Planta (1983)159:382-385

P l a n t a 9 Springer-Verlag 1983

Short communication

Gas chromatographic-mass spectroscopic identification and quantification of cyclic guanosine-3' :5'-monophosphate in maize seedlings (Zea mays) Boris Janistyn Institut ffir Pharmazeutische Biologic der Universit~t, Sch/inzlestrasse 1, D-7800 Freiburg, Federal Republic of Germany

Abstract. Gas chromatographic-mass spectroscopic evidence is presented for the presence of guanosine-31:5'-monophosphate (cGMP) in maize seedlings. The amount of cGMP (35-72 pmol g- 1 fresh weight) was quantified as a tetra-silyl derivative using gas-chromatographic detection with reference to a silylated standard of authentic cGMP. Gas-chromatographic separation of tri-silyl adenosine-31:5'-monophosphate and tetra-silyl cGMP is demonstrated. Key words: Cyclic guanosine-Y:5'-monophosphate (cGMP) - Zea (cGMP).

Introduction

Cyclic guanosine-3':5'-monophosphate has been found to occur in higher plants first by means of various bioassays but the amounts thus detected were small (Haddox et al. 1974; Lundeen et al. 1973). In contrast, more recent reports based on the findings of Cyong et al. (1982) and Cyong and Takahashi (1982) indicate the presence of much higher cGMP levels in the fruits of Evodia rutaecarpa, E. officinalis (10-35 nmol g-1 dry weight) and Zizyphus jujuba (30-50 nmol g-1 dry weight). The bioassays which had been used before had yielded quantitatively widely varying results. This has been mainly attributed to the presence, even in purified plant extracts, of materials interfering with the determination of cyclic nucleotides (Lin 1974) and (Amrhein 1974). Therefore, the problem required further study and I have used gas chromaAbbreviations: cAMP = cyclic adenosine-3' : 5'-monophosphate; cGMP = cyclic guanosine-3': 5'-monophosphate; GC-MS = gas chromatography - mass spectroscopy

tography-mass spectroscopy (GC-MS) in an attempt to quantify cGMP in a higher plant. Material and methods Maize seeds (Zea mays L., Golden Bantam 8 row and Bear Hybrid WF 9/38 Vaughan's Seed Co.) were kept under running water for 24 h and spread out on steam sterilized moist filter paper in plastic boxes. The seeds were sprayed with a 0,1% aqueous solution of penicillin G, Na-salt and kept at 20 ~ C in the dark. The seedlings were harvested after five days under green safelight. The seedlings (20.45 kg fresh weight) were subjected to a procedure reportet previously for the isolation of cyclic adenosine-Y: 5'-monophosphate (cAMP) (Janistyn 1981); in the same material, c-GMP had been found in a subfraction of one of the cAMP purification steps. The freeze-dried tissue was first extracted in petroleum ether (boiling point 4(~60 ~ C) which was then discarded. Thereafter the tissue was extracted in ethyl acetate which was also discarded. A third extraction using ethanol:water (80:20 v/v) removed the nucleotides and this extract was purified using charcoal followed by an anion-exchange column (MN2100.ECTEOLA-Cellulose; Machery, Nagel and Co., Dfiren;

Fig. 1. Mass spectrum of the isolated and silylated cGMP. Ordinate: relative abundance (%). abscissa: m/e. Conditions: the mass spectra were recorded on a Finnigan GC (9610)-MS Model 4000 instrument (Finnigan Instruments, Sunnyvale, Calif., USA) with sample introduction through the gas chromatograph inlet; 1% OV-17 on Gas Chrom Q, 100/120 mesh; 120mm long, 2 m m i.d. glass column, presilylated with SILYL-8 (Pierce); He, 60 ml min-1, 260 ~ C, column temperature 220 ~ C at 4 ~ rain 1 to 250~ (flame-ionization detector 200 ~ C); glass-jet separator 250 ~ C; ion-source temperature 250 ~ C; accelerating voltage 2 kV and I.E. 70 eV. The peak of the isolated and silylated cGMP was observed at the retention time of 9 min and derivated authentic cGMP co-chromatographeed at the same retention time. Under the given conditions, silylated cGMP and c-AMP (6 min 48 s) could be separated clearly Fig. 2. Mass spectrum of the silylated authentic cGMP. Ordinate: relative abundance (%). Abscissa: m/e. Conditions: see legend of Fig. 1

