytic subunits ; CREB and CREM, cAMP-responsive elements.

albeit with weaker technology ’ being ‘ few in number ’ and making ‘ occasional forays to semi-respectable journals ’. The objective of the present review is to survey and discuss the evidence and to attempt to convert a majority of the sceptics to acceptance of the existence in higher plants of cyclic nucleotides and enzymes connected with them, and the likelihood that cyclic nucleotides function in signal transduction and regulation in plants. cAMP has been established as a signalling molecule in both eukaryotes and prokaryotes, including lower plants, thus posing the question ‘ why should higher plants be different ? ’. Before attempting to answer this we must first consider the mechanisms of action of cAMP in these other organisms. From the initial discovery of cAMP by the Nobel Prize winner Earl Sutherland (Rall et al., 1957), and the subsequent demonstration of its role in mediating the action of mammalian hormones on carbohydrate metabolism in the liver, the secondary-messenger concept developed. According to this concept, mammalian hormones and neurotransmitters, acting as primary messengers, remain outside the cell and transmit their signal to the interior via receptors and a membrane-sited enzyme system, which releases the secondary messenger inside the cell. The

secondary-messenger concept has been extended beyond cyclic nucleotides to include the action of inositol phosphates and Ca#+. The original secondary-messenger concept involving receptors directly linked to a membrane-bound adenylyl cyclase has been developed into the present model for the mechanism of action of cAMP in eukaryotic signal transduction depicted in Fig. 1. Two sets of receptor units are associated with a single adenylyl cyclase catalytic subunit in the membrane. Both sets are of the ‘ seven-pass ’ structure, comprising a series of three-and-a-half loops across the membrane : one set is stimulatory and is designated Rs ; the other is inhibitory and is designated Ri. The binding of a ligand to a specific receptor induces a conformational change in the receptor, enabling it to interact with a GTP-binding protein (G-protein) and influence its activity. Two forms of G-protein are also present : Gs, which stimulates adenylyl cyclase, and Gi, which inhibits that enzyme. Both undergo a cycle in which they exist as heterotrimers, to which either GDP or no guanosine nucleotide is bound. These heterotrimers dissociate on binding GTP and the free Gsα subunit undergoes a conformational change enabling it to interact with and stimulate the catalytic unit of

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Cyclic nucleotides in higher plants

429 Light

Natriuretic peptides

GTP

Rhodopsin transducin

Guanylyl cyclase

Guanylin

cGMP

PDE

GMP

Nitric oxide

G-PK

lon channels

Fig. 2. Role of cGMP in mammals. Diagram of mechanisms of action of cGMP in the mammalian cell. PDE, phosphodiesterase ; G-PK, cGMP-dependent protein kinase.

adenylyl cyclase. The dissociation of the Gs heterotrimer is transient ; after the hydrolysis of GTP to GDP, reassociation takes place and a further dissociation occurs only after the GDP has been replaced by GTP. The dissociated Giα subunit exerts an inhibitory effect on adenylyl cyclase (Taussig et al., 1993 ; Taussig & Gilman, 1995) ; however, some types of adenylyl cyclase are not sensitive to Giα. Adenylyl cyclase I is inhibited by both Giα and Gβγ ; others (types V and VI) are stimulated by the βγ subunit of Gi (Taussig & Gilman, 1995). After stimulation, adenylyl cyclase, already functioning at a basal level in the absence of agonist, catalyses the conversion of ATP to cAMP, which is then released into the interior of the cell. The cAMP signal is switched off by another set of enzymes, the phosphodiesterases, which hydrolyse cAMP to AMP. Compared with the enormous research effort concentrated on the adenylyl cyclase system, little interest was shown initially in these enzymes. However, more recently they also have been found to be subject to tight regulation ; a number of phosphodiesterase ‘ families ’, varying in substrate specificity and effector sensitivity, have been investigated (Beavo, 1990 ; Conti et al., 1995). On release into the cytosol, cAMP elicits a response in two main ways. The first established mechanism is via the stimulation of two isoforms of cAMP-dependent protein kinase. Binding of cAMP to this kinase, which is composed of two types of subunit, causes the kinase to dissociate into a regulatory dimer, to which four molecules of cAMP are bound, and two catalytic monomers, which are then capable of phosphorylating a wide range of protein substrates. Phosphorylation alters the activity of the substrate as a result of a change in

surface charge and the subsequent change in conformation. An immediate cellular response is the result. However, phosphorylation by cAMP-dependent protein kinases does not exclusively target cytoplasmatic proteins. cAMP also exerts a second, intranuclear, effect. The catalytic subunits migrate to the nucleus, where they regulate the gene expression of cAMP-regulated genes through a set of transcription factors, called cAMP-response element (CRE)-binding (CREB) proteins (Montminy et al., 1986). In this scheme, de novo synthesis of proteins provokes the cellular response. Although much interest was initially focused on kinase-effected phosphorylation, it later became apparent that a series of phosphatases catalysing the dephosphorylation of the kinase substrates were also subject to regulation and were an integral part of the control mechanism (Cohen, 1989 ; Mumby & Walter, 1993 ; Shenolikar, 1994). In mammalian tissues, with the exception of the enucleate red blood cell, cAMP has been established as ubiquitous, and mediates the action of a wide range of hormones and neurotransmitters. Also in mammals a second cyclic nucleotide, guanosine 3h,5hcyclic monophosphate (cGMP), has been shown to have a more restricted role (Fig. 2), for example : altering the permeability of the cell membrane in the retinal rods to Na+ ions in response to activation of the visual pigment by light, via a G-protein interaction with cGMP phosphodiesterase (Fesenko et al., 1985 ; Koch & Kaupp, 1985 ; Stryer, 1986) ; regulating the movement of Na+ ions and water across membranes in response to guanylin and natriuretic peptides (Hofmann et al., 1992) ; and mediating the response to nitric oxide in smooth muscle (Moncada et al., 1992). Although significant differences occur in the physiological effects of the

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two nucleotides, enzymes for cGMP analogous to those for cAMP are present, namely guanylyl cyclase, cGMP-dependent protein kinase and cGMP phosphodiesterases. The action of cAMP in the mammalian system is not mediated solely by phosphorylation\dephosphorylation phenomena. In the process of olfaction, cAMP mediates the cellular response through the activation of ion channels in the plasma membrane in a way that is very similar to the action of cGMP, i.e. through a direct interaction with an ion channel. Airor water-borne chemicals are recognized at specific receptors in the plasma membrane of olfactory cilia that are connected to adenylyl cyclase through specific Golf-proteins. cAMP is released into the cytoplasm, the subsequent activation of cyclicnucleotide-gated channels results in the depolarization of the cell membrane, bringing about the propagation of an electrical signal through the olfactory nerve. Ca#+ that enters the cells through the opened channels modulates the response by increasing the activity of Ca#+\calmodulin-activated phosphodiesterases (Dhallan et al., 1990 ; Zufall et al., 1994 ; Finn et al., 1996). In most non-mammalian eukaryotic organisms other than higher plants, cAMP signal transduction has been shown to exist that is very similar to that in the mammalian model system. The basic components, adenylyl cyclase, phosphodiesterase and cAMP-dependent protein kinase, are almost ubiquitous but are very diverse. In contrast to that in Schizosaccharomyces pombe, the Saccharomyces cerevisiae adenylyl cyclase, for instance, is different from the prototypical mammalian adenylyl cyclase in that it is a peripheral membrane protein (Masson et al., 1984 ; Kataoka et al., 1985) that is regulated not by heterotrimeric G-proteins but by the monomeric GTP-binding Ras proteins (Toda et al., 1987). The slime mould Dictyostelium discoideum, in which cAMP acts as both a primary and a secondary messenger, has two kinds of adenylyl cyclase. One is very similar to the mammalian enzyme with 12 membrane-spanning helices and is connected to a Gprotein-coupled cAMP receptor (‘ cAR ’). The other contains a single transmembrane span and is expressed only during germination (Parent & Devreotes, 1995). The Dictyostelium discoideum protein kinase A is also different from mammalian cAMPdependent protein kinases. It exists as a dimer and probably contains only one functional cAMP-binding site (Mutzel et al., 1987). Despite these marked differences in building blocks, the overall scheme of eukaryotic signal transduction is well conserved. For decades, cAMP signal transduction has been presented as having two prototypical modes of action : the eukaryotic system described above, which acts mainly through protein phosphorylation, and the prokaryotic scheme, for which the model system is that of Escherichia coli. The model of prokaryotic

cAMP signalling is catabolite repression, a process that ensures adequate utilization of carbohydrate resources. cAMP content in Escherichia coli is highly regulated by the presence of glucose in the growth medium (Rickenberg, 1974). In its absence, [cAMP] is high and drives gene expression of the lac operon, enabling the bacterium to use lactose as an energy source. cAMP acts through direct binding to a transcription factor known as catabolite activator protein (‘ CAP ’) or cAMP receptor protein (‘ CRP ’). After binding to cAMP, CRP changes its conformation and binds to the promoter region of lac, enabling RNA polymerase to start transcription. In the presence of glucose, the intracellular [cAMP] decreases, cAMP-CRP complexes are no longer formed and transcription is stopped. Although this is probably the most important mode of action of cAMP in prokaryotes, there are indications that ‘ eukaryotic-type ’ signal transduction chains also exist in prokaryotes. Many bacteria possess serine\threonine kinases resembling eukaryotic protein kinases. The eubacteria Myxococcus xanthus (Mun4 oz-Dorado et al., 1993), Yersinia pseudotuberculosis (Galyov et al., 1993), Streptomyces coelicolor (Urabe & Ogawara, 1995) and Thermomonospora curvata (Janda et al., 1996) possess protein kinases with a eukaryotic character. Myxococcus xanthus has at least eight genes that have homology with eukaryotic kinases. Sequencing of the genome of the cyanobacterium Synechocystis has revealed the presence of a regulatory subunit of a cAMP-dependent protein kinase (Kaneko et al., 1996). Some eubacteria also have GTP-binding proteins reminiscent of heterotrimeric G-proteins. The hydrolysis of cGMP in Halobacterium halobium is enhanced by the addition of GTP, guanosine 5h-[γ-thio]triphosphate (‘ GTPγS ’) and AlF , which are activators of G-proteins. Besides $ having an effect on behavioural changes, light also influences the endogenous [cGMP] in Halobacterium : the process is possibly analogous to visual perception in mammals, because a G-proteinregulated cGMP-phosphodiesterase is present. On the basis of these enzymatic and additional immunological data, Schimz et al. (1989) postulate the existence of a Gα subunit in this bacterium. A number of systems exist in the lower plants for which the physiological role of cAMP and its metabolism are reasonably well understood. cAMP is an important signalling molecule during the sexual interaction between mt+ and mt- gametes of the diflagellate green alga Chlamydomonas. Both in Chlamydomonas reinhardtii (Saito et al., 1993 ; Zhang & Snell, 1994) and in Chlamydomonas eugametos (Kooijman et al., 1990) the intracellular [cAMP] increases after the agglutination of compatible mating types. The elevated [cAMP] stimulates a sequence of mating responses, such as the excretion of serine proteases, cell wall breakdown and actin

