Ever since the first reports on cyclic nucleotide metabolism, its occurrence and function in higher plants have been controversial (Assmann, 1995; Bolwell, 1995; Trewavas, 1997; Newton et al., 1999a; Newton & Smith, 2004). Using mass spectrometry-based identification the natural occurrence of cyclic nucleotides has been repeatedly demonstrated in various plant model systems (Newton et al., 1989; Witters et al., 1996). Biochemical proof for anabolic and catabolic cyclic nucleotide-related enzyme activity in higher plants have been reported in different model species (Newton et al., 1999a; Witters et al., 2004). The recently discovered cDNA, encoding for a soluble enzyme exposing adenylyl cyclase (AC) activity, provided the first evidence at the genetic level for enzymic adenosine cyclic monophosphate production (Moutinho et al., 2001). Similarly, using a molecular biology strategy, a novel protein with guanylyl cyclase activity has been cloned and proven to act as a monomer as well as a homo-oligomer, thereby representing a novel class of guanylyl cyclases (Ludidi & Gehring, 2003). Recent studies describing cyclic nucleotide gated channels (Köhler et al., 1999; Leng et al., 1999; Maathuis & Sanders, 2001; Hua et al., 2003) contributed greatly to the physiological relevance of cyclic nucleotides. With the evidence gathered over the past decade, although it still is fragmentary, renewed interest is emerging. An important consideration one has to make when studying the metabolism of signal molecules is their inherent fast and transient activity profile, making sampling time and spatial resolution crucial. Most data on AC activity in literature have been acquired using biochemical assays, often using tissue homogenates from various parts of the plant that are not necessary homogeneous with respect to their physiological status. As a result the basal metabolic activity can mask or obscure to a great extent the change in activity of the elicited cells. In catalytic histochemistry, both at the tissue level and the cellular level, enzymes are ideally detected in their natural environment at their original site. Cytoenzymological data can therefore provide the means to integrate functional metabolic aspects and morphology. The additional strength of cytoenzymology is its resolution, as it not only can reveal the site of action within a specific cell, but can also show what cells among others in a tissue are affected. The qualitative and to a certain extend also the quantitative information from catalytic cytoenzymological studies can thus be used as a complement to biochemical investigations. Early publications report on the appearance of AC activity at the side of root nodules in Trifolium spp. (Tu, 1974) using ATP as a substrate and lead as capturing agent. Using adenylylimidodiphosphate as a more specific substrate and lead as capturing agent, AC activity in Zea mays root tips was localized at the plasma membrane, endoplasmic reticulum and nuclear membranes (Al-Azzawi & Hall, 1976), at internal membranes of cytoplasmic vacuoles in Pisum sativum (Hilton & Nesius, 1978), at the external side of the host plasma membrane and membranes surrounding the endophyte in root nodules of Alnus glutinosa (Gardner et al., 1979), and at the external side of the plasma membrane of Pisum sativum (Nougarède et al., 1984). Using strontium as a capturing agent and adenylylimidodiphosphate as substrate, AC activity was associated to membrane structures and the cell wall on both pollen and stigma side during pollination (Rougier et al., 1988). They postulated that AC activity was a determinative factor in the compatibility of pollen tube formation in Populus spp. Curvetto & Delmastro (1990) showed that AC activity was localized in Vicia faba guard cells and that it was selectively stimulated by various effectors of the cyclic nucleotide metabolism.
As is the case with histochemistry, immunolocalization can reveal the topographical distribution of proteins at the ultrastructural level but does not give any activity information. Whereas immunolocalization of large biomolecules such as proteins is relatively straightforward, epitope retention and dedicated sample preparation for avoiding redistribution of small diffusible polar compounds such as cyclic nucleotides is far more challenging (Linner et al., 1986). Since those analytes are not encrusted in a matrix such as membranes, microfibres or cell wall, chemical fixation always imposes dilution and redistribution of the solvated molecules as the chemofixative enters the cytosol. Furthermore, chemical fixation is to be considered as a relatively slow process depending on diffusion rates and as a consequence of the covalent binding of the target molecules to the cell matrix, loss of antigenicity by blocking or chemically altering the antigenic recognition site can lead to loss of response. By contrast, cryofixation ensures almost instantaneous biochemical arrest of metabolic processes, does not alter the antigenic recognition sites and dilution and redistribution is kept to a very minimum. Among freeze fixation techniques such as propane-jet freezing, cold metal block slamming, plunge freezing and high-pressure freezing, the last is known to give the best results on ultrastructural preservation of ‘thick’ specimens (Gilkey & Staehelin, 1986). The next crucial step in the preparation procedure is substitution of the water phase with a resin. Resin impregnation via cryosubstitution is excluded a priori, since it involves subzero liquid-phase transfer redistribution, possibly resulting in dilution of unfixed antigens. In order to deal with these particular problems, molecular distillation drying is the method of choice (Linner et al., 1986). This technique enables the removal of amorphous water without recrystallization. Reports on immunolocalization of small diffusible molecules in literature are sparse and most of the reports on topochemical studies of cyclic nucleotides in literature are restricted to 3′:5′-guanosine cyclic monophosphate (3′:5′-cGMP) (Chan-Palay & Palay, 1979; De Vente et al., 1987) and 3′:5′-adenosine cyclic monophosphate (3′:5′-cAMP) (Wedner et al., 1972; Steiner et al., 1976) in paraformaldehyde-fixed animal tissue. Until now, to our knowledge, no reports describe immunolocalization of those compounds in plant tissue. In this study we report on the presence of active AC in chloroplasts as well as the immunolocalization of its product, adenosine 3′:5′-cyclic monophosphate, for the first time in higher plants.