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@ 1996 Kluwer Academic Publishers. Printed in the Netherlands. Short communication

Measurement of formaldehyde and some fully N-methylated substancesin tissue cultures of Datura innoxia *
E. Tyih& & 8. Sz6ke2
Plant Protection Institute, Hungarian Academy of Sciences, H-1525 Budapest, PO Box 102, Hungary; 2Semmelweis University of Medicine, Institute of Pharmacognosy, H-1085 Budapest, fill%, lit 26, Hungary
Received 26 July 1996, accepted 29 July 1996

Key words: aging, cell proliferation, choline, Datura innoxia Mill., formaldehyde, methylation-demethylation, Ntrimethyl-L-lysine, OPLC, tissue culture, TLC, trigonelline

Abstract Formaldehyde (HCHO) and choline, trigonelline and NE- trimethyl-L-lysine (TML) were detected and measured in tissue cultures of Datura innoxia Mill. by TLC and OPLC. The higher level of HCHO in young tissues appears to originate from the methylation reactions. This is in correlation with the high level of fully N-methylated substances. In spite of this the level of the fully N-methylated substances decreased with the aging of tissue. HCHO level also decreased with aging. However, in old tissues the amount of HCHO again increased. This HCHO originates mainly from demethylation reactions. It seems that there is a dynamic relationship between methylation/ demethylation processes based on HCHO reactions and cell proliferation. Abbreviations: HCHO - formaldehyde, HPTLC -high-performance thin-layer chromatography, HS -heat shock, OPLC - overpressured layer chromatography, SAM - S-adenosyl-L-methionine, TML-N-trimethyl-L-lysine, TLC- thin-layer chromatography 1. Introduction Betaines are derivatives of amino or imino acids containing a fully methylated pentavalent nitrogen moiety [13]. Many of these quaternary ammonium compounds have been recorded for plants. These special compounds have different biological activities and functions [l], e. g. TML exercises an important cell proliferation promoting effect as well as a protective effect against abiotic stresses [9]. Trigonelline as a phytohormone can retard plant cells in the G:! phase [lo]. Choline is found throughout the plant and animal kingdom, often as a constituent of phospholipids such as lecithin [ 11. According to recent investigations all biological systems have HCHO-yielding potential which can
* This work was supported by National Science Foundation (OTKA T-14141, -17394, -17351 and ETT-119, AMFK-465). grant

originate from demethylation and methylation processes, alike. During the demethylation process HCHO and demethylated compound can be formed as has been previously demonstrated [4]. As a result of a disease stress (plant viral infection), the amount of HCHO considerably increased in virus infected tobacco leaves and it was suggested that this was the result of enhanced enzymatic demetbylation processes. In the presence of L-metbionine and S-adenosyl-L-methionine (SAM) as substrates the demethylase activity was significantly high [2]. Recent experiments indicate that the measurable HCHO level is dramatically elevated in parts of water melon (Citrullus vulgaris L.) plants immediately after a non-lethal infection with Fusarium oxysporum. At the same time the level of some quaternary ammonium compounds as potential HCHO generators are considerably decreased [8]. A strong correlation exists between the external temperature and the amount of measurable HCHO in