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Cyclic nucleotides in higher plants polymerization into a mating structure, that eventually lead to cell fusion. These responses can also be evoked in cells from one single mating type by the addition of dibutyryl-cAMP. A number of compounds known to inhibit cAMP accumulation also inhibit the mating response. At the same time, cAMP is assumed to govern the motility of flagella of vegetative cells. The regulation of both non-related phenomena is believed to occur at the level of adenylyl cyclase activity (Zhang & Snell, 1993). Chlamydomonas reinhardtii possesses two adenylyl cyclase activities with distinctive properties. One is expressed only in gametes and is strongly regulated during the sexual interaction. In a fast response (approx. 15 s) this adenylyl cyclase activity rises 2–3fold as soon as compatible gametes are mixed. Activity does not seem to be regulated by Gproteins. It is dependent on regulation by phosphorylation\dephosphorylation through an antagonistic action of a constitutive inhibitor kinase and a facultative activator kinase. Ca#+ might be important in the process. The vegetative adenylyl cyclase is not regulated by G-proteins either, but differs from the gametic adenylyl cyclase in that it shows a 3–5-fold lower activity. It is not stimulated by Mn#+, it is not inhibited by Ca#+ and ATP, and it is not sensitive to the addition of staurosporine. It is still not known whether there are two different gene products or one adenylyl cyclase that is differently regulated at different stadia during differentiation. One of the best studied cAMP signal transduction systems of plant origin is that of Euglena gracilis. Both cAMP and cGMP are crucial in the regulation of cell division by day-night rhythm in this flagellate alga. All indispensable components of cAMPmetabolism have been found in this organism (Edmunds, 1994). Changes in cAMP were found to occur during the cell cycle and cAMP is believed to be the link between the internal clock and the cell cycle, in which it permits transition through the G \S and G \M boundaries. Experiments in which " # intracellular cAMP concentrations were manipulated through the application of cAMP, isobutylmethylxanthine or forskolin at very low doses (1–5 nM) have helped in elucidating the mode of action of cAMP in this phenomenon (Edmunds, 1994). In the light of the crucial roles of cyclic nucleotides in other organisms described above, why should higher plants not possess analogous functions for these compounds ? In comparison with mammals and many other animals, an immediate difference is the absence of neurotransmitters ; the presence of plant cell walls might seem to compromise the adenylyl cyclase model depicted in Fig. 1. However, the cell wall probably does not constitute a problem because it does not interfere in phytohormone action, and reports exist describing putative G-proteincoupled receptors with the seven-transmembraneregion signature (Josefsson & Rask, 1997 ; Plakidou-

431 Dymock et al., 1997). Both reports describe a putative seven-transmembrane-region G-proteincoupled receptor bearing greatest homology with the Dictyostelium discoideum cAMP receptor ; in the former case, a role in cytokinin signal transduction is postulated. Thus, at first glance, a comparison of cAMP systems in higher plants with those of lower organisms raises few intrinsic problems, particularly given the established role of cAMP in bacteria and algae together with the possible endosymbiotic origin of plant cell organelles ; this is also true of possible analogies relating to cGMP action in animals and higher plants. In summary, we can conclude that the existence and established functions of cAMP in mammals and other organisms do not allow the simple prediction that it is certain to have analogous functions in higher plants. What, therefore, is the evidence that it does, and\or that other cyclic nucleotides are involved in signal transduction in higher plants ? I I .                        c      The early reports on the existence of cAMP in plants were criticized on the basis that they were either presumptive deductions from the observed physiological effects of endogenously supplied cAMP or cAMP analogues, or conclusions based solely on insufficiently rigorous chromatographic identification. As an example of the former, Salomon & Mascarenhas (1971) reported that cAMP delayed petiole abscission in Coleus, but did not demonstrate that this was a process in vivo, merely that the cyclic nucleotide could replicate the action of auxin. In the latter category, Pollard (1970) obtained a radiolabelled product from the incubation of [8-"%C] adenine with germinating barley seeds that chromatographed together with cAMP in ten chromatographic systems. The latter report was criticized on the basis that the chromatographic systems would not resolve the putative secondary messenger cAMP from the RNA catabolism intermediate, adenosine 2h,3h-cyclic monophosphate. To overcome such criticism the putative radiolabelled cAMP was hydrolysed to AMP by cAMP phosphodiesterase, which was then determined enzymatically (Narayanan et al., 1970) ; however this expedient was criticized in that the phosphodiesterase was not of demonstrated absolute specificity for cAMP. Concomitantly with these reports of cAMP, reports of the existence of cAMP-based signalling enzymes were made ; these reports were also criticized, those of phosphodiesterase activity on the basis of substrate specificity (see section V) and those of adenylyl cyclase on the basis of product identification (see section IV). In an attempt to identify cAMP conclusively as an endogenous component of

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plant cells, a sequential chromatographic and electrophoretic procedure for the extraction and isolation of cAMP was developed (Brown & Newton, 1973). The identity of the putative cAMP was confirmed by cochromatography with an authentic sample in five paper and three thin-layer chromatography systems and by high-voltage electrophoresis in three different buffers. Collectively, these steps were capable of separating cAMP from all then known naturally occurring adenine nucleotides, including 2h,3h-cyclic AMP. Nevertheless, some authors still claimed that the evidence was equivocal (Keates, 1973 ; Amrhein, 1974a ; Lin, 1974), considering that hitherto unidentified adenine compounds, with identical chromatographic properties to those of cAMP in these systems, existed in higher plants. During and after this initial phase of investigation in this area, a considerable number of reports quantifying cAMP in various plant species were made, with concentrations of a similar order ranging from 2.1–3.5 pmol cAMP g−" wet wt in Zea (Tarantowicz-Marek & Kleczkowski, 1978) to 220–280 pmol g−" wet wt in Lactuca (Kessler & Levenstein, 1974) : a comprehensive listing of concentrations then reported is reviewed in Newton & Brown (1986). Nevertheless, some authors reported that concentrations of cAMP were close to and below the sensitivity of their methods (Niles & Mount, 1973 ; Amrhein, 1974a ; Bressan et al., 1976) and as a consequence several reviews at the time concluded that cAMP was not present in plants (Keates, 1973 ; Lin, 1974 ; Amrhein, 1974a, 1977) ; others suggested that any cAMP present was a result of bacterial infection (Bonnafous et al., 1975). The contamination concept was refuted by Ashton & Polya (1977), who calculated that less than 0.1 % was contributed by bacteria and demonstrated the presence of cAMP in axenic cell cultures of rye grass (Ashton & Polya, 1978), supporting earlier reports of cAMP in axenic cultures of soybean callus tissue (Brewin & Northcote, 1973) and of tobacco cells (Lundeen et al., 1973). Although a range of physiological processes and enzymatic reactions in plants were suggested to be responsive to cAMP (Brown & Newton, 1981 ; Newton & Brown, 1986), for credibility to be sustainable it was essential to demonstrate unequivocally the identity of the putative cAMP obtained in tissue and cell extracts and as the product of incubations with adenylyl cyclase. This was successfully accomplished by the use of physical techniques including MS and NMR spectroscopy, as detailed in section III ; the inclusion of suitable controls has also been used to demonstrate that the identified cAMP is not an artefact. As a consequence, although there were a few reports after the production of MS evidence of the identity of cAMP in plant extracts that claimed the absence of cAMP from plant cells (Spiteri et al., 1989), several reviews have appeared that show a shift in the balance of

opinion. Although most reviews in the initial phase expressed the opinion that cAMP did not, or was unlikely to, function in higher plants (Keates, 1973 ; Lin, 1974 ; Amrhein, 1974a, 1977), these have been superseded by commentaries suggesting potential functions (Brown & Newton, 1981 ; Francko, 1983 ; Newton & Brown, 1986 ; Assmann, 1995 ; Bolwell, 1995 ; Trewavas, 1997). As will be detailed in the sections below, conclusive evidence of the existence of cAMP, adenylyl cyclase, phosphodiesterase and cAMP-binding proteins is now available and systematic studies of the function of the cyclic nucleotide are appearing, for example in studies of its role in the cell cycle, in stress response systems and in the regulation of ion channels. III.                 c           In the 1970s, studies on the occurrence and effect of cAMP in higher plants were a mere extrapolation of the investigation going on in animal and fungal systems ; in retrospect it seems naive to assume that plant 3h,5h-cAMP-mediated signal transduction is virtually identical to that in other kingdoms. Early papers (Pollard, 1970 ; Oota, 1972 ; Keates, 1973 ; Truelsen et al., 1974) were based on observations of various physiological and metabolic responses after the exogenous application of cAMP, cAMP analogues and phosphodiesterase inhibitors known at that time. This shotgun-like approach was performed without any knowledge of the underlying metabolism in plants. A major point of interest in this respect is the discrepancy between the endogenous concentrations present in plants and in animals. Animal and microbial cells contain cAMP concentrations in the nanomolar to micromolar range, whereas in plants the [cAMP] is much lower. This suggests that either the global metabolic activity of adenylyl cyclase and phosphodiesterase is low or that it is subject to a strict temporal and spatial regulation (Brown & Newton, 1992). However, the term ‘ low concentration ’ might be misleading ; although the cAMP concentration per unit weight is much higher in animals than in plants, the picture might be distorted by differences in cell structure (such as the presence of vacuoles and a cell wall). The ratio of cAMP to ATP, for example, is similar in animals and plants : between 1 : 100 and 1 : 10000. This low [cAMP] definitely constitutes a major problem in cAMP research in plants. The initial application of the same extraction, purification and detection techniques as those described for animal tissues without considering the problems peculiar to plant matrices led to diverse results, which were often interpreted as contradictory. Without modification of the separation techniques (if indeed any separation was included, because the original work of Cailla et al. (1973) prescribed no requirement for

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Cyclic nucleotides in higher plants the purification of cAMP by radioimmunoassay at the femtomole level), ubiquitous interfering plant metabolites co-eluted and compromised the data. Furthermore, because the limit of detection had to be made very low, extra precautions had to be taken to prevent bacterial contamination (Bonnafous et al., 1975) and physicochemical cyclization of ATP to cAMP (Cook et al., 1957) catalysed by the presence of bivalent ions during extraction at basic pH (Brooker et al., 1979). In the late 1970s and early 1980s, the experimental set-up for cAMP analysis was adapted to a great extent to cope with plant matrices. Taking care to minimize the artefactual origin of cAMP, various research groups produced more reproducible and coherent data (Brown et al., 1977 ; Katsumata et al., 1978 ; Hilton & Nesius, 1978 ; Tu, 1979). In the 1980s, chromatographic techniques in plant cAMP analysis such as adsorption, ion-exchange, paper and thin-layer chromatographies were largely reduced to preparative steps and replaced by the far superior HPLC, which is able to separate the 3h,5h-cyclic nucleotides and their 2h,3h isomers (Van Onckelen et al., 1982 ; Brown, 1983). Although widely applied and among the most sensitive methods over the past decades, immunosorbent assays for cAMP, although having a femtomole dynamic range, lack accuracy. For most of these assays cAMP needs to be derivatized ; interference by unknown contaminants can be ruled out only if extensive controls are built in into the assay. Moreover, the presence of the cyclic nucleotide derivatives 2h-deoxyadenosine-3h,5h-cyclic monophosphate, 2h-deoxyguanosine-3h,5h-cyclic monophosphate, 2h-O-glutamyl-3h,5h-cyclic AMP, 2h-Oaspartyl-3h,5h-cyclic AMP, 2h-O-glutamyl-3h,5h-cyclic GMP, 5h-phosphoadenosine-2h,3h-cyclic monophosphate and 2h-phosphoadenosine-3h,5h-cyclic pyrophosphate was demonstrated in Porphyra umbilicalis used as a plant model system (Newton et al., 1995) ; such derivatives of cyclic nucleotides have been shown to interfere in radioimmunoassays of cyclic nucleotides (Newton et al., 1994), illustrating the inadequacy of the method as a sole detection technique. However, the use of anti-cAMP antibodies in an immunopurification step results in a very powerful sample clean-up because most interfering compounds in further quantitative steps are thereby disposed of. Polyclonal chicken egg yolk anti-cAMP antibodies in combination with UVPDA (photo diode array) HPLC has proved to be a very powerful analytical method for cAMP quantification in higher-plant matrices such as plasma membranes, chloroplasts and protoplasts (Roef et al., 1996 ; Witters et al., 1996). Despite the significant technical improvements, UV absorption spectra or fluorescence spectra together with chromatographic retention times were still considered ambiguous identification criteria. It

