An extract prepared from the apical meristematic region of etiolated pea seedligs was able to catalyze the incorporation of putrescine into trchloroacetic acid precipitable material. The enzyme was found to be soluble and followed a typical Michaelis-Menten kinetics when N-Ndimethyl casein was used as a substrate. Its activity was promoted by Ca2 We present here, for the first time, evidence indicating that an enzyme with characteristics similar to those found in most transglutaminases described so far is found in plant meristematic tissues. MATERIALS AND METHODSSeeds of Pisum sativum (var 'Kelvedon Wonder') were soaked for 6 h in tap water, planted in pots containing moist vermiculite, and grown in the darkness at 23°C and 80% RH. The apical meristematic hook regions from 7-d-old etiolated pea seedlings were excised and homogenized in a chilled mortar and pestle with 4 to 5 volumes of Tris 0.1 M (pH 8.5). The homogenate was centrifuged at 2000 rpm at 4°C for 10 min and the supernatant was assayed for transglutaminase activity. Transglutaminase activity was assayed by following the rate of incorporation of 3[HI putrescine into TCA precipitable protein. The assay was conducted following the modified filter paper method described by Lorand et al. (1 1). The assay contained, in a total volume of 150 ,ul, 45 mM putrescine containing 2 uCi of [3H]putrescine (18.6 iACi/mmole), up to 600 ,ug of plant protein in 75 ,l homogenization buffer, up to 0.95 mg N-N-dimethylated casein, 1 ,mole CaCl2, and 62.5 ,mol Caps' (pH 8.5). Extracts were incubated at 35°C for 30 min, then 60-ul aliquots were spotted on Whatman No. 1 paper presoaked in 10% TCA and immediately immersed in ice-cold TCA (10%). The filters were washed twice in 5% TCA and once in an ethanol/acetone 1:1 mixture, and once in acetone. The filters were then dried, placed in scintillation vials containing 3 ml Aqualuma (Lumac B.V., Holland), and counted in a Kontron scintillation counter. The transglutaminase activity was expressed as nmol of3[H]putrescine incorporated per mg ofplant protein per h. The activity was linear up to 45 min at 35°C in the range of protein content between 5 and 600 usg per assay. Blank values (up to 200 dpm) of acid-treated or boiled enzymes preparation were always subtracted. The data presented are from single experiments, which are representative of a group of three experiments, each involving triplicate sets of experimental treatments and controls.

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Intraperitoneal injection of 30 pg of luteinizing hormone (LH) into 20-to 22-day-old rats resulted in an increase in the specific activity of ornithine decarboxylase in the 38,OOOg,,,, supernatant fraction of ovarian extracts. A significant increase was apparent 1 hr after injection; the peak value (a 10-16-fold increase) occurred at 4 hr, and thereafter activity declined but at 8 hr was still significantly above the basal level. Adult rats also responded to exogenous L H by an increase in specific activity of ornithine decarboxylase. This rise was seen at every stage of the estrous cycle, even between 1400 and 1800 hr on the day of proestrus when there is a physiological rise in ornithine decarboxylase activity following the endogenous 0 rnithine decarboxylase (EC 4.1.1.17) catalyzes the conversion of ornithine to putrescine, which in turn is used in the biosynthesis of the polyamines spermidine and spermine. Present knowledge of polyamine biosynthesis and suggestions for a role of polyamines in the stabilization of nucleic acids and the biosynthesis of nucleic acids and proteins have been reviewed in a recent monograph (Cohen, 1971) and in the proceedings of a recent meeting (Herbst and Bachrach, 1970).Several hormones capable of stimulating growth and cell division cause a rise in the activity of ornithine decarboxylase at a n early stage in their action. Thus, growth hormone stimulates putrescine and spermidine synthesis in rat liver (Janne d . , 1968); epidermal growth factor produces an increase in ornithine decarboxylase activity when added to epiderriial cells grown in cell culture ; testosterone injection into castrated rats increases the specific activity of ornithine decarboxylase in the ventral prostate (Pegg d , , 1970); and injection of 17P-estradiol into ovariectomized (Cohen rr d . , 1970) or immature (Kaye rt d . , 1971) rats stimulates ornithine decarboxylase activity in the uterus.Kobayashi et ul. (1971) reported that there was an increase in ornithine decarboxylase activity in the ovary associated with the release of luteinizing hormone (LH)' from the pituitary during the estrous cycle. These authors as well as JBnne and Williams-Ashman (1971) and Williams-Ashman rt c d . (1972) also found an increase in ornithine decarboxylase activity in the ovaries of LH or gonadotropin treated adult rats. In the present paper, we describe some characteristics of

