Artefactual Origins of Cyclic AMP in Higher Plant Tissues

Abstract

A highly sensitive radioimmunoassay has been used to determine the levels of adenosine 3',5'-cyclic monophosphate (cAMP) in five higher plants (Lactuca sativa, Helianthus annuus, Oryza sativa, Pinus pinaster, Nicotiana tabacum). Particular attention was paid to the three main sources of errors in the charactenzation of cAMP in plants: presence of interfering substances in plant tissues; possible artefactual formation of cAMP from endogenous ATP during extraction, purification, and assay; and microbial origin of cAMP. In all the tested tissues, the cAMP level was below the detection limit of 0.5 picomole per gram fresh weight, a value much lower than those reported for similar materials of the same species in many previous studies. This result is not in favor of cAMP-dependent regulations in higher plants.et al. (7,8) to check the presence of cAMP in different plants chosen among gymnosperms, monocotyledons, and dicotyledons. Great care was taken to ensure the elimination of interfering compounds and the absence of de novo synthesis of cAMP during the assay, together with maximal recovery of endogenous cAMP. In all tissues, the cAMP level was* below the detection limit ofthe assay: the discrepancy between these results and published ones on similar materials is discussed in the light of potential sources of errors. MATERIALS AND METHODS Biological MaterialCyclic AMP is present in animal tissues and in many microorganisms such as bacteria, fungi, and green algae. Its occurrence in higher plants and its role in the regulation of their metabolism are still a matter of debate. The main evidence supporting the existence of the cAMP' system in plant tissues is based on cAMP estimations, but a large diversity exists among reported determinations of cAMP in higher plants: for example, in the case of tobacco callus, published values range from 900 (15) to less than 0.5 pmol. g-' (2). On the other hand, there is only scant evidence for enzymes involved in cAMP metabolism (9). Most data in this field can be found in reviews (9) Lettuce (Lactuca sativa L., cv Val d'Orge) was obtained from Societe Clause (France). Lettuce seeds were sterilized by soaking for 20 min in commercial sodium hypochlorite (150 g ofchlorine per L). After extensive washing with sterile water, seeds were either immediately frozen in liquid nitrogen or germinated for 2 h at 20C.Sunflower (Helianthus annuus L., cv Rodeo) was obtained from Cetiom (France). Different materials were used: immature seeds were removed from plant heads 7 and 14 d after self-pollination in a growth cabinet and nondesiccated mature seeds after 28 d; these seeds, as well as mature dry seeds, were soaked for 15 min in one-fourth diluted commercial sodium hypochlorite, dehusked, and further sterilized in one-tenth hypochlorite dilution; young seedlings were obtained 3 and 8 d after the beginning of germination under sterile conditions. Rice (Oryza sativa L., var Cigalon) was obtained from Station d'Amelioration des Plantes (INRA Montpellier, France). Dehusked dry seeds wer...

In silico comparative analysis and expression profile of antioxidant proteins in plants

“…This finding is consistent with previous reports in which catalase is required in one of the steps of tyrosine metabolism, namely, the conversion of p-hydroxy-phenylpyruvic acid to homogentisic acid (Moorhead et al, 2009). A. thaliana has a few protein Tyr-specific phosphatases and 22 dualspecificity phosphatases (Spiteri et al, 1989;Rayapureddi et al, 2005). The role of tyrosine phosphatases in stomatal regulation provides critical evidence that tyrosine phosphatases not only exist but also play an important role in higher plants.…”

Methylxanthines reversibly inhibit tracheary-element differentiation in suspension cultures of Zinnia elegans L.

“…Failure to inhibit TE differentiation with low concentrations of 8-phenyltheophylline and enprofylline, potent inhibitors of adenosine receptors and cAMP phosphodiesterase in animal cells (Fredholm 1985), is consistent with reports arguing against a regulatory role for cAMP in plants (Amrhein 1974;Spiteri et al 1989). Nevertheless, some authors have implicated cAMP in the regulation of TE differentiation based on stimulation of differentiation in the absence of cytokinin by theophylline (with or without added cAMP) or the cAMP analog 8-bromo-cAMP (Basile et al 1973;Mizuno and Komamine 1978).…”

“…This was interpreted by the authors as evidence for cAMP involvement in transduction of the cytokinin signal. However, recent reports of the absence of cAMP in plant tissues (Spiteri et al 1989), along with the failure to document the existence of cAMP-mediated transduction mechanisms (Amrhein 1974;Hepler and Wayne 1985), cast doubt on the regulatory role of cAMP in plants.…”

Cyclic AMP as a Second Messenger in Higher Plants (Status and Future Prospects)

“…Since the report of Spiteri et al (1989), others have also employed the same assay techniques as that group and have in some cases detected significantly higher cAMP concentrations. Concentrations of 36 pmol cAMP/g fresh weight in Torenia stem segments (Ishioka and Tanimoto, 1990) and 5 pmol cAMP/g fresh weight in suspensioncultured cells of Pkaseolus vulgaris (Bolwell, 1992) have been described.…”