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B. Janistyn: Gas chromatographic-mass spectroscopic identification and quantification

1,5 cm diameter, 100 cm long) eluted with redistilled water. Further purification was by thin-layer chromatography (TLC) and then paper chromatography. The samples were then silylated prior to analysis by GC-MS. When the pooled and freeze-dried fractions from the anionexchange column were submitted to preparative thin-layer chromatography (TLC) on precoated silica-gel plates (F-254, E. Merck Darmstadt, FRG) with the solvent system n-butanol: methanol:ethyl acetate: 25% (w/w) aqueous ammonia, 7:3:4:4 (by vol.) the zone of cAMP was visible by UV light (254 nm). No zone was detected corresponding to the reference spot of authentic cGMP (Rf values: cAMP, 0.29; cGMP, 0.15). Because preliminary experiments showed only a little mobilization of authentic cGMP on ECTEOLA-Cellulose (MN 300) TLC plates with water as the solvent, the elution of the anionexchange-column was continued with 0.1 M aqueous ammonia. Fractions of 5 ml were collected. When the eluate became basic (pH 8), a significant absorbance at 252 nm was observed in the next 50 fractions (UV-spectralphotometer, DB 1200, Beckman, Munich, FRG). These 50 fractions were pooled and freeze-dried. A light brown residue of 110 mg containing some column material was obtained. This residue was submitted to preparative TLC. A weakly blue fluorescing zone was detected with UV-light (254 nm) corresponding to a reference spot of authentic cGMP with the R f 0.15. The Rf 0.15 zone was scraped off and eluted with ethanol:water, 8:2 (v/v). The eluate was, concentrated on a vacuum rotary evaporator, filtered and freeze-dried. 25 mg of substance was obtained in all. The silica gel containing sample was applied to ascending paper chromatography (No. 2043b Mgl., 580 by 600 mm; Schleicher and Schiill, Dassel, FRG) in isobutyric acid:water:25% (w/w) aqueous ammonia, 66: 33 : 1 (by vol.). The chromatograms were dried in a vacuum oven at 50~ and then scanned for UV absorption. The zones with an Rf of 0.28, corresponding to a reference spot of cGMP, were cut out and eluted with methanol. The eluates were evaporated under vacuum and dried over phosphorus pentoxide at 80 ~ C under vacuum for 12 h. A sample of 1.05 mg was obtained and stored at - 2 0 ~ C over blue silica gel. Since at every purification step (preliminary experiments showed the same behaviour of cAMP and cGMP for the extraction and the charcoal absorption) the cAMP/cGMP ratio was found to be 10:1 and the recovery of cAMP had been calculated to be 45% (Janistyn 1981), it was assumed that the recovery of cGMP was also about 45%. For the GC-MS determination, sample aliquots of 50-80 gg were silylated with 100 gl of a mixture of 50 gl absolute pyridine and 50 gl of N,0-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS) (Pierce Chemical Co., Rockford, III., USA, No. 38831) in a Teflon-capped reacti-vial (Pierce) at 70~ for 30 min. To remove some turbidity the silylated samples were centrifuged and the supernatants were used for the GC and GC-MS determinations. The content of cGMP in the isolated sample, determined as the tetrasilyl derivative using GC detection, was quantified with reference to a standard of silylated authentic cGMP.

