Red - Far Red Light Effects On the Adenylil Cyclase and Guanylil Cyclase Activity of Sorghum bicolor Seedlings.

Kerim G.Gasumov, Chizuko Shichijo*, Tohru Hashimoto*

Institute of Botany, Azerbaijan Academy of Sciences, Patamdar Shosse 40, Baku 370073, Azerbaijan Republic, *Department of Biology, Faculty of Science, Kobe University, Rokkodai, Nada-ku, Kobe 657, Japan,

Abstract - Using monochromatic and dichromatic radiation we have shown that reaction of adenylil cyclase (AC) and guanylil cyclase (GC) of Sorghum bicolor seedlings are differently at the different period of year. The activity of AC mainly very high in summer period and strongly reacted on the action of R light. At the early autumn it disappeared and it was appeared again in middle of winter. But the higher GC activity was registered at autumn period and it decreased at the winter when increased AC activity. Probably both of these cNMPs participate in the phytochrome photosignaling and during of year one may substitute for another, although there is possibility participation both of these cNMP in photosignal transduction processes in the same time.

INTRODUCTION
Phytochrome is a pigment responsible for photoperiodism and photomorfogenesis in plants. The discovering of phytochrome system in higher plants is dated with discovering of cyclic AMP in animal organism and during of last 30 years was actively studied. Although a lot of research has been done on the regulation by phytochrome, the mechanism of this photoreceptor action has not yet been fully understood. Among of many hypotheses on the mechanism of regulation by phytochrome the one that is probably closest to the truth is concerned with the participation of cyclic AMP system (Mohr, 1972; Rast et al., 1973). It was shown that the red (R) and far red (FR) lights absorbed by phytochrome regulate activity of cAMP related enzymes in maize seedlings and red illumination of etiolated seedlings results to increasing of cAMP level in cell (Fdenko et al., 1983, 1988). In a nitrogen-fixing cyanobacterium Anabaena cylindirica, an immediate light-to-dark transition caused a 9-fold increase in cAMP concentration within 1 min. (Ohmori, 1989,). The rise in cAMP may result from the activation of adenylil cyclase or inactivation cAMP phosphodiesterase or both (Ohmori et al., 1996). The possibility of participation of the cGMP in phytochrome photosygnal transduction processes have also been shown (Bowler et al., 1994). Using microinjection metodology, cGMP was found to mediate PHYA-dependent anthocyanin biosynthesis and also to participate in co-ordinating chloroplast development (Bowler et al., 1994). Isolated chloroplasts have been found to contain cAMP, cGMP, the highest subcellular adenylil cyclase and guanylil cyclase activities, and light/CaM-sensitive PDE. Reports of cNMP-mediation of phytochrome regulation of chloroplast development and function stimulates
systematic investigation of the functions of cNMPs in these organelles. The problem weather cAMP and cGMP systems acts as a second messenger in photosignal transduction in plants separately or at the same time is particularly prone to be worked. In this article we attempt go deep into this problem looking reaction of adenylil cyclase and guanylil cyclase enzymes on the monochromatic red and far red light effects.

MATERIALS AND METHODS.

In experiments Sorghum bicolor as a plant material was used. Seeds of Sorghum bicolor Moench, cv. Acme Broomcorn 1995 crop from Kobe University Farm at Kasai, were soaked for 24 h in water bath of 24^oC, in which temperature-adjusted tap water being supplemented circulated. From sowing to irradiation seedlings were grown in the dark at 20^oC for 5 days.

Irradiation of seedlings and light source
As a red light source RLED lmax = 670 nm, (half-bendwidth 655-685 nm) red light emitting diodes, (QB-1310CS-670-735; Quantum Revices, Inc., Wisconsin, USA) and far red light source FRLED l max-750 (half-bendwidth 730-760nm), far red light emitting diodes (QB-1310CS-735, Quentium Revices, Inc., Wisconsin, USA) were used. Green safe-light G526 was supplied from green-filter-coated fluorescent tubes (Coloured lamp, FL20S, G-F; Matsushitu Electric Co., Osaka), through a sheet of No109-1 yellow (Mitsubishi Rayon) polyacrylic resin film, lmax=526nm with a half-bandwidth 505-535 nm. Etiolated seedlings were irradiated according to Shichijo et al. (1993) by R in the intensity of 100 mmol m^-2 s^-1 x 200 s (20000 mmol m^-2) and FR 400 mmol m^-2 s^-1 x 30 s (12000 mmol m^-2) where intensity of R is plateau for anthocyanin sinthesis. All processes of irradiation were carried out under greensafelight.

Nucleotide cyclase preparation.
Nucleotide cyclase preparates were prepared from etiolated seedlings. Homogenisation of seedlings conducted by glass mortar for 1 g seedlings used 3ml tris buffer pH 7.4 containing: 25 mM Tris (hydroxymethyl)-amminometane, 5 mM MgCl2, 0.25 mM NaEDTA and 1mM Dithiothreitol (threo-1,4 Dimercapto-2,3-butanediol). Homogenisation of seedlings was conducted in ice bath (+2 - +3^oC), homogenate filtered through four layer cheesecloth. The filtered homogenate was subjected to centrifugation at 1000g for 10 min, and the pellet discarded. The supernatant was recentrifuged at 30,000g for 40 min +2- +3^oC. Pellet (membrane fraction, rich with nucleotidil cyclase activity) dissolved in 2 ml extraction buffer and was dialysed against 2L the same buffer during 18-20 hr and used for adenylil and guanylil cyclase activity assay.