433 was mass spectrometric analysis that unequivocally established cAMP as being endogenous to plant tissues (Newton et al., 1980 ; Johnson et al., 1981 ; Janistyn, 1983). These first observations were obtained by electron-impact GC-MS of volatile trimethylsilyl derivatives of cAMP, as reported by Lawson et al. (1971) with the chemically synthesized compound. Although electron-impact GC-MS is a sensitive method, its major pitfall is the requirement for the non-homogenous silylation of cAMP extracts. The advent of the soft-ionization mass spectrometric techniques of fast atom bombardment (FAB) and electrospray ionization (ESI) was a big step forward in nucleotide research, because it removed this requirement for derivatization. For an overview of the basic principles of MS and its application to biomolecular research, the reader is recommended to consult Newton & Walton (1996) and Caprioli et al. (1997). The potency of static FAB-MS in plant nucleotide research was first demonstrated by Newton and coworkers. FAB ionization readily provided molecular mass information of non-volatile polar compounds including nucleotides. However, because it is a soft ionization technique, the major drawback of the spectra obtained is the absence of diagnostic fragments. FAB mass spectra do not permit detailed structural analysis ; isomeric compounds, for example, cannot be differentiated because they produce very similar mass spectra. To overcome this problem, collision-induced dissociation (CID) of the protonated molecule selected from the FAB mass spectrum provides a mass-analysed kinetic energy (MIKE) spectrum that can be used to generate structural information, including the differentiation of cyclic nucleotide isomers (Kingston et al., 1984, 1985 ; Newton et al., 1984b, 1986, 1989). This tandem MS permits the identification of diagnostic fragments in the MIKE spectrum from the protonated molecule and has thereby allowed the unambiguous identification of 3h,5h-cAMP (Fig. 3) and 3h,5h-cGMP (section VIII). Furthermore, FABCID\MIKE analysis of partly purified extracts from meristematic and non-meristematic tissue from Pisum sativum has demonstrated the natural occurrence of inosine 3h,5h-cyclic monophosphate (cIMP), uridine 3h,5h-cyclic monophosphate (cUMP), cytidine 3h,5h-cyclic monophosphate (cCMP) and 2h-deoxythymidine 3h,5h-cyclic monophosphate (cdTMP) in addition to cAMP and cGMP (Newton et al., 1991). ESI-MS has become a very popular detection technique for the analysis of polar biomolecules and is replacing many of the FAB-MS applications by virtue of its greater sensitivity. As with FAB ionization, ESI produces mainly the quasi-molecular ion ; to acquire information on the molecular structure, tandem MS (MSMS) needs to be performed (Fig. 3). When combined with separation techniques

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Fig. 3. For legend see opposite.

such as capillary zone electrophoresis or capillary HPLC, ESI-MS becomes a very powerful analytical technique. The sensitivity is reached primarily by virtue of the ESI process, which behaves as a concentration-sensitive phenomenon (Hopfgartner et al., 1993). Reducing the dimensions of the LC setup enhances the sensitivity exponentially (Chervet et al., 1996 ; Vanhoutte et al., 1997 ; Witters et al.,

1997a) and the introduction of a capillary column switching method yields detection limits as low as 25 fmol (Witters et al., 1997b, 1998). As will be discussed in section VII, the use of this sensitive LC-ESI-MSMS set-up has enabled a cell cycleregulated cAMP accumulation to be demonstrated in a Nicotiana tabacum BY2 cell culture (Ehsan et al., 1998).

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Fig. 3. Mass spectrometric identification of cyclic nucleotides. (a) Positive-ion FAB mass spectrum of cAMP, showing a protonated molecule at m\z 330. (b) CID\MIKE spectrum of m\z 330 from cAMP including diagnostic fragments at m\z 136 (protonated base), m\z 164 (protonated base j 28) and m\z 178 (protonated base j 42). (c) Partial CID\MIKE spectra of m\z 330 from cAMP (solid line) and 2h,3h-cyclic AMP (dotted line), demonstrating differentiation between them. (d) Fragmentation of cAMP in a FAB-MS-CID\MIKE spectrum scan showing the origin of m\z 164 and m\z 178 peaks by S and S cleavage : 2h,3h-cyclic AMP is # " unable to produce m\z 178 because of the substitution at the 2h-O position. (e) Product ion spectra of 2h,3hcyclic AMP (first and third panels) and cAMP (second and fourth panels) in both negative (top two panels) and positive (bottom two panels) ionization modes. Both sets of spectra contain the base fragment as the major peak, at m\z 134 and 136 respectively. The absence of the PPi ion at m\z 79 for 2h,3h-cyclic AMP and its presence in the cAMP spectrum differentiates between the isomers in negative mode : in positive ionization mode the presence of peaks at m\z 177 and 312 in the cAMP spectrum and at m\z 195 in the 2h,3h-cyclic AMP spectrum, together with the relative heights of the peaks at m\z 97 and 99, is a further means of differentiation between the cyclic nucleotide isomers by ES-MSMS.

IV.   Adenylyl cyclase activity has been demonstrated in higher-plant material by the use of both histochemical and biochemical procedures. Histochemical methods are based predominantly on the standard Wachstein-Meisel lead phosphate precipitation technique (Wachstein & Meisel, 1957), with ATP as a substrate for adenylyl cyclase : an electron-dense precipitate of lead or cerium ions in the presence of PPi is released on the formation of cAMP from ATP and is detected by electron microscopy. Because cells harbour tremendous quantities of ATP-hydrolysing enzymes that subsequently release Pi, this procedure produced lots of false positive results that could not be overcome by the use of compounds to inhibit these contaminating activities. The specificity of the reaction was considerably improved by the replacement of ATP with a specific substrate analogue for adenylyl cyclase such as adenosine 5h-[β,γ-imido]

triphosphate (Yount et al., 1971) or adenosine 5h[α,β-methylene]triphosphate (Mayer et al., 1985), which are much less sensitive to ATPase and other phosphatase activities. (For a recent review on histochemical methodology the reader is referred to Richards & Richards (1998).) By such methods, early indications of the presence of adenylyl cyclase activity were found in plasma membrane, in endoplasmic reticulum and nuclear membranes in Zea mays root tips (Al-azzawi & Hall, 1976), on internal membranes of cytoplasmatic vacuoles in Pisum sativum (Hilton & Nesius, 1978), on the external side of the host plasma membrane and membranes surrounding the endophyte in root nodules of Alnus glutinosa (Gardner et al., 1979), and on the external side of the plasma membrane of Pisum sativum (Nougare' de et al., 1984). Physiological roles have been proposed for plant adenylyl cyclase : Rougier et al. (1988) postulated that adenylyl cyclase activity is a determining factor in the compatibility of pollen

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tube formation in Populus spp., whereas Curvetto & Delmastro (1990) located an adenylyl cyclase activity in Vicia faba guard cells that was selectively stimulated by IAA, Ca#+, caffeine, GTP, forskolin and, to a smaller extent, ABA. According to these authors cAMP is involved in the IAA signal transduction chain during stomatal movement through a G-protein-mediated mechanism. An adenylyl cyclase activity was also found on the external side of the plasma membrane and on thylakoid membranes in primary leaves of Phaseolus vulgaris, a finding corroborated by the immunolocalization of cAMP in the chloroplast and cell wall (Gadeyne, 1992). A histochemical approach is invaluable for pinpointing the exact location of adenylyl cyclase activity and is thus extremely helpful in providing clues to a physiological role. However, the methods available are not definitive. In spite of the fact that the substrate now used is insensitive to phosphatase activity, little is known of the impact of apyrases and diphosphohydrolases ; some caution has therefore to be observed in the interpretation of these data. Data obtained by a biochemical approach, in which the actual formation of radiolabelled cAMP from radiolabelled precursor (ATP or adenosine 5h-[β,γ-imido] triphosphate) is measured, are far more reliable in this respect. Nevertheless, the first such reports were heavily criticized for inadequate identification of the newly formed compound, but more rigorous separatory procedures provided more credible evidence. For example Carricarte et al. (1988) described a soluble adenylyl cyclase in roots of Medicago sativa with an estimated molecular mass of 84 kDa. The enzyme was active in the presence of Ca#+ and Mg#+. A DEAE-purified extract produced 204 pmol cAMP min−" mg−" protein in the presence of Ca#+, an activity that was stimulated 15-fold by bovine calmodulin. The effect of the addition of Spinacea oleracea calmodulin was less marked but was nevertheless significant (approx. 7-fold stimulation). The stimulation was abolished by the addition of EGTA and chlorpromazine, an inhibitor of calmodulin function. GTP, guanosine 5h-[β,γ-imido]triphosphate, forskolin, fluoride and cholera toxin were ineffective, indicating that the enzyme was not dependent on G-protein function. By contrast, Lusini et al. (1991) described a sedimentable adenylyl cyclase in the roots of Ricinis communis. An enzyme activity of approx. 20 pmol min−" mg−" protein was measured in the presence of 3 mM MnCl . In contrast with the alfalfa enzyme, # MgCl did not stimulate this adenylyl cyclase, but # NaF and GTP did ; thus this enzyme might well be G-protein regulated. Although this radiometric evidence was still not universally accepted, the advent of highly reliable MS techniques (as described in section III) capable of identifying cAMP unambiguously as the reaction product, has now

produced a number of reports proving unequivocally the presence of adenylyl cyclase activity in higher plants. A sedimentable adenylyl cyclase activity was identified in Pisum sativum (Pacini et al., 1993) by using mass spectrometric techniques for the first time, producing unambiguous identification of the reaction product. This enzyme utilizes Mg#+-ATP as a substrate and is stimulated by GTP at 100 nM. Higher concentrations of GTP (110 µM) inhibit the activity, probably owing to competition with ATP. The application of zeatin, GA , IAA and Ca#+\ $ calmodulin yielded surprising results. No simple dose-dependent response was observed ; all four effectors stimulated activity by up to 50 % at an optimum concentration, but higher concentrations were less effective and with zeatin and GA they $ even became inhibitory. A plausible explanation is the involvement of an additional regulatory component that can become saturated by these effectors or becomes limiting by another, effector-independent, mechanism. Medicago sativa cell cultures exposed to the elicitor of the phytopathogenic fungus Verticillium albo-atrum respond with an increased adenylyl cyclase activity (Cooke et al., 1994). Again, cAMP formation was confirmed unequivocally by mass spectrometric analysis. Adenylyl cyclase activity was dependent on Mg#+ and was stimulated by Ca#+. Basal activity was very low (maximum 400 fmol min−" mg−" protein) but increased by 300 % within a time span of 4 min on application of the elicitor. The transient rise in adenylyl cyclase activity was accompanied by an increase in intracellular [cAMP] and was followed by a transient increase in phosphodiesterase activity (with a maximum at 100 min). A role in the defence mechanism of higher plants on attack by pathogens is proposed (see section VII). More recently a MgCl -stimulated adenylyl cy# clase activity has been demonstrated by ESI-MS in plasma membrane preparations from apical hooks from Phaseolus vulgaris (Roef et al., 1996 ; Roef, 1997), confirming the histochemical and immunochemical data of Gadeyne (1992). This presence of adenylyl cyclase activity associated with plant plasma membrane preparations seems compatible with a mammalian-type secondary-messenger system. The first paper reporting a plant gene sequence showing high homology with that of mammalian adenylyl cyclase (Ichikawa et al., 1997) and detailing aspects of its regulation has now unfortunately been withdrawn (Ichikawa et al., 1998) as the data cannot be reproduced, the circumstances being discussed at length in Balter (1999). Although we feel obliged to indicate our knowledge of these three reports for completeness, we do not consider it appropriate to comment further at this stage, other than to state that an acceptable demonstration of such a plant gene sequence would have a very significant impact.