Results and discussion

As shown in Figs. 1 and 2 the mass spectrum of the isolated and silylated sample agreed with the corresponding spectra of the silylated authentic

Table 1. Gas-chromatographic determination of minimum and

maximum amounts of c-GMP extracted from maize seadlings c-GMP content (pmol g- 1 FW or DW)

FW DW

Minimum

Maximum

35 436

72 989

cGMP. Furthermore, no difference was found between the GC peaks when authentic silylated cGMP was added to the isolated silylated sample (see legend of Fig. 1). The mass of the molecular ion and all significant fragment ions of the derivated cGMP were derived from the GC-MS investigations of parts of the whole cGMP molecule by Lawson et al. (1971). Also, the molecular ion of m/e 633 or the primer fragment ion of 618 (loss of one methyl group) was found for the tetrasilyl cGMP. Referring to a standard of silylated authentic cGMP, the content of the sample of isolated cGMP was measured. Because of the loss of silylated cGMP during the recording of GC, a minimum and a maximum amount of cGMP were estimated as shown in Table 1. The data represent the maximal fluctuations observed in all our GC determinations. The data show that the concentration of cGMP found in maize seedlings is of the same order of magnitude as found in animal tissue (10 .8 M) (Goldberg and Haddox 1977). With the unambiguous identification of cGMP in maize seedlings using GC-MS the occurrence of another biologically important molecule which is known to be regulatory in animals (Goldberg and Haddox 1977) has now been established in a higher plant. This work was supported by the Deutsche Forschungsgemeinschaft. The technical assistance of Mrs. M. Weber is gratefully acknowledged. I am also obliged to Dr. R. Haas for critically reading the manuscript.

References Amrhein, N. (1974) Evidence against the occurrence of adenosine-Y:5'-monophosphate in higher plants. Planta l l g , 241-258 Cyong, J., Takahashi, M., Hanabusa, K., Otsuka, Y. (1982) Guanosine-3':5'-monophosphate in fruits of Evocadia rutaecarpa and E. officinalis. Phytochemistry 21, 772778 Cyong, J., Takahashi, M. (1982) Identification of guanosine3' : 5'-monophosphate in the fruits of Zizyphus jujuba. Phytochemistry 21, 1871-1874

B. Janistyn: Gas chromatographic-mass spectroscopic identification and quantification Goldberg, N.D., Haddox, M.K. (1977) Cyclic GMP metabolism and involvement in biological regulation. Annu. Rev. Biochem. 46, 823-896 Haddox, M.K., Stephenson, J.H., Goldberg, N.D. (1974) Cyclic GMP in meristematic and elongating regions of bean root. Fed. Proc. Fed. Am Soc. Exp. Biol. 33, 1755 Janistyn, B. (1981) Gas chromatographic, mass- and infraredspectrometric identification of cyclic adenosine-3': 5'-monophosphate (c-AMP) in maize seedlings (Zea mays). Z. Naturforsch. Tell C 36, 193-196 Lawson, A.M., Stillwell, R.N., Tacker, M.M., Tsuboyama, K.,

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McClosky, J.A. (1971) Mass spectrometry of nucleic acid components. Trimethylsilyl derivatives of nucleotides. J. Am. Chem. Soc. 93, 1014-1023 Lin, P. P.-C. (1974) Cyclic nucleotides in higher plants? Adv. Cyclic Nucl. Res. 4, 439-461 Lundeen, C.V., Wood, H.N., Braun, A.C. (1973) Intracellular levels of cyclic nucleotides during cell enlargment and cell division in excised tobacco pith tissues. Differentiation 1, 255 260 Received 10 December 1982; accepted 31 March 1983

Gas chromatographic-mass spectroscopic identification and quantification of cyclic guanosine-3': 5'-monophosphate in maize seedlings (Zea mays).

Gas chromatographic-mass spectroscopic evidence is presented for the presence of guanosine-3': 5'-monophosphate (cGMP) in maize seedlings. The amount ...
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