318 Pinto bean leaf tissues [ 121. The highest amount was detected after a heat shock (HS) treatment to leaves. As a result of HS, the level of 3 potential HCHO generators (trigonelline, choline and N-trimethyl-L-lysine) decreased moderately. The higher activity of demethylases including HS, at elevated temperatures, is cause of an increase in HCHO [5]. Recently, it has been shown that the formation of HCHO from the S-methyl-group of SAM is linked to an enzymic transmethylation [6]. It is increasingly evident that there is a HCHO cycle in biological systems [l 11, that is, the enzymic methylation reactions take place through HCHO and at the same time all methyl groups are potential HCHO precursors. There exist a number of rapid HCHO pathways through the methylol groups of different binding force. The rate and intensity of these pathways - the HCHO-donating activity - influence the stress tolerance of biological systems. It seems that HCHO plays a role in dynamic methylation-demethylation processes [6, 131. This paper describes the level of HCHO and some fully N-methylated substances in tissue cultures of different age from Daruru innoxia Mill. 2.3 Synthesis offormaldemethone Formaldemethone was prepared for identification purposes by adding formaldehyde solution (20 mL of 48% solution) to a solution of dimedone (5 g) in hot ethanol (30 mL) and water (5 mL). The white precipitate which separated was filtered and recrystallized from aqueous ethanol; m.p. 191. 2.4 Consecutive separation of HCHO in dimedone adductform and its main potential generators Solutions of samples were applied with a Hamilton syringe (Bonaduz, Switzerland). TLC-HPTLC separations were performed in unsaturated chambers in the ascending mode. OPLC separations were carried out with a CHROMPRES 10 OPLC instrument (Factory of Laboratory Instruments Co. Ltd, Budapest, Hungary). The densitograms were recorded with Shimadzu Cs 930 scanner (Shimadzu Co., Kyoto, Japan), at A = 265 nm and 525 nm after spraying with modified Dragendorffs reagent according to Vagujfalvi [ 141. Calibration curves were used. Separation of formaldemethone by TLUHPTLC or OPLC was carried out using a chloroform - methylene chloride eluent mixture (35: 65, V/V) and TLC or HPTLC silica gel 60 F254 chromatoplate (1 st separation step). The plate was dried in a stream of cold air for 2 min. Quantitation was conducted in the reflectance mode at X = 265 nm. The chromatoplate was then used for the separation and determination of residual dimedone using acetone as eluent (this is also a cleanup step) (2nd separation step). The chromatoplate was then suitable for the separation and measurement of the quatemary ammonium compounds using eluent mixture containing i-propanol-methanol-O. 1 M Na acetate (20: 3 : 30, V/V) (3rd separation step).

2. Material and methods 2.1 Test callus tissue The test material used in our investigations was a secondary callus tissue isolated from root of Daturu innoxia Mill. The callus tissues were grown in light (2500 Lux, 10 hours day-) at 26 C on a basic nutrient medium of Murashige-Skoog [7], containing 1 mg L-l of kinetin and 1 mg L- of 2, 4-D [3]. We measured the fresh weight of the tissues and calculated the daily growth rate (end weight-initial weight/ number of days) and the relative growth value (end weight initial weight/ initial weight) [33]. 2.2 Chemical preparation of callus tissues The fresh callus tissues were frozen with liquid nitrogen, powdered and treated with dimedone solution (e.g. 0.25 g tissue powder 0.7 cme3 of 0.01% dimedone solution). This suspension was centrifuged at 1000 g for 10 minutes 4C. The clear supematants were used for TLC/ HPTLC or OPLC separations [ 121.

3. Results and discussion Table 1 shows the age of culture, growth value, daily growth value and the level of HCHO calculated per g frozen-tissue. The level of HCHO decreased with aging. However, in old tissues (6 week) the amount of HCHO was again increased. The higher level of HCHO in young tissues (l-2 week) originated mainly from methylation processes [ 131 while the increase of HCHO in old tissues is an aftermath of an increased demethylation process. The daily growth of tissues decreases dramatically with the aging. It

Table 1. Quantitative changes in the level of HCHO tures of Datura innmia Mill. associated with aging Age of culture (week) 0 1 2 3 4 5 6 Growth Growth value Daily growth, (mg day-) HCHO (,og g-i 1.20 1.83 1.58 1.05 1.06 f f f f f

in callus

cul-

frozen 0.13 0.15 0.19 0.10 0.14

tissue)

8.6 23.9 27.8 29.9 30.6 28.9 and HCHO values

182 518 612 32 28 0 are means

Demethylatioa

Methylation

1.03 f 0.12 1.14f 0.13 of 3 replicates.