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Cyclic nucleotides in higher plants V .                  Even before the first reports of cyclic-nucleotidecontaining extracts from higher plants, the enzyme phosphodiesterase, which is capable of hydrolysing cAMP to AMP, was reported in pea seedlings (M. Liberman & A. T. Kunishi, unpublished) ; this was quickly followed by demonstrations of activity in such diverse sources as tobacco (Wood et al., 1972), barley seeds (Vandepeute et al., 1972), carrot leaves (Venere, 1972), potato (Shimoyama et al., 1972 ; Ashton & Polya, 1975) and Jerusalem artichoke tubers (Giannattasio et al., 1974b). The occurrence of phosphodiesterase activity in these plants was interpreted by several of these authors as indicating that the substrate, cAMP, must be an endogenous component of the tissue and that it would possess functions analogous to those of cAMP in other organisms. However, a conflicting view was expressed by Lin & Varner (1972), who reported that unlike its mammalian counterpart the phosphodiesterase from pea seedlings had an acidic pH optimum, was insensitive to methylxanthines, yielded 3h-AMP rather than 5h-AMP as the major hydrolytic product, and, most significantly, had substantially greater activity with the RNA breakdown intermediate 2h,3h-cyclic AMP as substrate than with the putative secondary-messenger isomer, 3h,5h-cAMP. Because at that time the known mammalian phosphodiesterases functioning in the cAMP secondary-messenger cascade produced only the 5hmononucleotide product and would not hydrolyse 2h,3h-cyclic AMP to any significant extent, Lin & Varner (1972) concluded that the pea phosphodiesterase was functioning not in a plant signal transduction system but as part of a catabolic sequence of RNA. This view had an immediate negative impact on theories relating to a regulatory role for cAMP in plants, which were dealt a further blow by a survey of phosphodiesterases from a range of plant species and tissues that concluded, on the basis of pH optima and substrate specificity, that 3h,5h-cAMP was not their natural substrate (Amrhein, 1974b). This view was endorsed by evidence that the phosphodiesterase preparations from barley seeds (Vandepeute et al., 1972), carrot leaves (Niles & Mount, 1974) and tobacco (Brennicke & Frey, 1977) had at least an equal activity with a 2h,3h-cyclic AMP substrate as with the 3h,5h isomer. However, further examination of more purified plant phosphodiesterases indicated that more than one form is present. French Dwarf bean seedlings were found to contain a phosphodiesterase that, when partly purified, possessed properties more similar than the plant phosphodiesterases discussed above to those of the mammalian phosphodiesterases (Brown et al., 1975, 1977). It was active towards several 3h,5h-cyclic nucleotides as substrate but inactive with 2h,3h-cyclic nucleotides, produced 5h-

437 mononucleotides as the major product, and the Km, pH optimum and sensitivity to methylxanthines were also more like those of the mammalian enzyme than those reported by Lin & Varner (1972) and Amrhein (1974b). Interestingly, the enzyme was stimulated by an endogenous protein with which it was able to form a complex ; this protein was only partly purified and was not characterized further ; it had been obtained before the demonstration of calmodulin in higher plants, but it was also found to stimulate bovine brain calmodulin-sensitive phosphodiesterase, suggesting a further parallel between the French Dwarf bean and mammalian phosphodiesterases. Examinations of the subcellular distribution of phosphodiesterase activity have confirmed the existence of more than one phosphodiesterase type in plant cells. In spinach, three forms of phosphodiesterase were observed : one, designated Ic, had its major subcellular site in the chloroplast, and a second, predominantly outside the chloroplast, had its major yield in the microsomal fraction designated Im. Type Im phosphodiesterase conformed to the profile described by Lin & Varner (1972) and Amrhein (1974b) in other plant species, having an acidic pH optimum of 4.9, relative insensitivity to methylxanthine inhibitors, and greater activity with 2h,3h- rather than 3h,5h-cyclic nucleotide substrates (Brown et al., 1979b). In contrast, the Ic phosphodiesterase had highest activity with 3h,5h-cGMP and 3h,5h-cAMP and little activity with their 2h,3h isomers, had a less acidic pH optimum of 6.1, was sensitive to inhibition by methylxanthines, and liberated 5h-mononucleotides as the main product ; it also displayed sensitivity to endogenous protein effectors and was activated by Ca#+ (Brown et al., 1979b). Further examination of Ic phosphodiesterase revealed that it occurred in multienzyme complexes of molecular mass 187 and 370 kDa in association with acid phosphatase, ribonuclease, nucleotidase and ATPase (Brown et al., 1980a). A multiplicity of phosphodiesterases has also been reported in other species. In potato (Ashton & Polya, 1975) three phosphodiesterases are present : one with greatest activity with 3h,5h-cyclic nucleotide substrates, one with greatest activity with 2h,3h-cyclic nucleotides, and one with NAD pyrophosphate as the major activity. Similarly, in Portuluca (Endress, 1979) three phosphodiesterases are present : two show Michaelis-Menten kinetics, one having greater activity with 3h,5h-substrates and one with 2h,3hsubstrates, and the third is arguably the most interesting because it shows positive cooperativity and is sensitive to allosteric regulation by nucleotides, with for example the presence of cGMP stimulating a high activity towards 3h,5h-cAMP and 3h,5h-cGMP as substrates, a kinetic process analogous to that in one of the established mammalian phosphodiesterase families (Manganiello et al.,

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1990). In carrot cell cultures two phosphodiesterases with distinct kinetic parameters have been reported : constitutive phosphodiesterase activity did not depend on either Ca#+ or calmodulin, but a calmodulindependent isoform could be induced by increased [Ca#+] (Kurosaki & Kaburaki, 1995). Kinetic analyses suggested that the constitutive phosphodiesterase has a role in the maintenance of the resting state of the carrot cells by keeping cellular [cAMP] and [Ca#+] very low, whereas the calmodulin-sensitive phosphodiesterase induced in the excited cells hydrolyses cAMP rapidly under conditions of high [cAMP] and [Ca#+] as one of the response-decay mechanisms. The presence of several forms of phosphodiesterase offers a ready explanation of the apparent incompatibility of the data obtained and conclusions drawn by different groups in the earlier reports of plant phosphodiesterases, with different extraction and purification protocols selecting for one or other of the phosphodiesterase types. In addition, the observation that in at least one plant species the phosphodiesterase is present in a complex also containing nucleotidase suggests that identification of one or other mononucleotide isomer as the major product of phosphodiesterase activity might not be as clearcut as it seems at first. For example a 3h,5hcAMP phosphodiesterase activity from Phaseolus vulgaris seedlings 7 d old had an acidic pH optimum, was strongly stimulated by Mn#+, Mg#+ and Ca#+ and imidazole, was inhibited by NaF, PPi and Fe$+ and was insensitive to butylmethylxanthine ; purification away from a contaminating monoesterase activity revealed that the protein hydrolysed the 3h-ester linkage exclusively (Dupon et al., 1987). The existence of multiple forms complicates the interpretation of phosphodiesterase function ; this is compounded by the complex kinetics of the individual enzymes. Several of the phosphodiesterases examined had activity not only with cAMP but also with cGMP. The greater activity with 3h,5h-cAMP as substrate than with the 2h,3 isomer can be considered indicative of the function of the enzyme’s being hydrolysis of the putative signalling molecule. However, greater activity with cGMP might suggest that the latter is in fact the natural substrate, for example with a Phaseolus chloroplast phosphodiesterase, which has a low Km for cGMP of 77 µM and is more than 3-fold more active with cGMP as substrate than with cAMP (Newton et al., 1984a). The activity of at least some plant phosphodiesterases is not confined to purine cyclic nucleotide substrates : a lettuce phosphodiesterase with a molecular mass of 62 kDa (and thus a smaller entity than the enzymes from spinach and French Dwarf bean) showed significant similarity to the multifunctional phosphodiesterase isolated initially from pig liver (Helfman et al., 1981). This lettuce phosphodiesterase differs from other plant 3h,5h-cyclic nucleo-

tide phosphodiesterases in that it exhibits comparable activity with both pyrimidine and purine cyclic nucleotide substrates, hydrolysing cytidine 3h,5h-cyclic monophosphate (cCMP) at a similar rate to cAMP and cGMP, with Km values of 1.1, 0.71 and 0.64 mM and Vmax\Km values of 5.1i10$, 3.7i10$ and 3.4i10$ l min−" mg−" protein for cAMP, cGMP and cCMP, respectively (Chiatante et al., 1986). A unique feature of this enzyme among plant phosphodiesterases is that it is stimulated to the greatest extent by Fe$+ ions, a feature previously observed only in the mammalian multifunctional (Kuo et al., 1978) and cCMP-specific (Newton et al., 1990) phosphodiesterases. This lettuce enzyme was able to hydrolyse both 3h,5h- and 2h,3h-cyclic nucleotide substrates. With the 3h,5h substrates both 5h- and 3hmononucleotide products were released and the 5h isomer was the major form. With the 2h,3h-cyclic nucleotide substrates the point of cleavage was affected by the nature of the base : 3h-CMP was the major product from 2h,3h-cyclic CMP, 2h-GMP was the sole product of 2h,3h-cyclic GMP hydrolysis, and equimolar proportions of 2h- and 3h-AMP were liberated from 2h,3h-cyclic AMP (Chiatante et al., 1987). Kinetic analysis of this enzyme revealed a complex picture in which the presence of one cyclic nucleotide affects the hydrolysis of another : with a 3h,5h-cAMP substrate other 3h,5h- and 2h,3h-cyclic nucleotides exhibit mixed-type inhibition, with the Ki values of for example cGMP and 2h,3h-cyclic AMP with cAMP as substrate being two orders of magnitude lower than the Km values of the former two compounds when they are sole substrates. This suggests that there is more than one binding site for each cyclic nucleotide, although no cooperative effect seems to exist for a single cyclic nucleotide as substrate. In contrast to the mixed inhibition above, the hydrolysis of cGMP was stimulated by the presence of cAMP and cCMP. In this instance at least it seems that a major factor regulating the hydrolysis of one cyclic nucleotide is the presence of others. With a highly purified preparation, from which endogenous nucleotidase activity had been removed, this enzyme was found to have greater activity with 3h,5h-cAMP than 2h,3h-cyclic AMP and produced 5h-AMP as the major product from the former. With cGMP and cCMP the 2h,3h isomers were the preferred substrates, but kinetic data confirmed that there were distinct catalytic sites for the 2h,3h- and 3h,5h-cyclic nucleotides (Chiatante et al., 1988). Although the phosphodiesterase activity in mammals seems tightly regulated and integrated to maintain a consistent turnover of cyclic nucleotides, this pattern alters under various conditions, for example a phosphodiesterase rebound activation after stimulation of nucleotidyl cyclase, during the cell cycle and, for cGMP phosphodiesterase, in the visual cycle. In plants a phosphodiesterase rebound

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Cyclic nucleotides in higher plants is observed after the stimulation of adenylyl cyclase by an elicitor as part of the plant cell’s defence against pathogens, as discussed in section VII. In the context of the suggested involvement of phosphodiesterases in the regulation of the cell cycle (Levi et al., 1981) the presence of two major forms of phosphodiesterase in the meristems of peas is of relevance. One form hydrolysed cAMP, cGMP and cCMP ; the second was unique in that it had a preference for cCMP over cAMP but was devoid of activity with cGMP. It was stimulated by Fe$+ but not by calmodulin and was inhibited by methylxanthines ; most interestingly in respect of cell cycle regulation, the enzyme was inhibited by the cytokinins kinetin and kinetin riboside, which were also demonstrated to inhibit the growth of the pea roots (Chiatante et al., 1990). The other apparent effector of plant phosphodiesterase activity identified so far is light. As described in section I, in mammals a light-sensitive cGMP phosphodiesterase is an integral component of the visual cycle (Gillespie, 1990). In plants a lightdependent response of this enzyme was first shown in the spinach chloroplast : the chloroplast phosphodiesterase had similar Vmax values in light- and darkgrown seedlings, but in the dark-grown plants the Km was 27 µM in comparison with 870 µM in the light, suggesting greater activity in the dark ; However, in the light the enzyme was sensitized to Ca#+\calmodulin : the enzyme in the light-exposed chloroplast was stimulated 3-fold by calmodulin but the enzyme from the dark-grown plant showed no response (Brown et al., 1989). A non-transient activation of cAMP phosphodiesterase activity by red light in etiolated corn sprouts has also been reported (Kasumov et al., 1991) and light is again involved in the calmodulin response in this species : one phosphodiesterase from the dark-grown seedlings has a calmodulin sensitivity that is dependent on [Mg#+] and the season (Fedenko et al., 1992). In spring it is activated by calmodulin irrespective of [Mg#+] ; in autumn at high [Mg#+] it is inhibited by calmodulin but activated at low [Mg#+], with the high-[Mg#+] autumn inhibition being converted to activation if the corn seedlings are pre-illuminated with phytochrome-absorbed red light. In maize sprouts the effect of the GTPase-resistant GTP analogue guanosine 5h-[β,γ-imido]triphosphate in inhibiting only dark-grown seedlings exposed to red light led the authors to propose a role for G-proteins and phosphodiesterase in light signal transduction in this plant (Fedenko & Kasumov, 1993). V I . c   -                        ,    , c    -                                 -              To identify conclusively any physiological role(s) for cAMP in higher plants it is necessary to establish