Adenilil and Guanilil cyclases assay.
The standard assay was done in epindorf tube in the 50 ml mixture containing 40 mM tris-HCl, 5mM MgCl2, 0.25mM Na-EDTA, 0.5mM DTT, 20mM NaF (for adenilil cyclase only), 5mM cafeine, 4mM phosphocreatine, 8 mg/ml creatine phosphokinase, 0.2mM ATP or GTP, 14C-ATP 0.1mCi or, 3H-GTP 1.0mCi in sample, protein 6-9mg. Incubation was conducted at the 33^oC for 20 min and was terminated by adding of 5ml 20mM cAMP or cGMP and boiling for 2 min. After boiling the samples cold in ice bath. After incubation tubes was centrifuged for 5 min 12000 RPM. Separation of nucleotides was carried out by thin layer chromotography on the silicagel manufactured by "Merck". Before spotting of samples on plate we spotted 2ml each point solution of "nucleotides witnesses" containing 10 mM of each nucleotides - ATP, ADP, AMP, cAMP, adenosine and adenine, or GTP, GDP, GMP, cGMP, guanosine, and guanine. On the spotted nucleotides-witnesses we spotted 6ml incubation mixture for adenilate cyclase or 5ml for guanilate cyclase. Chromotography was developped in the system of solution: n-butanol:acetone:ammonia (25%) in relation 8:2:7 and again the same system in relation 8:2:2. Nucleotides visualised under UV light, cut and counted in toluone sinntillation liqued containing in 1L toluone 0.2g Dimethyl-POPOP and 4g PPO (2,5-Diphenyloxasol) on the "Becman LS 6000" liquid scintillation spectrometer. All chemicals used for enzyme isolation and assay were produced by "Wako" and "Sigma" chemical companies. Protein concentration was determined by method of Lowry and as a marker was used bovine albumine.

RESULTS
Separation of adenylil and guanylil cyclases.

We have isolated membrane fraction of protein riched by cyclase activity from 5 days etiolated Sorghum bicolor seedlings and this fraction was subjected to adenylil cyclase (AC) and guanylil cyclase (GC) activities assay. We have found the higher cyclases activity in membrane fraction of seedlings and optimal enzyme activity was occurred in moderately alkaline medium - pH 7.8. We determined that, the higher AC activity appeared in summer and early autumn period. But in the middle of autumn the activity of AC disappeared, and it was again appeared only in the middle of winter. On the contrary of AC the activity of GC in summer and all period of autumn was very higher. And beginning with middle of winter it was getting down the GC activity when starts of increasing of the AC activity.

Red and far red light effects on AC and GC.

Before isolation of enzyme fractions we irradiated seedlings separately by red (R) and far red (FR) lights and by their combination - irradiation of seedlings by far red light just after red illumination (RFR). Immediately after illumination of seedlings they subjected for isolation membrane fraction of cyclases activity. Irradiation of seedlings by R and FR lights led to significantly alteration of AC and GC activities. We determined that the alteration of AC and GC activity, caused by monochromatic lights was differently in the summer and winter season. Beginning with of summer AC of the etiolated Sorghum seedlings grown in 20^o was activated by FR strongly (July -early September) and this activation was observed by irradiation of seedlings with RFR too. Irradiation of seedlings by R in this period did not show any effect on AC activity (Fig.1A). But in the middle of winter when appeared AC activity again we determined activation of AC by all versions of irradiation - R, FR and RFR (Fig.1B). As compared with AC, the GC isolated from etiolated seedlings significantly activated by R and FR lights which behavior of GC may be predictable, based on current knowledge of the actions of phytochrome A and phytochrome B (Smith et al., 1997). The activation of GC by R and FR lights we observed repeatedly in all autumn experiments (Fig.2A). Moreover in this period D and FR irradiated activity of GC in compare of summer period was much higher, and higher activity of GC has been continued until of winter. But beginning with of January it became decreasing, where in this period we determined increasing of AC activity of D and irradiated seedlings. Although the total activity of GC in middle of winter was decreased but its reaction to monochromatic lights effect was determined (Fig.2B).

DISCUSSION.

Alteration of activity of the AC and GC enzymes caused by monochromatic lights testifies the red and far red light fotoreceptors' - phytochromes participation in this regulation processes. Activation of AC in etiolated seedlings by FR light appeared more strongly in summer and early autumn. It is obviously that the same feel of AC to light treatment duration of Summer and Autumn is a result interaction of this enzyme with phytochrome A. With coming late of winter and early spring the sensitiveness AC was increased to R and FR light too. It seems to be AC this period responsible to the action of phytocromes A and B i.e. activated phytochromes absorbing R and FR lights cause some photoinversion of AC, although there is a possibility participation of multiform of AC which differed by responsiblity to light effects. And also there is probability of the  GC participation in the transduction of photosignal from phytochromes. Higher GC activity in the autumn, when there is absent AC activity, and reaction of GC to red and far red light effects testifies that cGMP system also as a cAMP may become secondary messanger of photosignal transduction. Probably both of these cNMPs participate in the phytochrome photosignaling and during of year one may substitute for another, although it is possible both of these cNMP in the same time participate in photosignal transduction processes. At the end of winter and early spring observed activation of GC by red light, although total activity of enzyme was lower than autumn season testify that, GC like as a AC can be interacted with different forms of phytochromes and two cNMP systems differ in the relation with phytochromes in different period of year.
 

Acknowledgements -The authors gratefully acknowledge Japan Society for the Promotion of Science for the funding of this work and the Experimental Farm, Kobe University, Kasai for seeds.
 

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