BIOLOGIA PLA_NTARD2r (PRAHA) 16 (2) : 86--93, 1974 The Depression of the Synthesis of Pea Diamine Oxidase due to Light and the Verification of its Participation in Growth Processes using Competitive inhibitors L. MACHOLi~ and J. Mr~s Departments of Biochemistry and Plant Biology, J. E. Purkynii University, Brno, Czechoslovakia* Received January 2, 1973 Abstraet. The time courses of the synthesis of diamine oxidase in pea plants grown for 14 days either in the light or in the dark are similar with the highest increase in activity occurring in the cotyledons and in the shoots during the first 6 to 8 days. Plants grown in the dark showed a 2- to 3-fold higher enzyme activity than plants grown in the light. Pea diamine oxidase could be in rive efficiently inhibited by substrate analogues 1,4-diamino-2-butanono and 1,5-diamino-3-pentanone. The first compound inhibited proportionally to its concentration the growth of etiolated pea plants, but its instability makes an unoqulvocal interpretation of the results difficult. On the other hand, 1,5-diamino-3-pontanono a stable and more efficient diamino oxldaso inhibitor depressed the growth of pea seedlings only at concentrations as high as 5 m)5 and 10 raM, at which the growth of cress seedlings not containing diamine oxidaso was also strongly depressed. The results obtained indicate that tryptamine oxidation catalyzed by diamine oxidase is not involved in the main metabolic pathway leading from tryptophan to indoleacetate in pea plants. Young pea plants contain high diamine oxidase activity [diamine: oxygen oxidoreduetase (deaminating), EC 1.4.3.6.) which is sometimes simply referred to as amine oxidase. The synthesis of the enzyme starts in the first hours of germination and in normal green plants reaches the highest activity after the first few days (K~TE~ and MA~ 1952, W~.~L~ et al. 1959, 1961). Because amine oxidases are able to oxidize tryptamine, some authors supposed that it could be involved in the formation of indoleacetate (KENTE~ and MA~ 1952, CL)_~KE and MA~ 1957, LAI~TICA~ and MUIR 1967). Ex- periments carried out so far mostly with the inhibitors of hydrazine type lead, however, to contradictory conclusions. REED et al. (1965) and recently KI~URA and TA~AK)~ (1971) found a relation between depressed growth of pea plants and the inhibition of tryptamine oxidation caused by the herbicide B-995 or by monoamine oxidase inhibitors safrazine and nialamide. On the Address: Kotls 2, 611 37 Brno, Czechoslovakia. 86 SYNTHESIS OF PEA D~NE OXIDASE 87 contrary, R. Moss from the Gordon's laboratory (see LARSE~ 1967, page 433) found that indoleacotate production was not changed in the pea stem tissues in which tryptamine oxidase activity was nearly completely inhibited. Aminoguanidine, isonicotinio acid hydrazide, and isopropylhydrazino in- activating diamine oxidase activity in vivo inhibited the germination not only in pea but even in the plant species not containing this enzyme (WEI~LE et al. 1959). In this paper we therefore try to verify if the selective inhibition of diamine oxidase can influence the germination and growth of pea seedlings. Nearly untoxical substrate analogues 1,4-diamino-2-butanone and 1,5-dia- mino-3-pontanone were used as efficient competitive inhibitors of diamine oxidase; they do not affect mitochondrial monoamine oxidase (MAc~oLA~ et al. 1967, MACHOLA~r 1974). Another aim of this paper was to follow the dynamics of the formation of diamine oxidase in pea plants grown either in the light or in the dark. Material and Methods 1,4-diamino-2-butanone dihydrochlerido was prepared after MICltALSK~ etal. (1953) and 1,5-diamino-3-pentanone was synthesized according to a method devised in our laboratory (MAc~OL%_lq 1974). Cultivation of the Plants The experiments were performed with pea seedlings (Pisum sativum L.) cv. Liliput, and, cross plants (Lepidium sativum L.) were used for bioassays. Selected seeds were surface sterilized for 5 rain. with 96% ethanol, rinsed with water, and then soaked for 24 h in distilled water or in inhibiter solution of pH approx. 