ABSTRACIPolyamines were identified by high performance liquid chromatography (benzoylation) and by thin layer chromatography (dansylation)

ABSTRAC1The incorporation of [l4qsginine and I'4qornithine into various polyamines was studied in mung bean ( Vigna radiata IL.] Wilczek) hypocotyl cuttings with respect to the effect of indole-3-butyric acid on adventitious root formation.Both '4Qujginine and I'4Cornithine are rapidly incorporated into putrescine, spermidine, and spermine, with similar kinetics, during 5-to 24-hour incubation periods. The incorporation of arginine into putrescine is generally higher than that of ornithine. The biosynthesis of putrescine and spermidine from the precursors, in the hypocotyls, is closely related to the pattern of root formation: a first peak at 0 to 24 hours corresponding to the period of root primordia development, and a second peak of putrescine biosynthesis at 48 to 72 hours corresponding to root growth and elongation. Indole-3-butyric acid considerably enhances putrescine biosynthesis in both phases, resulting in an increase of the putrescine/ spermidine ratio.It is concluded that the promotive effect of indole-3-butyric acid on putrescine biosynthesis, from both arginine and ornithine, supports the hypothesis that auxin-induced root formation may require the promotion of polyamine biosynthesis.The biosynthesis and function of naturally occurring PA3 have been studied in detail in mammalian tissues and in procaryotes (4, 7, 19), whereas relatively little is known about PA metabolism in plants. Where the addition of propylamino residues derived from S-adenosyl methionine (1, 4).In recent years, it has been demonstrated that PA. play a significant role in plant responses to hormonal and environmental stimuli, both with respect to growth and active cell division, and to stress and senescence (2,3,6,12). Thus, induction of cell division and growth was studied recently in several plant systems and its close relationship with PA metabolism and biosynthetic enzymes has been demonstrated (14,15,17).In a previous study (10) we suggested that PA are involved in IBA-induced root formation in mung bean cuttings. This conclusion was based on the effect ofexogenous compounds, including PA precursors and some analogs and metabolic inhibitors, and on changes in the titer of endogenous PA. This was recently corroborated by similar data (13) showing that IBA leads to enhanced levels of PA in the hypocotyl where roots are formed (although we could not show that exogenous Spm enhanced root formation). The present investigation was undertaken in order to elucidate the role of PA in auxin-induced root formation by studying the incorporation of L-arginine and L-ornithine into various PA. In the following, we show that these two precursors are rapidly incorporated into Put, Spd, and Spm, with similar kinetics, and that the induction of adventitious roots by IBA is preceded and accompanied by an increase in the incorporation. MATERIALS AND METHODS

The effect of several polyamines (putrescine, spermidine, and spermine), their precursors (L-arginine and L-omithine), and some analogs and metabolic inhibitors (L-canavanine, L-canaline, and methylglyoxal-bis IguanyIhydrazonel) on root formation have been studied in mung bean (Vigna radiata IL.1 Wilczek) hypocotyl cuttings.Exogenously applied polyamines did not promote adventitious root formation. Rooting was inhibited by L-canavanine and L-canaline, and this inhibition was reversed by the corresponding amino acids L-arginine and Lornithine. Methylglyoxal-bis (guanylhydrazone), an inhibitor of S-adenosylmethioine decarboxylase and polyamine biosynthesis, was also found to inhibit root forMatn All cMpoUnds at concentratios of >10-4 molarity completely inhibited natural root formation, whereas at <10-5 mohrity only the indole-butyric acid-induced root formation was inhibited.Indole-butyric acid-induced root formation was accompanied by a considerable increase in polyamine levels, more than 2-fold of the control.Whereas senescing (unrooted) cuttings evinced a rapid decfine in polyamine content during 48 hours, indole-butyric acid treatment resulted in elevated levels of putrescine and increased putrescine to spermidine ratio. The changes in polyamines were dependent on indole-butyric acid concentration and were organ specific. a significant role in plant growth and senescence (4), similar to their activity in mammalian tissues and procaryotes (7,10,31). It has been shown that active growth of germinating seeds, of habituated and crown gall tissues, of pollen tubes, of potato buds and tomato fruits (6,14,18,29,33), as well as embryo and organ differentiation (4,21,22), are correlated with significant changes in PA content and metabolism. In addition, PA have been shown to respond to environmental and hormonal stimuli (12, 18), and to play an important role in protoplast, tissue, and organ senescence (2,3,17).It, therefore, seems that cuttings may serve as an important experimental system to elucidate the involvement of PA in their senescence or in auxin-induced root formation. In addition to the effect of exogenous PA, endogenous changes in PA, and possible inhibitors of PA biosynthesis were also investigated. Both L-canavanine and L-canaline, the structural analogs of L-arginine and L-ornithine, respectively (25, 26), may affect putrescine formation from these two amino acids. Similarly, MGBG can block spermidine and spermine biosynthesis by inhibiting S-adenosylmethionine decarboxylase (24). L-Canavanine has been shown previously to inhibit IAA-dependent elongation of Avena coleoptiles (9), and growth of Phaseolus roots (34) and soybean cell suspensions (11). The present paper is the first in a series of studies on polyamines and root formation.Cuttings excised from plants either senesce