439 cellular targets for cAMP action. It has long been established that cAMP action in eukaryotes is predominantly mediated by the phosphorylation of target proteins via cAMP-dependent protein kinase ; it was only comparatively recently that cAMP was shown to exert some of its effects through a direct interaction with ion channels (Zufall et al., 1994). Consequently, the search for cAMP targets in plants has concentrated primarily on the quest for cAMPdependent protein kinases. At present there have been no reports of the purification of a plant cAMPdependent protein kinase to homogeneity ; most indications of the existence of cAMP-dependent protein kinase result from experiments in which the phosphorylation of specific substrates is regulated by cAMP. Three cyclic-nucleotide-responsive protein kinases have been reported in Lemna paucicostata (Kato et al., 1983). Each could phosphorylate histone : one was stimulated by 10 µM cAMP, cGMP and cIMP, a second was inhibited by these nucleotides, and the third was cAMP-independent but sensitive to cGMP and cIMP. The protein extract showing cAMPstimulated protein kinase activity was also found to contain a cAMP-binding protein, but a possible interaction between the two proteins was not reported. cAMP-dependent protein kinase was also shown in Zea mays seedlings (Janistyn, 1988). This electrophoretically purified protein of molecular mass 36 kDa had a strong dependence on MnSO : replace% ment of MnSO by NiSO , CoSO or FeSO % % % % abolished the cAMP dependence of the kinase activity. In contrast to the data from Lemna (Kato et al., 1983) other cyclic nucleotides (cGMP, cIMP, cCMP and cUMP) did not exhibit any stimulating effect on the protein kinase activity. Janistyn (1989) has also described the cAMP-dependent phosphorylation of protein components present in dialysed coconut milk. At 1–10 µM, cAMP enhances the phosphorylation of two endogenous proteins of molecular masses 60 and 70 kDa, this phosphorylation being inhibited by a protein component (molecular mass 9 kDa) present in non-dialysed coconut milk. The apparent complexity of the coconut milk system is illustrated by the fact that cAMP also inhibited the phosphorylation of other endogenous proteins with molecular masses between 27 and 30 kDa, implying that more than one cAMPresponsive kinase was present, that the specificity of a single kinase was altered by cAMP, or that cAMP in some way directly or indirectly stimulates protein phosphatase activity. More recently, Komatsu & Hirano (1993) identified a cAMP-stimulated protein kinase activity in rice leaves and roots. At 10 nM, cAMP enhanced the phosphorylation of histone II-A, and the phosphorylation of three endogenous proteins (molecular masses 40, 50 and 55 kDa) of rice seedlings 11 d old

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was specifically stimulated by the addition of cAMP. cGMP, phorbol ester or Ca#+ did not induce the same effect. A similar cAMP-responsive phosphorylating activity was present in the rice embryo and roots. In each of above cases the kinase substrate was either a histone or an endogenous, unidentified, protein. The evidence relating to a partly purified protein from Petunia petals (Polya et al., 1991) is perhaps more convincing on the basis of substrate information. A basic protein fraction (molecular mass 30 kDa) phosphorylated both histone III-S and Kemptide, a specific substrate for cAMPdependent protein kinase. Its activity was inhibited by both the Walsh-Krebs inhibitor peptide and the regulatory subunit of cAMP-dependent protein kinase from beef heart (50 % inhibition at 0.4 µg\ml), with the latter inhibition being completely abolished by the addition of 3 µM cAMP. The apparent Km value of the Petunia Kemptide kinase for Kemptide was 24 µM as opposed to 3.6 µM for its mammalian counterpart in beef. The authors did not find a regulatory subunit and therefore concluded that the Petunia Kemptide kinase was regulated differently from the animal protein kinase. Although it is conceivable that regulatory and catalytic subunits have separated during the course of the purification, it is also possible, given the apparent broad specificity of for example the Lemna cyclic-nucleotide-responsive protein kinase, that the plant kinase is more similar to the mammalian cGMP-dependent kinase, which does not dissociate into separate subunits. However, their finding that a mammalian regulatory subunit inhibits activity, an effect that can be alleviated by the addition of cAMP, possibly activating dissociation, would strongly suggest a model similar to the animal cAMP-dependent protein kinase. The application of modern molecular biology techniques has provided further evidence of cyclicnucleotide-responsive protein kinases in plants. A number of cAMP-dependent protein kinases from various organisms have been cloned and characterized and show a well-conserved primary structure. Recently, molecular biological evidence was put forward that indicated the presence in higher plants of protein kinases with high homology with cyclicnucleotide-dependent protein kinases from other organisms. Although the indications discussed below are based solely on sequence similarity and still need to be consolidated by biochemical evidence, they nevertheless constitute a significant step forward. Lawton et al. (1989) isolated some candidate serine\threonine protein kinase genes from Phaseolus vulgaris and Oryza sativa cDNA libraries. PVPK-1 (bean) and G11A (rice) have high homology with the catalytic subunit of both protein kinase A and protein kinase C. The open reading frames of these cDNA species contain all except one signature of serine\

threonine protein kinase ; all invariant amino acids necessary for ATP binding and phosphotransfer are present. Thr-197, which is autophosphorylated in cAMP-dependent protein kinase, was replaced by a serine residue, which can serve the same purpose. The presence of the sequence DLKPEN (singleletter amino acid codes) (DLKLDN in protein kinase C) and a GTHEYLAPE sequence (GTPEYLAPE in cyclic-nucleotide-dependent protein kinases and GTPDYIAPE in protein kinase C) points to a greater resemblance to the cyclic nucleotide kinases than to protein kinase C. The plant sequence carries an additional strand of 79–81 nucleotides between these sequences. This insertion was also found in Saccharomyces cerevisiae without apparent loss of catalytic activity. The amino termini of the highly conserved region comprising these sequences in both cDNA species carry 238 additional amino acid residues. These regions differ significantly between both enzymes and probably contain regulatory sequences. This region contained no similarity to other enzymes. Highly homologous protein kinases were found in Zea mays, 90.7 (Biermann et al., 1990) ; Pisum sativum PsPK1 (Lin et al., 1991) and Arabidopsis thaliana Atpk7 (Hayashida et al., 1992), Atpk64 (Mizoguchi et al., 1992) and Atpk5 (Hayashida et al., 1993). Their DNA sequences contain one open reading frame carrying all conserved residues occurring in serine\threonine kinases except that corresponding to Thr-197 ; there, as in Phaseolus vulgaris and Oryza sativa, they carry a serine residue. All show the amino-terminal candidate regulatory domain comprising 108–238 amino acid residues, rich in threonine and serine residues as potential phosphorylation sites. They show the highest degree of homology with cAMP-dependent protein kinase and are categorized as cAMP-dependent protein kinases solely on this basis (Hunter, 1991). Although there is thus an increasing literature indicating cyclic-nucleotide-responsive protein kinases in plants, it is the identification of the protein substrates that will provide the best clues as to which physiological processes are governed by cAMP. The phosphorylation of a substantial number of plant proteins is influenced by cAMP. For example, Li et al. (1994) have recently described an outwardrectifying K+ channel in Vicia faba that is regulated by cAMP. This regulation is dependent on a phosphorylation event ; additional evidence for a cAMP-dependent protein kinase is that the event can be mimicked by addition of a mammalian protein kinase A. In fact, a number of plant proteins are substrates for mammalian protein kinase A. Phytochrome (Wong et al., 1986), phosphoenolpyruvate carboxylase (Terada et al., 1990) and sucrose phosphate synthase (Huber & Huber, 1991) are examples of proteins that are influenced in their function by such phosphorylation. Though they are

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Cyclic nucleotides in higher plants not in themselves sufficient to postulate the existence of the same mechanism in plants, these data provide a valuable indication of which processes are susceptible to regulation through a cAMP-dependent phosphorylation. As cited in section 1, cAMP regulates a considerable number of mammalian physiological processes by interfering with gene expression. Induction is generally fast and independent of intermediate protein synthesis de novo. In a search for cis-acting sequences governing the sensitivity of somatostatin gene expression to cAMP, a short palindromic sequence motif (5h-TGACGTCA-3h) was found that was highly conserved in the promoter region of many cAMP-induced genes (Montminy et al., 1986). This CRE is a classic enhancer sequence in that it stimulates transcription irrespectively of its orientation and distance from the transcription starting point ; when placed upstream of a normally noninduced gene, cAMP inducibility is introduced. Further analysis of cAMP-inducible promoters revealed the presence of a smaller functional motif (5h-CGTCA-3h) that is present in two perfect copies on one or both of the DNA strings (Fink et al., 1988). The potential of CREs to act synergistically when placed in tandem indicates that the number of CGTCA motifs in the promoter region is an important factor for the degree of transcriptional inducibility by cAMP. In animal cells, CREs are recognized by members of the DNA-binding protein family called CREB\ ATF (CREB\activating transcription factor). Proteins belonging to this family consist of two domains. A carboxy-terminal domain with a basic leucine zipper (‘ bZIP ’) motif is involved in the sequencespecific binding of DNA and dimerization, whereas the activation domain possesses regions interacting with other components of the transcription machinery and other signal transduction chains. CREB\ATF proteins bind as dimers to conserved CREs. The formation of heterodimers between CREB (Yamamoto et al., 1988 ; Gonzalez & Montminy, 1989), ATF-1 (Hurst et al., 1991) and CREmodulator protein (‘ CREM ’) (Foulkes et al., 1991) yields a number of different transcription regulators affecting transcription in a positive or a negative manner. CREB\ATF proteins also dimerize with members of the AP-1 transcription factor family such as Jun and Fos (Hai et al., 1989). Eventually, an abundant number of new tissue-specific and functionally different homodimer and heterodimer complexes can be formed, leading to extremely accurate cell-specific regulation. Even in mammals the exact mode of action of CREB is still not completely known. An important prerequisite for its action is a cAMP-dependent phosphorylation of CREB at Ser-133 (Gonzalez & Montminy, 1989). This phosphorylation is necessary for interaction with a CREB-binding protein (CBP)

441 (Kwok et al., 1994) and this CREB-CBP complex then activates transcription, although an an additional cAMP-dependent phosphorylation of CBP might be necessary for the interaction with CREB (Parker et al., 1996). The first indications of the presence of CREB in higher plants come from a study by Inamdar et al. (1991). Radioactively labelled oligonucleotides bearing the CRE motif were recognized by DNA-binding proteins in pea, soybean, cauliflower and wheat. Methylation of the oligonucleotide in the CRE motif resulted in a marked loss of binding capacity that was more pronounced than that in animal systems (Iguchi-Ariga & Schaffner, 1989). For this reason this protein was named MIB-1 (methylation-inhibited binding protein 1). In 1992 the same group isolated a cDNA clone from Vicia faba showing extreme resemblance to the animal CREB protein, both in sequence and in biochemical properties (Ehrlich et al., 1992). As in the animal protein, the amino acid sequence derived from the VBP1 protein contains a basic domain located next to a leucine zipper motif. The protein shows greatest homology with a tobacco DNA-binding protein, TGA1a, that was originally isolated as a CREB homologue (Katagiri et al., 1989), but in contrast with TGA1a the preferred DNA-binding motif of VBP1 was a perfect -TGACGTCA- palindrome. Methylation of the CRE motif destroyed the binding capacity of VBP1 almost completely. Although VBP1 lacks the PKA-phosphorylation site characteristic of CREB (R-R-XS\T (Gonzalez et al., 1989)), it still is a good candidate for a modulator of cAMP-mediated gene expression in higher plants. It contains three degenerate phosphorylation sites for a cAMP dependent protein kinase (R-X-S\T (Kennelly & Krebs, 1991)). Recently, two cDNA fragments were amplified by PCR in Cichorium intybus having extensive homology with TGA1 and VBP1 (Messiaen & Van Cutsem, 1996). Plant genes have been shown to carry CRE motifs in their promoter region : the motif in a soybean proline-rich protein has a perfect palindrome (Hong et al., 1987) ; the sweet-potato α-amylase gene carries a tandem repeat CGTCA motif in its promoter region, and an analysis of its binding factor indicates that it actually prefers the perfect palindrome (Ishiguro et al., 1993). Unfortunately, at present, a role for cAMP in their regulation is merely speculative because data on the induction of these genes by cAMP are lacking. As stated earlier, cAMP does not act solely through the phosphorylation of proteins. Olfactory perception, for example, is governed by a direct binding of cAMP to cyclic-nucleotide-gated channels. A search for cAMP-binding activity in plants readily yielded cAMP-specific binding activities without accompanying protein kinase activity. A Helianthus binding protein was very specific for