5.0. The seeds were then transferred onto a layer of granulated polyethylene underlayed with diluted (1 : 4) Richtor's nutrient solution supplemented with microolements in Hoaglan4 A--Z solution. The germination and further cultivation occurred at 23 ~ either in the dark or in continuous light (white and rosy fluorescent tubes 1 : 1, 7 000 lx at the level of the plants) in a chamber with controlled conditions. The volume of the nutrient solution was kept constant by replenishing with distilled water. In the experiments with the inhibitors, groups of 250 seedlings, 3 to 4 days old, were transferred into 1 000 ml flat plastic cultivation vessels containing 500 ml of diluted (1 : 1) nutrient solution either with the inhibitor or without it. Enzyme Extraction and Assays 10 to 22 pea seedlings were carefully washed, and then cotyledons, roots, and shoots wore sepa- rated and weighted. Tostao wore removed from the cotyledons and each plant part was separately homogenized in a mortar with chilled 0.1 M phosphate buffer pH 7.0 and sdica sand (0.2 g of sand per g of plant material). The homogonato was sqeeze4 through a nylon cloth and the remaining pellet was repeatedly extracted with the buffer solution. The filtrate was made up to 50 or 100 ml with the buffer solution and immediately assayed. Protein was determined in the supornatant after centrifuging the homogenate at 10 000 g for 15 rain u~ing a modifigd biurot method with the subtraction of the turbidity after the addition of KCN (SzARI~OWSKX and KLI~'GENBERG 1963). Bovine sorumalbumin was used as standard protein. Diamino oxidaue activity was determined spectrophotomotrically ([-IoI, MSTEDT etal. 1961, M~-C~OLs 1966) at 25 ~ The assay mixture (final volume 5 ml) consisted of 80 mM phosphate buffer pl-I 7.0, 2.5 mM c-aminobenzaldehyde, 20 ~g catalase, 10 mM 1,4-diaminobutano, and 0.5 ml plant extract. One unit (lJ) represents the amount of enzyme catalyzing the formation of one fxmole Al-pyrrolino per rain. Specific activity is expressed in units per mg protein. Results Diamine Oxhlase Level in Pea Plants Grown Either in the Light or in the Dark Diamine oxidase activity was followed in extracts from different parts of plants grown in continuous light or in the dark during the first 14 days. The 88 h. ~[ACHOL~i.N, J. MINAI~ COTYLEDONS SHOOT DAB o4d eed O~ o9e D o<I.- o I ~ I I / DAB oee oed D O11d c DAP o Yd 10d o13d 01 , , ~ ,-- n 2 4 6 8 10 12 2 4 6 8 10 0 2 4 6 8 10 TIME [d] INHIBITOR CONCENTRATION [10 .3 M] Fig. 1. Diamine oxidase activity in pea plants grown in the light and in the dark. Enzyme activity in the cotyledons (~), in the shoots @, and in the roots[~, as expressed per g fresh weight -- A, per plant -- B, and per mg soluble protein -- C. Full symbols: plants grown in the dark, open symbols: plants grown in the light. <1, Diamine oxidase activity in above-ground parts of etiolated plants which were transferred at the time denoted by an arrow into the continuous light. Fig. 2. The inhibition of diamine oxidase in the cotyledons and in the above-ground parts of pea plants grown in the dark by 1,4-diamino-2-butanone (DAB) and 1,5-diamino-3-pentanone (DAP). The inhibitor was applied in the concentrations given in the abscissa either only when soaking the seeds (A), or both to seeds and roots in the diluted (1 : 1) l~ichter's nutrient solution (B, C)o found values were expressed per g fresh weight (Fig. 1A), per plant (Fig. IB), or per mg total soluble protein (Fig. 1C). The synthesis of the enzyme starts soon after the swelling of the seeds and its activity rises sharply in the follow- ing 6 to 8 days, mainly in the cotyledons. This increase in activity is most conspicuous when the activity is expressed per plant. We can see that after an initial sharp increase, the activity changes only slowly in both normal and etiolated plants. When the activity was expressed per fresh weight unit a decrease in diamine oxidase activity was established in the above-ground SYNTHESIS OF PEA DIAMINE OXIDASE 89 parts and in the roots which was obviously connected with the growth of these organs. In the cotyledons which gradually loose their weight, the level of diamine oxidase continuously slowly rises. Specific activity of diamine oxidase progressively rises in the cotyledons but decreases after an initial increase in the above-ground parts during the development of the seedlings. The plants grown in the dark always showed a higher diamine oxidase activity then those grown in the light. At the end of the 14th day of culti- vation approx, a twofold activity was found in the cotyledons and approx. a threefold activity was found in the above-ground parts when expressed per plant (Fig. 1B). When 10 days old etiolated plants were transferred into the light diamine oxidase activity remained at the same level as in control plants kept continuously in the dark (Fig. 1A). This indicates that only the initial phase of the synthesis of diamine oxidase is controlled by the light. The Influence of the Inhibitors The competitive inhibitors of diamine oxidase, 1,4-diamino-2-butanone and 1,5-diamino-3-pentanone, were tested on pea plants grown in water cultures in the dark. 1,4-diamino-2-butanone was applied at the beginning of the experiments to the soaking seeds in 0.625 to 10mM water solutions.