General properties and relative activities of L-arginine decarboxylase (ADC) (EC 4.1.1.19) and L-ornithine decarboxylase (ODC) (EC 4.1.1.17), two important enzymes in putrescine and polyamine biosynthesis, were investigated in mung bean (Vigna radiata L.) tissues. Both activities increase linearly with increasing concentrations of crude enzyme, but the increase in ADC activity is considerably greater. The decarboxylation reaction is linear for up to 30 to 60 minutes, and both enzymes have a pH optimum of 7.2. a-Difluoromethyl-ornithine inhibits ODC activity of excised roots, while increasing ADC activity.High specific activity of both enzymes is detected in terminal buds and leaves, while root and hypocotyl activity is low. Different ADC-to-ODC activity ratios are found in various tissues of mung bean plants. Substantial increase in the activity of both enzymes is detected in incubated sections as compared with intact plants. A The importance of polyamines in various growth and physiological processes in plants has been inferred from both application of exogenous polyamines and from changes in endogenous polyamines and related metabolites (1,2,4,9,13 2Abbreviations: ADC, L-arginie decarboxylase; ODC, L-ormithine de-carboxylase.ADC and ODC are active and involved in polyamine biosynthesis (16,19,21). In recent studies (10), ODC activity of developing tomato fruits was found to increase dramatically up to the 3rd day after pollination, while ADC activity was very low, and both arginine and ornithine were equally well decarboxylated in tobacco cell cultures (5).In view of the contradictory results regarding the activity and importance of ADC and ODC in plants, a comparative study of these two polyamine-biosynthetic enzymes was undertaken. We report the kinetics and several properties of ADC and ODC as well as their relative activity in tissues of mung bean seedlings.MATERIALS AND METHODS Plant Material. Mung bean (Vigna radiata L.) seeds were germinated and grown in vermiculite in a controlled phytotron (27°C-day and 22°C-night temperature, 16-h photoperiod, 78% RH). One cm subapical sections of epicotyls were harvested from 7-to 8-day-old seedlings and were extracted for determination of enzyme activities. Occasionally, other plant parts were extracted, as mentioned. Epicotyl sections of French bean (Phaseolus vulgaris L. var. Brittle Wax) and cotton (Gossypium hirsutum L. SJ-2), grown for 8 days under the same conditions, were also used in several experiments.Extraction and Assay of ADC and ODC. Sections were harvested, weighed, and ground in a prechilled mortar with a pestle (250-600 mg fresh weight/2 ml medium). Extraction medium consisted of 10 mm phosphate buffer (pH 7.2), 0.1 mm DTT, 1 mm pyridoxal-5'-phosphate, and 20 mm Na-EDTA. The extracts were centrifuged at 12,000g for 20 min, and the supernatant, hereafter referred to as crude enzyme, was used immediately.

No abstract