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cAMP and 8-bromo-cAMP. 5h-AMP and other 3h,5hcyclic nucleotides did not bind to this protein (Giannattasio et al., 1974a). High-affinity binding activities were also found in Phaseolus and Hordeum (Brown et al., 1979a ; Smith et al., 1979). The partly purified binding protein from Hordeum (Brown et al., 1980b) was highly specific for cAMP ; it had a molecular mass of 170 kDa, a pH optimum of 6.5 and a Kd of 8 nM ; this last value is very similar to that for mammalian cAMP-binding proteins (2–3 nM). The cAMP-binding activity was found in a protein fraction that also contained glucose-6-phosphatase, ATPase, 5h-nucleotidase and fructose-1,6diphosphatase activity ; cAMP binding had no effect on glucose phosphatase or fructose diphosphatase activities, but acted as a weak competitive inhibitor of ATPase and a mixed inhibitor of nucleotidase. Previously, 5h-nucleotidases had been isolated from Triticum vulgare and Solanum tuberosum that were competitively inhibited by cAMP with micromolar Ki values (Polya & Ashton, 1973 ; Polya, 1975). The same group have also described phosphatase activities in Beta vulgaris and Solanum tuberosum that were inhibited by cyclic nucleotides (Polya & Hunziker, 1987 ; Polya & Wettenhall, 1992). Both enzymes, with molecular masses of approx. 30 kDa, were inhibited by cGMP and cAMP. The Kd values of the Beta enzyme for cGMP and cAMP were 0.4 and 3.3 µM respectively, and those of the Solanum enzyme were 0.8 and 2.1 µM respectively. Both enzymes catalyse the dephosphorylation of nucleoside monophosphates and O-phosphotyrosine, but not O-phosphoserine and O-phosphothreonine ; they are thought to be involved in the dephosphorylation of tyrosine-phosphorylated proteins. The use of techniques of molecular biology has also indicated binding sites for cAMP relating to ion channels, for example two channels in Arabidopsis thaliana (KAT1 and AKT1) belonging to the Shaker superfamily of K+-channels. Within this family, KAT1 (Anderson et al., 1992) and AKT1 (Sentenac et al., 1992) show greatest similarity to the eag channel in Drosophila (Warmke et al., 1991), which itself bears a strong sequence similarity to cyclicnucleotide-modulated channels (Guy et al., 1991). The eag channel is regulated by intracellular cAMP (Bru$ ggemann et al., 1993). Apart from a hydrophobic domain consisting of six putative transmembrane stretches also found in the animal K+ channels, both plant genes possess a putative cyclicnucleotide-binding domain. Although not yet proved, a direct interaction of cGMP with this binding domain might explain the sensitivity of the channels to cGMP (Hoshi, 1995 ; Gaymard et al., 1996). Although KAT1 activity is thus influenced by cGMP (see section VIII), cAMP and protein kinase A did not produce the same effect (Hoshi, 1995) ; however, the data from a K+ channel in carrot (Kurosaki, 1997) do suggest an involvement of

cAMP. Treatment of carrot cells with dibutyrylcAMP or the adenylyl cyclase activator forskolin produced an appreciable, but transient, decrease in extracellular [K+] which could be inhibited by K+channel blockers. An increase in intracellular [Ca#+] in response to cAMP was also inhibited by K+channel blockers but was stimulated by a K+ current evoked by an ionophore. These data have led to the proposal that the K+ channel has a role in cross-talk between cAMP and Ca#+, with the gating of some of the inward K+ channels located at the plasma membrane controlled by intracellular [cAMP], and the increased K+ current in response to elevated [cAMP] eliciting a Ca#+ influx into the cells possibly by the activation of voltage-dependent Ca#+ channels (Kurosaki, 1997). V I I .                    C   A large number of physiological processes in plants are potentially sensitive to alterations in [cAMP], as suggested by reports of the effects of cell-permeating cAMP analogues and of alterations in [cAMP] and cAMP-related enzyme activities during physiological events (Brown & Newton, 1981 ; Newton & Brown, 1986 ; Assmann, 1995 ; Bolwell, 1995). However, it is questionable whether many of these observations have any relevance in vivo. Reliable indications of cAMP functions in several plant processes are beginning to emerge, for example a role in ion transport (section VI). It can also be argued that a role in the chloroplast is likely, given the apparent presence of the complete cAMP machinery in this organelle. In this context it is interesting that in the experiments demonstrating that cGMP has a pivotal role in phytochrome phototransduction (as discussed in section VIII) the effect of the microinjection of cAMP and Ca#+ into phytochromedeficient mutants was greater than that of cGMP and Ca#+, and that the effects of cAMP derivatives analogous to Rp-cGMPS (Rp-Guanosine 3h,5h cyclic monophosphothioate) and GTPγS (Guanosine 5h-O3-thiotriphosphate), which are instrumental to the demonstration of the involvement of cGMP, were not examined (Bowler et al., 1994a). Other systems have emerged as being very promising with regard to a physiological role for cAMP. Ehsan et al. (1998) report on fluctuations in [cAMP] that are tightly connected to cell cycle progression in tobacco BY-2 cells. Peaks of [cAMP] were observed during S-phase and to a smaller extent in G (Fig. 4). " The addition of indomethacin, a drug that inhibits adenylyl cyclase activity (Wang et al., 1978), resulted in the loss of the peak in S-phase, an event that was accompanied by a marked decrease in mitotic activity. The authors postulate the existence of an adenylyl cylase activated by prostaglandin or prostaglandin-like compound (e.g. jasmonic acid) that is highly regulated during the cell cycle.

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[cAMP] (pmol g–1 f.wt)

H4 mRNA and mitotic index (%)

Cyclic nucleotides in higher plants

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2 4 6 8 10 Time after aphidicolin release (h) S

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Fig. 4. Fluctuation of cAMP levels during the cell cycle of aphidicolin-synchronized tobacco BY-2 cells. (Modified from Ehsan et al. (1998).) Circles, H4 mRNA ; squares, mitotic index ; bars, [cAMP].

The significance of these cAMP fluctuations becomes apparent on comparison with cell cycle features in other organisms. cAMP is prominent in cell cycle control in animal and fungal systems ; its concentration fluctuates during cell cycle progression (Negishi et al., 1982) and, depending on the cell type, it exhibits stimulatory or inhibitory effects on cell proliferation (Dumont et al., 1989 ; Magnaldo et al., 1989 ; Roger et al., 1995). A transient rise in cAMP before the onset of S-phase is part of a series of events leading to DNA synthesis (Boynton & Whitfield, 1983). The expression of key regulators of the cell cycle such as cyclin A, cyclin D and cyclin E is affected by cAMP (Desdouets et al., 1995 ; Barlat et al., 1995 ; Ward et al., 1996 ; L’Allemain et al., 1997). Forskolin and 8-Br-cAMP inhibit cyclin Aand cyclin E-dependent histone H1 kinase activity in an astrocytic cell line (Gagelin et al., 1994). With cyclin D a direct phosphorylation in the cyclin box " by a cAMP-dependent protein kinase is thought to regulate its activity (Sewing & Mu$ ller, 1994). The Saccharomyces cerevisiae cell cycle is also highly regulated by the Ras\cAMP signal transduction pathway (Baroni et al., 1994 ; Tokiwa et al., 1994). The same seems to apply to organisms close to higher plants. Sakuanrungsirikul et al. (1996) isolated three Chlamydomonas reinhardtii mutants showing cell cycle arrest in G when grown at a non" permissive temperature. This cell cycle arrest could be attributed to a decreased adenylyl cyclase activity and cAMP content of the cells. Blocked cells could be rescued by the addition of cAMP, indicating a function of the cyclic nucleotide in cell division. cAMP was also shown to be a key regulator of the circadian-rhythm-driven cell cycle of the unicellular alga Euglena gracilis (Carre! & Edmunds, 1992, 1993 ; Edmunds, 1994). It is believed to form the link between the internal clock and the cell cycle by negotiating the transition through the G \S and "

G \M boundaries. A study of adenylyl cyclase and # phosphodiesterase revealed that both activities showed oscillating changes occurring in counterphase (Tong et al., 1991). The changing adenylyl cyclase activity is probably due to the regulation of a constant enzyme pool by an oscillating modulator, rather than through a change in the amount of enzyme. At all times of the day the adenylyl cyclase could be stimulated to the same maximal activity with forskolin. The addition of forskolin also resulted in a dampening of the amplitude of the cAMP oscillations and a loss of rhythmicity in cell division. Experiments with isobutylmethylxanthine showed that phosphodiesterase activity was inhibited in a manner dependent on the time of application. However, isobutylmethylxanthine is an inhibitor that influences the activity of various phosphodiesterases, each to a different extent. The difference in efficiency of isobutylmethylxanthine addition was therefore a result of the presence of heterogeneous types of phosphodiesterase during the cell cycle. One specific phosphodiesterase (or a small subset) might be responsible for the oscillations in cAMP content. In a search for the clock directing cAMP content, Tong & Edmunds (1993) studied the role of Ca#+, calmodulin, inositol 1,4,5-trisphosphate (Ins(1,4,5) P ) and cGMP in the regulation of adenylyl cyclase $ and phosphodiesterase. The cGMP content showed oscillations that preceded the oscillations in cAMP by 2 h. The cGMP analogue 8-Br-cGMP and the guanylyl cyclase inhibitor LY83583 (6-aniloquinoline-5,8-quinone) also had an effect on the activities of adenylyl cyclase and phosphodiesterase. cGMP is therefore a good candidate for a mediator of cAMP metabolism. The effect of cAMP on the cell cycle is dependent on the time of addition. The cell cycle was delayed by the addition of cAMP during the subjective day and enhanced during the night. This can be explained by the presence of different receptors for cAMP that selectively regulate one or the other of two regulatory pathways. Two types of cAMPdependent protein kinase have been revealed in Euglena (cPKA and cPKB) with differing affinities for cAMP and some analogues (Carre! & Edmunds, 1992). The use of specific cAMP analogues activating the two kinases in an uneven fashion revealed that the two kinases had different effects on the cell cycle : cPKA delayed the cell cycle ; cPKB promoted it (Carre! & Edmunds, 1993). According to Edmunds (1994), an increase in [cAMP] inhibits DNA synthesis, keeping the cell in G , thereby resulting in the # inhibition of cell division during the day. As soon as [cAMP] decreases this block is abolished. The G \M # transition, or maybe mitosis itself, is stimulated by a second peak of [cAMP]. In this way, mitosis is put into phase with night. The delaying effect of cAMP during the subjective day is regulated by the activation of cPKA ; the stimulation of mitosis during

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the night is regulated by cPKB. The activation of one of the two results in the phosphorylation of a specific set of target proteins. Possibly the expression of cPKA and cPKB differs during the cell cycle, with cPKA being expressed during the day and cPKB during the night, but it is equally possible that the expression of the protein targets varies in such a way that cPKA activation can exert its effect only during the day and cPKB only during the night. The search for targets for cAMP-dependent phosphorylation is under way (Edmunds, 1994). There is a strong argument that it is very unlikely that a compound with such an impact on a function of the cell as fundamental as division would have been lost during evolution from lower plants to higher plants. Indeed, most key regulators of the (supposedly more distant) animal cell cycle seem to be conserved in higher plants. The occurrence of cyclins and cyclin-dependent protein kinases in higher plants similar to those found in animals is now well established (Ferreira et al., 1991, 1994 ; Hemerly et al., 1992 ; Reichheld et al., 1995, 1996), and retinoblastoma protein homologues (Murray, 1997 ; Murray et al., 1997 ; Huntley et al., 1997) and cdc25 homologues (Sabelli et al., 1998) have been isolated recently. Given this overall conservation of cell cycle machinery and circumstantial evidence such as the fact that in a number of cyclins the primary structure reveals signatures for post-translational modifications dependent on cAMP (analogous to cyclin D ), the observation made by Ehsan " et al. (1998) demands a further search for a role for cAMP in the plant cell cycle. There is also strong evidence that cAMP is involved in the plant defence response that produces phytoalexins. This stress response system has clear analogies to the mammalian secondary-messenger system, involving an extracellular signal, a receptor, a signal transduction system and a metabolic response (Smith, 1996). Several extracellular elicitors have been identified, including polysaccharides, oligosaccharides, oligogalacturonides, fatty acids, proteins and glycoproteins ; few receptors for these elicitors have been identified, but those known so far are proteins located on the plasma membrane. The response to the stimulus at these receptors is the activation of specific defence response genes, including those leading to the production of enzymes required for phytoalexin synthesis. The mechanism by which the perceived signal at the plasma membrane is relayed to the nuclear genes has been variously cited to involve Ca#+, jasmonic acid, active oxygen, diacylglycerol and inositol phosphates, and cAMP (Smith, 1996). The first evidence implicating the involvement of cAMP in a cellular defence mechanism was the activation of phytoalexin synthesis in Ipomoea (Oguni et al., 1976) ; similar effects were later reported in carrot (Kurosaki et al., 1987 ; Kurosaki &

