Guanosine-3',5'-Cyclic Monophosphate (cGMP) in Plants

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Although cGMP was recognized as an important second messenger in animal cells in the early 1970s, plant biologists were slow in recognizing its importance in plant cell signal transduction. The reason for this is traceable to the cAMP debacle. Beginning in 1969, plant biologists began churning out a spate of hastily conceived and sloppily executed research concerning cAMP and plants. At the same time, they were also gathering reports from various researchers that questioned whether cAMP was even present in plants. These negative reports were marshaled by Amrhein (1977) in a very influential review article. Amrhein's (1977) review, while salutary to the field in general, did more than just prune the untamed growth of early cAMP research: it hacked the young shrub to the crown. Indeed, in the wake of Amrhein's (1977) review, the mere mention of cyclic nucleotides was enough to elicit snickers and the rolling of eyes in the seminar room and instant rejections from funding agencies. Although in the ensuing years, a few under-appreciated researchers tried to restore interest in cyclic nucleotides and plants, their arguments fell largely on deaf ears (Brown and Newton, 1981; Francko, 1983).

	Early Reports of cGMP Effects in Plants

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Even during the Dark Ages of plant cyclic nucleotide research, there were a handful of scattered reports implicating cGMP in a variety of plant physiological processes. These processes included the thigmotropic responses of Portulaca grandiflora stamens (Jaffe et al., 1977), pollen germination in Pinus densiflora pollen (Takahashi et al., 1978), tumorigenesis in Nicotiana tabacum (Ames et al., 1980), the autophosphorylation of elongation factor I from wheat (Triticum aestivum) embryos (Ejiri and Honda, 1985), and the flowering and circadian rhythmicity of Lemna paucicostata (Hasunuma et al., 1988). These early reports were ahead of their time and have not received the attention they deserve.

	cGMP Becomes Accepted as a Second Messenger in Plants

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Cyclic GMP was not widely embraced by the plant research community as a legitimate second messenger in plants until the publication in 1994 of three seminal papers. First, Pfeiffer et al. (1994) found that the exposure of spruce (Picea abies) needles to gaseous nitric oxide (NO) led to a strong (up to 10,000-fold) and rapid increase in cGMP levels. These findings suggested very strongly that plants possess a NO-sensitive guanylate cyclase similar to that occurring in animal cells. Second, Bowler et al. (1994) found that the microinjection of cGMP into phytochrome A-deficient aurea tomato (Lycopersicon esculentum) leaf cells mimicked the phytochrome A-mediate anthocyanin biosynthesis found in wild-type cells. Third, Li et al. (1994) presented evidence that cAMP affected the gating properties of Vicia faba guard cells. Although these authors found that cGMP was without effect in their system, their findings rendered irrelevant the foremost objection against cAMP being a second messenger in plants, namely the paucity of evidence for cAMP-dependent protein kinases. cAMP and, by inference, other cyclic nucleotides could be acting directly upon the gating properties of plant ion channels.

	Effects of cGMP on Plant Ion Channels

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Leng et al. (1999) reported the cloning and first functional characterization of a cyclic nucleotide gated (cng) ion channel from a plant. The Arabidopsis cDNA AtCNGC2 was reported to encode for a polypeptide that was homologous to the -subunit of several animal cng channels. Moreover, AtCNGC2 facilitated cyclic nucleotide-dependent cation currents upon expression in three different heterologous systems.

More recently, Maathuis and Sanders (2001) characterized voltage independent channels (VICs) in Arabidopsis roots, the opening probabilities of which were dramatically decreased by the presence of micromolar concentrations of cytoplasmic cAMP or cGMP. They also found that cyclic nucleotides including cGMP reduced the unidirectional influx of Na⁺ into the roots.

	cGMP and Plant Defense

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Durner et al. (1998) found that the infection of resistant, but not susceptible, tobacco with tobacco mosaic virus resulted in enhanced NO synthase (NOS) activity. Furthermore, administration of NO donors or recombinant mammalian NOS to tobacco plants or tobacco suspension cells triggered expression of two defense-related genes. These genes were also induced by cyclic GMP and cADP-Rib. Consistent with cGMP acting as a second messenger in tobacco, NO treatment induced a dramatic and transient increase in cGMP levels. More recently, Klessig et al. (2000) implicated cGMP and NO in the induction of the hypersensitive response in plants.

	cGMP and Programmed Cell Death in Plants

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Clarke et al. (2000) found that Arabidopsis suspension cultures generate elevated levels of NO in response to challenge by avirulent bacteria, and that these levels of NO were sufficient to induce cell death in Arabidopsis cells independently of reactive oxygen species (ROS). They concluded that NO-induced cell death is a form of programmed cell death (PCD), requiring gene expression, and has a number of characteristics of PCD of mammalian cells. NO-induced chromatin condensation and caspase-like activity in Arabidopsis cells, and NO-induced death could be blocked by a caspase-1 inhibitor. A specific inhibitor of guanylate cyclase also blocked NO-induced cell death in Arabidopsis cells, and this inhibition was reversed by a cell-permeable cGMP analog. The cGMP analog alone, however, did not potentiate NO-induced cell death. Thus, cGMP synthesis is required but not sufficient for NO-induced cell death in Arabidopsis.

	Characterization of a Plant Guanylyl Cyclase

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Guanylyl cyclases (GCs) catalyze the formation of cGMP from GTP. A comparison of the Arabidopsis genome with GC sequences from cyanobacteria and various eukaryotes revealed no homologous proteins (Ludidi and Gehring, 2003). However, a motif search of the Arabidopsis genome based on conserved and functionally assigned amino acids in the catalytic center of annotated GCs returned one candidate (AtGC1) that contained the adjacent Gly-rich domain typical for GCs. When AtGC1 was expressed in Escherichia coli, cell extracts yielded >2.5 times more cGMP than controls. This increase, however, was not NO-dependent. AtGC1 is thus not only the first functional plant GC but also, due to its unusual domain organization, a member of a new class of GCs.

	cGMP and Plant Hormones

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cCMP has been implicated in a number of hormone signal transduction chains in plant cells. Penson et al. (1996) found that cGMP levels increased transiently after incubation in gibberellic acid but not abscisic acid. An inhibitor of GC prevented the GA-induced increase in cGMP and inhibited GA-induced -amylase synthesis and secretion. Cousson (2001) provided pharmacological evidence that cGMP may partially mediate auxin-induced stomatal opening in Commelina communis, while Gehring (1999) has suggested cGMP may play a role in mediating the effects of plant "natriuretic peptides" on stomatal opening.