Cytoenzymological analysis of adenylyl cyclase activity and 3′:5′‐cAMP immunolocalization in chloroplasts of Nicotiana tabacum

Abstract

Summary• In this study a combination of cytoenzymological and immunocytochemical techniques was used in order to demonstrate the presence of cyclic nucleotide metabolism in chloroplasts of higher plants.• Catalytic cytochemistry was used to localize adenylyl cyclase activity by means of electron microscope investigation on Nicotiana tabacum cv. Petit Havana leaf fragments. Various immunocytochemical techniques were explored to visualize the presence of the second messenger adenosine 3 ′ :5 ′ -cyclic monophosphate.• Making use of adenylyl imidodiphosphate as a substrate, the enzyme activity was predominantly located at the intermembrane space of the chloroplast envelope. In order to provide further topographical information, intact, isolated chloroplasts were submitted to the same cytoenzymological procedure and revealed stromal adenylyl cyclase activity. Using high-pressure freezing as a physical fixative to obtain an instantaneous metabolic arrest the cellular vitrified water phase was sublimed under ultra-high vacuum by means of molecular distillation drying, avoiding recrystallization and hence redistribution of small highly diffusible molecules. This sequential combination preserved 3 ′ :5 ′ -cAMP epitope retention in chloroplasts as was demonstrated by immunogold labelling.• These results further substantiate in a unique way the growing evidence of the presence of an organelle-specific cAMP metabolism in higher plants. Furthermore the data presented support the status of chloroplasts as an excellent model to further investigate cAMP metabolism and to correlate it with a variety of physiological functions.

Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling

“…cGMP was also implicated in chloroplast development ( Bowler et al, 1994a ), which could be inhibited by high level of cGMP ( Bowler et al, 1994b ; Wu et al, 1996 ). Immunochemical and cytoenzymological detection of cAMP and adenylate cyclase (AC) activity, respectively, was demonstrated in the developing tobacco chloroplasts ( Witters et al, 2005 ), providing additional evidence for a general role of cNMPs in photosynthetic activity of higher plants, resembling well-characterized functions of these secondary messengers in lower plants. In addition to gibberellic acid (GA)-dependent elevation of cAMP concentration required for seeds germination, observed in common broomrape ( Orobanche minor ) ( Uematsu et al, 2007 ) and phacelia ( Phacelia tanacetifolia ) seeds ( Uematsu and Fukui, 2008 ), cGMP was identified as a positive regulator of Arabidopsis seed germination ( Teng et al, 2010 ).…”

Molecular Targets and Biological Functions of cAMP Signaling in Arabidopsis

“…Plant tissue extracts displayed both mAC and sAC activities, and these activities were present across various cellular organelles including the nucleus, chloroplasts, mitochondria, and vacuoles [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ]. Comparable sAC activities were observed between plants and animals, and the activities of both mAC and sAC in plants were significantly impacted by external factors, such as far-red and red light, temperature, and exogenous phytohormones, as well as specific triggering compounds of fungal and bacterial origin [ 23 ].…”

Cyclic AMP: A Polyhedral Signalling Molecule in Plants

“…Moreover, the treatment of Medicago cells with cAMP enhanced PAL activity and the synthesis of the phytoalexin medicarpin [ 106 ]. Consistently, in Arabidopsis, the treatment of seedlings with the permeable cAMP analogue 8-Br-cAMP increased, up to 40-fold and 2-fold, respectively, the expression of PAL2 and PAL1 [ 87 ]. PAL, the expression of which increased in response to diverse pathogens and elicitors, also plays a key role in SA synthesis [ 111 , 112 , 113 ] ( Figure 3 ).…”

“…cAMP involvement in photosynthetic pathways was also described by Donaldoson and colleagues [ 43 ], who reported the interaction between cAMP and enzymes involved in Calvin cycle and photorespiration pathway. This is of interest since in Nicotiana tabacum , through a quantitative method based on mass spectrometric analysis, AC activity was observed in chloroplasts [ 87 ]. Moreover, in oat seedlings, it was shown that light influenced cAMP accumulation, pointing out that cAMP could take part in the phytochrome signalling pathway [ 88 ].…”