Nishi, 1993) in which not only did cAMP enhance phytoalexin accumulation, but also activators of adenylyl cyclase, cholera toxin and forskolin, and a phosphodiesterase inhibitor, theophylline, also enhanced phytoalexin synthesis with a concomitant rise in intracellular [cAMP]. Compelling evidence for the involvement of cAMP is available from a stress reaction of Medicago, in which the phytoalexin medicarpin is synthesized in response to a challenge from a glycoprotein elicitor from the fungus Verticillium albo-atrum (Smith, 1991). There is also a significant increase in the activity of phenylalanine ammonia-lyase (PAL), which catalyses an early reaction in the committed phase of phytoalexin synthesis. Treatment of Medicago seedlings with dibutyryl-cAMP brought about an enhancement of PAL activity and an induction of medicarpin synthesis (Cooke et al., 1989, 1994) ; in cell suspension cultures a 4–5-fold increase in cAMP, unequivocally identified by FAB\MIKE spectrum analysis, was observed after challenge by the elicitor. Although in agreement with the trend of the data from carrot cells (Kurosaki et al., 1987) the response was much higher and more rapid in Medicago, reaching a maximum 3–5 min after challenge, in contrast to the 30 min required in carrot and the 15 min maximum response time in French bean (Bolwell, 1992). In Medicago, the time course of adenylyl cyclase stimulation and decay followed by a phosphodiesterase elevation (Fig. 5a) is a classic example of a switch-on\switch-off system ; arguably the most significant finding is that there is a dosedependent response to the elicitor (Fig. 5b) (Cooke et al., 1994). This dose-dependent response of a particulate adenylyl cyclase preparation in Medicago indicates a direct effect by the elicitor either on the cyclase itself or on an adjacent regulator. The effect of cholera toxin in stimulating phytoalexin synthesis in carrot (Kurosaki et al., 1987) and PAL activity in bean cultures (Bolwell, 1992) suggests that a G-protein might well be involved. Although the data above give a clear indication that cAMP is involved in the phytoalexin defence response, it is not yet known how cAMP triggers off a change in phytoalexin synthesis. In several plant species inositol phosphates are also implicated in this process (Smith, 1996) : in Medicago a very rapid rise, within 1 min of challenge, in [Ins(1,4,5)P ] in $ response to the V. albo-atrum elicitor was observed, returning to control levels within 3 min. This is consistent with changes in [Ca#+] in elicitor-treated cells (Walton et al., 1993). A release of Ins(1,4,5)P $ is potentially capable of releasing Ca#+ from internal stores ; there could therefore conceivably be a mechanism involving Ca#+\calmodulin-dependent protein kinases and protein kinase C. The great sensitivity of the adenylyl cyclase to Ca#+ levels (Cooke et al., 1994), together with the effect of cAMP on ion fluxes (Kurosaki, 1997), suggests that

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445 Adenylyl cyclase (pmol min–1 mg–1)

[cAMP] and enzyme activity (%)

Cyclic nucleotides in higher plants

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Duration of incubation (min)

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Carbohydrate elicitor (lg)

Fig. 5. cAMP in the stress response of Medicago. (a) Response of cAMP (solid line), adenylyl cyclase (broken line) and cyclic nucleotide phosphodiesterase (dotted line) in Medicago cell culture to challenge by fungal elicitor. (Modified from Cooke et al. (1994).) (b) Dose-response curve of partly purified Medicago adenylyl cyclase to fungal elicitor. (Modified from Cooke et al., 1994.)

at least part of the cAMP effect might result from cross-talk between the adenylyl cyclase\cAMP and phophoinositide\Ca#+ signalling pathways. Although there is no apparent change in cAMP-responsive protein kinase in response to elicitor in carrot cells, there is a transient activation of Ca#+ and calmodulindependent kinase activity in response to dibutyrylcAMP or forskolin, leading to a suggested mechanism in which the elicitor-induced synthesis of cAMP leads to an influx of Ca#+, which in turn stimulates a response to elicitor by increasing kinase activity (Kurosaki & Nishi, 1993 ; Kurosaki, 1997). V I I I . c                  When other cyclic nucleotides are considered it is found that, unlike the comparisons between cAMP in mammalian and higher-plant systems, there are clear, established and apparently accepted parallels between the functions of cGMP in plants and mammals. Furthermore it could be argued that a similar paucity of knowledge is available for cyclic nucleotides other than cAMP and cGMP in mammals and higher plants. The first evidence for the occurrence of a second cyclic nucleotide in a higher plant was the report that changes in cGMP concentration took place during cell enlargement and division in excised pith tissues of Nicotiana (Lundeen et al., 1973). cGMP was further reported in bean root tissue (Haddox et al., 1974), pine pollen (Katsumata et al., 1978), in tumour-prone Nicotiana amphyloid (Ames et al., 1980) and in relatively high concentrations in Evodia and Zizyphus (Cyong & Takahashi, 1982a,b,c ; Cyong et al., 1982). However, the means of identification of the putative cGMP was open to the same criticism as was levelled at the identification of cAMP discussed above. To establish the identity of the putative plant

cGMP unequivocally, large-scale extracts from French Dwarf bean seedlings were purified by sequential chromatography and identified by physical analyses (Newton et al., 1984a). The UV absorbance spectrum of the isolated compound had the characteristic lmax values of 255, 252 and 259 nm at pH 1, 7 and 11 respectively ; the NMR spectrum was essentially identical to that of a cGMP standard, with the large shift between the protons at positions 8 and 1 that is characteristic of a cyclic nucleotide. These data strongly supported the identification of the analyte as cGMP but did not establish it unambiguously ; however, the use of FAB-MS with CID\MIKE spectrum scanning provided conclusive evidence (Newton et al., 1984a). The protonated molecule of the extracted putative cGMP (m\z 346) yielded a CID\MIKE spectrum containing the diagnostic fragments at m\z 152, 180 and 194 (Kingston et al., 1984), essentially identical to that of the cGMP standard. It was clearly distinguishable from that of the 2h,3h-cyclic GMP isomer, from which the m\z 194 peak was absent and there were different peaks at m\z 195, 214 and 217, the two cGMP isomers undergoing analogous fragmentations to their cAMP counterparts. Evidence was produced at the same time of cGMP in maize seedlings by GC-MS (Janistyn, 1983). The quantification of the cGMP extracted from French Dwarf bean seedlings illustrated the pitfalls of plant cyclic-nucleotide estimations, in that whereas samples purified to remove non-nucleotide and non-cyclic-nucleotide material gave the correct linear binding curves in the RIA (radioimmuneassay), less purified extracts gave falsely high positive readings (Newton et al., 1984a). With a purified extract the chloroplast was calculated to contain 3.3 pmol g−" cGMP, giving a ratio of substrate concentration to Km for the chloroplast cGMP phosphodiesterase (see section VI) comparable to that found in vertebrate cells. In addition, the chloroplast

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contained guanylyl cyclase activity capable of converting radiolabelled GTP to cGMP (Newton et al., 1984a) ; the product of the enzyme activity has been identified by its FAB\MIKE spectrum (Newton, 1996). Although comparatively few investigations of the function of cGMP in higher plants have been carried out, they have met with significant success. As detailed above, cGMP has a substantial role in the responses to light within the mammalian eye, and evidence has accumulated of an analogous role for cGMP in the responses of higher plants to light. Initially, dibutyryl-cGMP was found to induce changes in the plastid terpenoid components of darkgrown Spinacea that mimicked the effects of phytochrome and were not induced by dibutyryl-cAMP (Brown et al., 1989). Similarly, illuminating Lemna with far-red light increased the level of cGMP, and dibutyryl-cGMP stimulated flowering (Hasunuma et al., 1988). These data conflict with evidence from lichens that suggests, for example, that in Cladonia verticillaris the phytochrome-induced accumulation of phenolics such as fumarprotocetraric acid is strongly enhanced by red light or exogenous cAMP, with red light increasing the endogenous synthesis of cAMP (Mateos et al., 1993). These observations led to the proposal of a mechanism involving the activation of adenylyl cyclase by phytochrome, in which the subsequent increase in intracellular [cAMP] initiated a cascade process similar to that effected by rhodopsin in the mammalian eye, with cAMP rather than cGMP opening ion channels by the activation of a protein kinase (Vicente, 1993). However, strong evidence that cGMP is involved in the action of phytochrome has been produced by single-cell assays in a phytochrome-deficient tomato mutant (Bowler et al., 1994a). After microinjection of putative signalling molecules into hypocotyl cells of the tomato mutant aurea, which lacks type 1 phytochrome but has normal Phytochrome B activity (Sharma et al., 1993), products of phytochrome signalling could be monitored. Although these cells did not develop chloroplasts or synthesize anthocyanins in response to light, microinjection of exogenous type 1 phytochrome restored chloroplast development and anthocyanin production (Neuhaus et al., 1993) and it was possible to activate the expression of a photoregulated reporter gene injected with the photoreceptor (Bowler et al., 1994a). Stimulation of anthocyanin biosynthesis and chloroplast development involved the participation of one or more heterotrimeric G-proteins, together with Ca#+ and calmodulin acting further down the pathway. Although G-protein activation mediated a full cellular response, in its absence Ca#+ and calmodulin alone did not activate anthocyanin biosynthesis and produced incompletely developed chloroplasts. It is therefore cGMP that triggers the

production of anthocyanins ; together with Ca#+ it activates the full development of chloroplasts and it has been demonstrated by means of reporter genes that cGMP and Ca#+ act primarily by modulating gene expression (Bowler et al., 1994a). Subsequently three signal transduction pathways, dependent on cGMP or Ca#+, or both, were identified as the means by which phytochrome controls the expression of genes required for anthocyanin biosynthesis and chloroplast development (Bowler et al., 1994b) ; chs is controlled by a cGMP-dependent pathway, cab by a Ca#+-dependent pathway and fur by a cGMP and Ca#+-dependent pathway. Cross-talk occurs between the pathways : cGMP concentration changes mediate the induction and desensitization of the chs gene to light, and high cGMP levels downregulate both the Ca#+-dependent and the Ca#+\cGMP pathways (Bowler et al., 1994a,b). A further gene, asparagine synthetase, has been shown to be downregulated by light, being expressed in the dark and repressed in the light. The repression of asparagine synthetase in the light seems to be controlled by the Ca#+\cGMP pathway, which activates other light responses (Neuhaus et al., 1997), indicating that the same signal transduction pathway can both activate and repress different responses to phytochrome. By the use of complimentary loss-of-function and gain-offunction studies a 17 base pair cis element within the asparagine synthetase promoter has been identified that is the target for a highly conserved phytochrome-generated repressor regulated by both Ca#+ and cGMP (Neuhaus et al., 1997). In addition to its action in mediating phytochrome responses, cGMP has been proposed to have a role in GA -induced gene expression in barley aleurone $ layers. In a manner consistent with the suggestion that cGMP was involved in ion channel regulation (Hoshi, 1995), the K+ channels KAT1 (Anderson et al., 1992) and AKT1 (Sentenac et al., 1992) in Arabidopsis and the cation channel HvCBT-1 in barley aleurone layers (Schuurink et al., 1998) have a cGMP-binding motif in the C-terminal region. The barley aleurone layer, as a secretory tissue metabolically regulated by hormones with many examples of GA-ABA antagonism, has provided an excellent system for exploring the role of cGMP in plant signal transduction. Several studies have demonstrated that the site of GA perception is at the plasma membrane (Hooley et al., 1991 ; Gilroy & Jones, 1994) and that elevations in cytosolic [Ca#+] (Bush, 1996) and [calmodulin] (Schuurink et al., 1996) are early events in signal transduction. GA $ was further shown to elevate [cGMP], which was unaffected by ABA, and a guanylyl cyclase inhibitor (LY83583) blocked both GA-induced [cGMP] elevation and α-amylase secretion (Penson et al., 1996a,b). This effect of LY83583 on α-amylase secretion could be reversed by cell-permeating derivatives of cGMP, and whereas LY83583 in-

