Changes in the activities of three enzymes (nitrate reductase, L-phenylalanine ammonia-lyase, and a dehydronicotinamide adenine dinucleotide-oxidase complex) were measured during development of water stress in young maize (Zea mays) plants.L-Phenylalanine ammonia-lyase and nitrate reductase activities decreased markedly with water deficits of 10 to 20%. 'Abbreviation: PAL, L-phenylalanine ammonia-lyase. MATERIALS AND METHODSThe dominant tall form of the d1 dwarf maize (Zea mays) was grown in vermiculite in the greenhouse. Nutrient requirements were provided with a modified Hoagland's solution 1 (9). Moisture stress was initiated by withholding water for varying periods beginning approximately 10 days after germination. In early experiments, the plants remained in the greenhouse until sampled for assay, but in later experiments they were transferred to a growth chamber when water stress was initiated. The chamber environment was 27 C, 30 to 50% relative humidity, and had a light period of 12 hr/day of 1000 ft-c. The plants were sampled at the middle of the light period. In the rehydration experiments, the vermiculite base was saturated with water 24 hr prior to sampling.Water deficit was estimated by the method of Weatherly (21). Duplicate or triplicate 5-cm sections were taken from the midportion of the third leaf of two or three plants. The original weight was determined immediately after harvest, and the tissue was floated in deionized water for 4 hr. At that time, the saturated weight was taken and the tissue was dried at 95 C for 24 hr for dry weight determination. The water deficit was expressed as the ratio of water taken up divided by the final water content, multiplied by 100.The midportion of the leaf blades from four to six seedlings was used as the source of tissue for nitrate reductase and NADH oxidase. PAL was prepared from the leaf sheaths of the same or comparable seedlings. Nitrate reductase activity was determined by the method of Hageman and Flesher (6), except that the tissue was ground in a chilled mortar with pestle and sand. PAL was prepared and assayed as described by Reid and Marsh (15). NADH oxidase activity was determined in the same extract used for nitrate reductase. The reaction mixture for the oxidase assay contained 200 ymoles of tris buffer, pH 8.2; 20 ,tmoles of (NH4)2SO4; 0.66 umole of NADH; plus enzyme in a final volume of 3 ml. The reaction was run at 30 C and was initiated by the addition of NADH. Changes in absorbancy at 340 nm were followed spectrophotometrically for 5 min.One unit of activity for all three enzymes was defined as the amount of enzyme which catalyzed the formation of 1 ,tmole of product in 1 min under standard conditions. Protein was determined by standard biuret or Lowry methods (12) with bovine serum albumin as a standard. RESULTSThe results of water deficit measurements (Fig. 1)

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L-Phenylalanine ammonia-lyase (E.C. 4.3.1.5) from maize is active with L-tyrosine and L-phenylalanine and exhibits atypical Michaelis-Menten kinetics with both substrates. With phenylalanine as a substrate, the pH optimum is 8.7 and with tyrosine, 7.7. The estimated Kin at high substrate concentrations is 0.27 mM for phenylalanine and 0.029 mM for tyrosine. However, the Vmax with phenylalanine is eight times higher than the Vmax with tyrosine when both are measured at pH 8.7, and 7 times higher when both are measured at their pH optima. The following evidence leads us to the conclusion that there is a common catalytic site for both substrates: (a) It is impossible to appreciably alter the ratio of the two activities during purification and isoelectric focusing. (b) The ratio of the products formed in mixed substrate experiments is in good agreement with the ratio predicted from the estimated Km values. (c) NaBH4 reduces both activities to the same degree and L-phenylalanine, L-tyrosine, cinnamate, and p-coumarate protect both activities against NaBH4 reduction to the same degree. In contrast, the enzyme isolated from potato, which does not act on L-tyrosine, is not protected against reduction by either L-tyrosine or p-coumarate. However, both enzymes appear to have a dehydroalanine-containing prosthetic group.The enzyme L-phenylalanine ammonia-lyase (EC 4.1.3.5), which converts L-phenylalanine to trans-cinnamate and ammonia has been isolated and purified to varying degrees from a number of sources (6,10,13,16,19,20,23,26 however, the experimental results would favor the view that one enzyme is capable of catalyzing the two reactions (23). In surveys made with a wide variety of organisms there are no examples in which an extract has activity with tyrosine but not with phenylalanine, and indeed, no cases in which higher activity is observed with tyrosine than with phenylalanine (1, 2, 21, 28, 29). Attempts to purify a specific tyrosine ammonialyase have resulted in preparations which are three to four times more active with phenylalanine than tyrosine when assayed at saturating substrate concentrations (15,22).Resolving the question of enzyme specificity or lack of it when working with a nonhomogeneous system is a very difficult one. Factors such as relative Vmax and Km, as well as pH optima, influence the results of certain types of inhibitor and mixed substrate experiments and these factors must be taken into account when the results are interpreted. In this paper we characterize the L-tyrosine ammonia-lyase activity of maize and present the several lines of evidence which lead us to believe that both L-tyrosine and L-phenylalanine react at the same active site. The question of specificity must be answered before we can understand the in vivo function and control of this enzyme. MATERIALS AND METHODSEnzyme Sources. L-Phenylalanine ammonia-lyase was purified through the Agarose step from light exposed potato tuber slices (12). The enzyme from maize was purified by a revised procedure (24) which will be p...

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Abstract. The effects of gibberellic acid on lignification in seedlings of a dwarf and a tall cultivar of pea (Pisum sativum) grown under red or white light or in the darkness, were studied. Gibberelilic acid (10-6-10-4 M) promoted stem elongation in both light and dark and increased the percentage of lignin in the stems of the light-grown dwarf pea. The gibberellin had no effect oil the lignin content of the tall pea although high concentrations (10-4 M) promoted growth of the itaill plants. Time course studies indicated ithat the enhanced lignification in the gibberellin-treated dwarf plants occurred only after a lag period of several days.It was concluded that gibberellic acid-enhanced lignification had no direct relation to gibberellic acid-promoted growth. The activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5) was higher in gibberellin-treated dwerf plants grown under white or red light than in untreated dwarf plants. Gibberellic acid had no detectable effect on the activity of this enzyme when the plants were grown in darkness, just as it had no effect on lignification under dark conditions.The data suggest that in gibberellin-deficient peas the activitv of phen)lalanine ammonia-slyase is one of the limiting factors in lignification.

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