Activity of arginine decarboxylase in etiolated pea seedlings appears 24 hours after seed imbibition, reaches its highest level on the 4th day, and levels off until the 7th day. This activity was found in the apical and subapical tissue of the roots and shoots where intensive DNA synthesis occurs. Exposure of the seedlings to ethylene greatly reduced the specific activity of this enzyme. The inhibition was observed within 30 min of the hormone application, and maximal effect-90% inhibition-after 18 hours. Ethylene at physiological concentrations affected the enzyme activity; 50% inhibitory rate was recorded at 0.12 microliters per liter ethylene and maximal response at 1.2 microliters per liter. Ethylene provoked a 5-fold increase in the K,," of arginine decarboxylase for its substrate and reduced the V.w by 10-fold. However, the enzyme recovered from the inhibition and regained control activity 7 hours after transferral of the seedlings to ethylene-free atmosphere. Reducing the endogenous level of ethylene in the tissue by hypobaric pressure, or by exposure to light, as well as interfering with ethylene action by treatment with silver thiosulfate or 2,5-norbornadiene, caused a gradual increase in the specific activity of arginine decarboxylase in the apical tissue of the etiolated seedlings. On the basis of these findings, the possible control of arginine decarboxylase activity by endogenous ethylene, and its implication for the hormone effect on plant growth, are discussed.arises from the study of Bagni (5), who first reported that growth ofdormant Helianthus tuberosus was initiated when either auxin or one of the polyamines was added to the medium and that growth was accompanied by an increase in polyamine titer. There are now recorded instances of increased polyamine biosynthesis and titer in plants following application of each of the growthpromoting plant hormones: auxin (6), GA3 (13), and cytokinin (28). On the other hand, retardation ofgrowth by ABA has been shown to be accompanied by decreased polyamine biosynthesis (28). Consequently, the role of 'second messenger' mediating the effect of plant hormones has been proposed for polyamines (14).Ethylene has been shown to inhibit cell division, DNA synthesis, and growth in meristems of roots, shoots, and axillary buds of etiolated pea seedlings (1). In the shoot apex, cell division was almost completely annulled by ethylene and only 60% inhibition was observed in the root apex (1). The gas inhibits cell division by blocking a stage before prophase (1). In the subapical regions of the seedling, the hormone inhibited cell expansion and DNA synthesis (2). In the various parts of the seedling, a qualitative relationship was found between the inhibition of DNA synthesis, cell division, and growth caused by ethylene (1).In view of the above, a study was undertaken to pursue a possible involvement of polyamines in the effects of ethylene on plant growth. In this communication, we show that ethylene provokes a pronounced and reversible inhibition of ADC2 ac...

Exposing etiolated pea seedlings to ethylene which inhibited the activity of arginine decarboxylase and S-adenosylmethionine decarboxylase caused an increase in the level of cadaverine. The elevated level of cadaverine resulted from an increase in lysine decarboxylase activity in the tissue exposed to ethylene. The hormone did not affect the apparent K,, of the enzyme, but the apparent V,,,, was increased by 96%. While lysine decarboxylase activity in the ethylene-treated plants increased in both the meristematic and the elongation zone tissue, cadaverine accumulation was observed in the latter only. The enhancement by ethylene of the enzyme activity was reversed completely 24 hours after transferring the plants to an ethylene-free atmosphere. It is postulated that the increase in lysine decarboxylase activity, and the consequent accumulation of cadaverine in ethylene-treated plants, is of a compensatory nature as a response to the inhibition of arginine and S-adenosylmethionine decarboxylase activity provoked by ethylene.Increasing evidence accumulated in recent years suggests that the naturally occurring polyamines, putrescine, spermidine, and spermine, act as modulators of some cellular and physiological processes during plant growth and development (11). Consequently, changes in the level of these polyamines and in the activity of their biosynthetic enzymes have been studied in a variety of plant species at different stages of development (1 1, 17). Palavan et al. (15) showed that ethephon inhibited the activity of ADC and ODC in pea terminal buds.We have recently shown (4-6) that ethylene which is known to influence many aspects of plant growth and development (1) inhibited the activity of ADC2 (7) and SAMDC (13), thereby reducing the rates of formation of the subsequent polyamines.The requirement for putrescine, spermidine, and spermine usually found in eukaryotes can apparently be fulfilled, at least for a short period, by closely related amines like cadaverine or aminopropyl cadaverine (2,3). In a recent study we have shown ' Contribution No. 1647-E, 1986 series, from the Agricultural Research Organization, The Volcani Center, P. 0. Box 6, Bet Dagan 50250, Israel.2Abbreviations: ADC, arginine decarboxylase; SAMDC, S-adenosylmethionine decarboxylase; LDC, lysine decarboxylase; PLP, pyridoxal phosphate.(8) that cadavarine was formed in Pisum sativum seedlings via a specific LDC activity. In addition, we found a higher level of cadaverine in the nonmeristematic subapical tissue of pea seedlings (8) where cell elongation, differentiation, and active DNA synthesis take place (4, 5).Because polyamines are required for various plant cellular processes (11), a study was undertaken to follow cadaverine formation in pea seedlings where ADC and SAMDC activity were inhibited by ethylene treatment.In this communication, we describe a stimulatory effect of ethylene on LDC activity in pea seedlings and accumulation of cadaverine in the elongation zone of the plant. MATERIALS AND METHODSPea seeds (Pisum sativum ...