Figure 1. The origin of HCHO reactions in summarized form.

from

methylation

and demethylation

Table 2. Changes in the level of fully N-methylated substances in callus cultures of Datum innoxia Mill. associated with a&z Age of culture (week) 1 2 3 4 5 6 Growth value TML Choline (fig g-t frozen Trigonelline tissue)

8.6 23.9 27.8 29.9 30.6 28.9

8.6 zt 1.6 7.5 LIZ 1.8 5.0 f 1.2 5.4f 1.0 5.1 f 3.2 f 1.1 1.4

124.8 f 102.7 I!Z 94.8 f 80.6Zt 75.7 k 45.6 zt

2.6 3.4 2.1 1.6 1.7 1.6

66.5 59.4 40.3 39.7* 36.5 25.5

f 1.5 f 2.6 f 1.5 1.4 + 1.7 f 1.9

cules interact immediately during the demethylation processes [ 131. It is suggested that this extremely reactive molecule can participate in the basic defense mechanisms of plant tissues. Indeed, excited HCHO, as a special reaction product, may be an important part of the resistance potential (e.g. natural disease resistance) and may participate in programmed cell death, that is in actual apoptosis.

References
1. Blunden G and Gordon SM ( 1986) Betaines and their sulphonio analogues in marine algae. In: Round-Chapman (ed) Progress in Phycological Research Vol. 4. Bioptess Ltd Burgyan J, Szarvas T and Tyihak E ( 1982) Increased formaldehyde production from L-methionine (S-14CHs) by crude enzyme of TMV-infected tobacco leaves. Acta Phytopathol Acad Sci Hung 17: 1 l-15 Dung NN, Szoke E and Verz&r-Petri G (1981) The growth dynamics of callus tissues of root and leaf origin in Darura innoxia Mill. Acta Bot Acad Sci Hung 27: 325-333 Fannin FF and Bush LP (1992) Nicotine demethylation in Nicoriam. Med Sci Res 20: 867-868 Gullner G and Tyihak E (1987) Hydrogen peroxide dependent N-demethylase activity in the leaves of normal and heatshocked bean plants. Plant Sci 52: 21-27 Huszti S and Tyih&k E (1986) Formation of formaldehyde from S-adenosyl-L-(methyl-3 H) methionine during enzymic transmethylation of histamine. FEBS Letters 209: 362-366 Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plain 15: 473-497 Stirdi 6 and Tyihak E (1992) Effect of Fusarium infection on the formaldehyde cycle in parts of water melon (Cirrullus vulgaris L.) plants. In: Tyihak E (ed) Proc. 3rd Intern Conf Role of Formaldehyde in Biological Systems. Methylation and Demethylation Processes, 18-22 May 1992, Sopron Hungary, pp 145-l 50. Budapest: Hung Biochem Sot Szende B, Lapis K and Simon K (1982) Combined effect of cytostatic drugs and E-N-L-trimethyllysine in healthy and transplantable tumor bearing mice. Neoplasma 29: 427-439 Tramontano WA, Harmett CM, Lynn DG and Evans LS (1982) Relationship between trigonelline concentration and promo-

Growth values and amount are means of 3 replicates.

of fully

N-methylatcd

substances 2.

can be seen that the level of trigonelline, choline and TML decreased in parallel with the aging of callus culture (Table 2). Thus, it follows that callus culture can be used for studying the relationship between the biotransformation steps of the HCHO cycle and cell proliferation. In methylation reactions (mainly in young tissues) we can measure HCHO which has a normal energy level while in old tissues there is a possibility for the formation of excited HCHO (*HCHO). This latter excited HCHO is a very reactive radical compound which has relatively long lifetime and is rich in energy r131. Figure 1 illustrates the origin of HCHO from methylation and demethylation reactions in summarized form. There seems to be a real possibility of the formation of excited HCHO in the reaction between HCHO and Hz02, which are normal components of plant tissues. Because Hz02 participates directly in the oxidative demethylation reactions so these two small mole-

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