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Cyclic nucleotides in higher plants hibited the GA-induced synthesis of GAMyb, an activator of the high-pI α-amylase promoter (Gubler et al., 1995), it had little effect on the ABA-induced accumulation of RAB21 (a characteristic response to ABA) (Mundy & Chua, 1988), and led to the conclusion that cGMP is an important early compound in the response of cereal aleurone layer to GA but not that to ABA (Penson et al., 1996a,b, $ 1997). A further apparent interaction between Ca#+ and cGMP in plants lies in the anaerobic signal transduction pathway and provides another interesting analogy with the role of cGMP in the mammalian anaerobic response (Depre & Hue, 1994). Ca#+ mobilization from intracellular stores into the cytoplasm is necessary for the mRNA transcription of anaerobic proteins such as alcohol dehydrogenase and sucrose synthetase (Subbaiah et al., 1994a,b), and in the anaerobic accumulation of γ-aminobutyrate (Aurisano et al., 1995, 1996). Whereas [cAMP] decreased, the imposition of anaerobic conditions resulted in a rapid transient increase in [cGMP] in both root and coleoptile of rice seedlings (Reggiani, 1997), which led to a proposed role for both cyclic nucleotides in shutting down ATPdependent ion channels during anoxia, a parallel to that observed in hypoxia-tolerant animals (Hochachka et al., 1988). A final analogy between mammalian and higherplant cGMP systems might lie in the response to nitric oxide. Nitric oxide was identified in mammals as the long-observed but unidentified endotheliumderived releasing factor, and has now been found to have extensive physiological functions involving cGMP (Moncada et al., 1992) : in plants, nitric oxide has been reported to stimulate cGMP formation in spruce (Pfeiffer et al., 1994). Recent evidence shows that nitric oxide potentiates the induction of hypersensitive cell death by reactive oxygen intermediates produced in soybean cells in response to pathogens and, independently of the oxygen intermediates, nitric oxide induces genes for the synthesis of protective natural products (Delledonne et al., 1998). This leads to the conclusion that it has a key role in plant cellular defence, a concept supported by the compromising of disease resistance by inhibitors of nitric oxide synthase. Among other effects, nitric oxide has been shown to drive the activation of PAL in a process, mediated by the guanylyl cyclasecatalysed synthesis of cGMP, which can be further stimulated by cADP-ribose (Durner et al., 1998). Information relating to cyclic nucleotides other than cAMP and GMP is almost as sparse in mammals as in plants. In mammals, the occurrence of cCMP, cIMP, cdTMP and cUMP has been demonstrated, together with enzymes capable of their synthesis and hydrolysis (Newton et al., 1984b, 1986) : their function has not been established, although initial evidence suggests that effects are not

447 solely due to action as agonists or antagonists of cAMP or cGMP (Brus et al., 1984), and that these four additional cyclic nucleotides have independent functions, with cCMP effects and fluctuations in concentration compatible with a role in the regulation of cell proliferation (Newton, 1995). In plants, as detailed above, cyclic nucleotides other than cCMP and cGMP can bind to cyclic-nucleotidebinding proteins ; in lettuce, a phosphodiesterase analogous to the mammalian multifunctional phosphodiesterase, which appears to function primarily in cCMP metabolism, has been found (Chiatante et al., 1986) ; for several of the phosphodiesterases a major factor in the rate of cAMP hydrolysis is the presence or absence of other competing cyclic nucleotide substrates, so the presence of further cyclic nucleotides in plant tissues can be seen to be of considerable significance. The occurrence of cCMP, cUMP, cIMP and cdTMP in Pisum roots has been established by FABMS and CID\MIKE technology (Newton et al., 1989) : the relative levels in meristematic and nonmeristematic tissues show significant differences. In the meristems, cAMP, cCMP, cGMP and cUMP are present, with cAMP, cGMP, cCMP, cIMP, cUMP and cdTMP in the non-meristematic regions, with a significantly higher concentration of cCMP in the meristems and conversely a higher relative level of cUMP in the non-meristematic region than the meristem. The greater quantity of cCMP in the meristem might reflect a role in the rapidly dividing cells, analogous to that proposed for cCMP in mammalian cells, whereas the presence of higher relative concentrations of cUMP in non-meristematic cells than in meristematic cells, taken together with the presence of cIMP and cdTMP in the nonmeristematic regions and their absence from the meristems, might also reflect the difference in proliferation rate of the two types of tissue. Other analogies suggest different roles for these cyclic nucleotides, for example a possible role in intercellular communication in higher plants, similar to the functional extrusion of cCMP, cIMP, cAMP and cdAMP from bacterial cells (Newton et al., 1998), because similar extrusion processes have already been reported for cAMP in lower plants (Francko & Wetzel, 1980a,b). IX.   This review has provided incontrovertible evidence that cAMP, adenylyl cyclase and cyclic nucleotide phosphodiesterase are indeed present in higher plants. The mass spectrometric evidence in particular overcomes the objections raised about the early reports over the identities of the putative cAMP in tissue extracts and as the product of adenylyl cyclase activity. This poses the question of the correctness of the original observations and

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Ion channel

Adenylyl cyclase

Membrane

Light

GA3

Anaerobiosis

G-protein

R

R Nitric oxide

Guanylyl cyclase

CaM ATP

M+(+)

cAMP

Cytoplasm

G-protein CaM

PDE

A-PK

PhyA

AMP GMP Phenylalanine ammonialyase

Protein Protein-PO4 Phosphatase

GTP

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Binding protein Nucleotidase

PDE

Anthocyanins

[G-PK] Phytoalexin a-amylase

(Cyclins) Cell cycle machinery (Cyclins)

Nucleus

VBP1

CRE sites

GAMyb

chs

fur

ASI

cab

CRE sites Ca2+

Fig. 6. Cyclic nucleotides in higher plants. Diagram of cAMP and cGMP metabolism and function in higher plants. Molecular conversions are represented by solid arrows, regulatory effects by dotted arrows. A-PK, cAMP-dependent protein kinase ; ASI, asparagine synthase ; CaM, calmodulin ; G-PK, cGMP-dependent protein kinase ; PDE, phosphodieasterase ; PhyA, type 1 phytochrome ; R, receptor.

theories, put forward in the 1970s, relating to cAMP function. In our view this recent evidence does not automatically render the earlier observations correct, but equally they should not be rejected out of hand. Instead there is an urgent need to re-examine them with the use of the modern, specific, techniques of affinity purification coupled with quantitative mass spectrometric analysis to determine fluctuations in cAMP and adenylyl cyclase in precise kinetic studies of physiological events to assess the actions of cAMP. Emphasis must be placed on the measurement of fluctuations in concentrations and activities. A consensus of the recent quantitative studies of cAMP in plants suggests that levels are appreciably lower than those reported in the 1970s. Although this disparity partly reflects the ambiguities and crossreactivities intrinsic to the older methodologies and procedures, it is our view that it also reflects that there is a low basal level of cAMP in plant cells that is significantly elevated for transient spells concomitant with a physiological event. After the unequivocal demonstration of the existence of the enzymes and other key components of cyclic nucleotide synthesis and breakdown, the ‘ missing link ’ is now the proof that cAMP is a

crucial element in the regulation of basic physiological processes in plants. A comparison of Fig. 6, showing the elements of cyclic nucleotide metabolism and action established in plants, with those of animals and other cell types depicted in Figs 1 and 2 shows the progress made and the gaps remaining to be filled. The results discussed in section VIII detail the evidence that leads to our opinion that cAMP has a key role in, for example, the cell cycle and cellular defence mechanisms in higher plants. Further elucidation of cyclic nucleotide functions will require a broad but systematic attack. In addition to monitoring of fluctuations of cyclic nucleotide and cyclicnucleotide-related enzymes during physiological processes, the investigation of cyclic-nucleotideresponsive protein kinases and cyclic-nucleotidebinding proteins and the identification of their targets will constitute a major step. Complementary to mass spectrometric and biochemical analyses, immunocytological techniques, incorporating highpressure freezing technology and molecular distillation, which permit the localization of small diffusible molecules such as cyclic nucleotides, will facilitate the identification of the subcellular dis-

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Cyclic nucleotides in higher plants tribution of cAMP and cAMP-binding sites ; cytochemical analyses will permit the localization of the enzymes such as adenylyl cyclase. Molecular biology techniques will permit the structural characterization of the genes for adenylyl cyclase and phosphodiesterase, and will be employed to prevent the transient elevation of cAMP by anti-sense manipulation of these enzymes in concert with the use of cAMP analogues to modulate the kinase or cyclic nucleotide-binding activities. As with any scientific research field, the study of cyclic nucleotides in higher plants contains particular problems relating to the techniques and sample sources involved ; however, the major stumbling block in this field has been the failure to attain a ‘ critical mass ’ in respect of the number of laboratories involved in such studies. A number of previous ‘ landmark ’ or ‘ seminal ’ reports have purported to demonstrate the non-existence of cAMP regulatory functions in plants and have had the effects of (i) reducing the number of laboratories wishing to be active in this area and (ii) dissuading the appropriate funding bodies from supporting those that do. In presenting the evidence above, which validates the long-held opinions of the laboratories in the ‘ small coterie of believers ’, it is our fervent hope that this review will have the precisely opposite effect, and stimulate a new momentum in cyclic nucleotide research in higher plants.  Al-azzawi MJ, Hall JL. 1976. Cytochemical localization of adenyl cyclase activity in maize roots. Plant Science Letters 6 : 285–289. Ames IH, Richman RA, Weiss JP. 1980. Is cyclic GMP involved in the regulation of tumorogenesis in the Nicotiana genetic system ? Plant Cell Physiology 21 : 367–372. Amrhein N. 1974a. Evidence against the occurrence of cyclic AMP in higher plants. Planta 118 : 241–258. Amrhein N. 1974b. Cyclic nucleotide phosphodiesterases in plants. Zeitschrift fuW r Pflanzenphysiologie 72 : 249–261. Amrhein N. 1977. The current status of cyclic AMP in higher plants. Annual Review of Plant Physiology 28 : 123–132. Anderson JA, Huprikar SS, Kochian LV, Lucas WJ, Gaber RF. 1992. Functional expression of a probable Arabidopsis thaliana potassium channel in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences, USA 89 : 3736–3740. Ashton AR, Polya GM. 1975. Higher plant cyclic nucleotide phosphodiesterases. Biochemical Journal 149 : 329–339. Ashton AR, Polya GM. 1977. Adenosine 3h,5h-cyclic monophosphate in higher plants. Biochemical Journal 165 : 27–32. Ashton AR, Polya GM. 1978. Cyclic AMP in axenic Rye grass endosperm cell culture. Plant Physiology 61 : 718–722. Assmann SM. 1995. Cyclic AMP as a second messenger in higher plants. Plant Physiology 108 : 885–889. Aurisano N, Bertani A, Reggiani R. 1995. Involvement of calcium and calmodulin in protein and amino acid metabolism in rice root under anoxia. Plant Cell Physiology 36 : 1525–1529. Aurisano N, Bertani A, Reggiani R. 1996. Evidence for the involvement of GTP-binding proteins in the process of anaerobic g-aminobutyrate accumulation in rice roots. Journal of Plant Physiology 149 : 517–519. Balter M. 1999. Plant science - data in key papers cannot be reproduced. Science 283 : 1987–1989. Barlat I, Henglein B, Plet A, Lamb N, Fernadez A, McKenzie F, Pouysse! gur J, Vie! A, Blanchard JM. 1995.

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Note added in proof The presence of both adenylyl cyclase and guanylylcyclase has now been demonstrated in spinach chloroplasts by quantitative mass spectrometry. Initial evidence indicates crosstalk between the enzymes (Newton RP, Bayliss MA, Langridge JA, Wilkins ACR, Games DE, Walton TJ, Brenton AG, Diffley PE, Harris FM, Smith CJ. 1999. Product identification and kinetic studies of nucleotidyl cyclase activity in isolated chloroplasts by quantitative fast-atom bombardment mass spectrometry. Rapid Communications in Mass Spectrometry 13 : 979–985.

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