Circadian Rhythmicity in the Activities of Phenylalanine

The oscillations in phenylalanine aunnnia-lyase activity from Spirodels pelyrhiza and phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities from Lemnas perpusilam disayed a circadian rhythm under continuous lght. Rhythmiclty in enzymic activity could not be detected in continuous darkness since under this condition phenylalanine ammonialyase activity remains at a fairly constantly low level. Results from our studies of the oscillatory pattern of the respective activities of phenylalanine and tyrosine ammon_a-lyase support their "inseparability." The study of biochemical oscillations, especially of enzyme systems, has become a very important area of research (11). Among the more extensively investigated examples of oscillatory or rhythmic behavior at the metabolic level are the enzymic reactions of glycolysis in yeasts (24, 26). There are examples of soluble enzyme systems in plants which exhibit rhythmicity (17, 19, 25), and a recent review of biological rhythms and physiological timing in plants (16) suggests that rhythms are common in plant metabolism. In the Lemnaceae, rhythmic behavior of respiratory gas exchange has received considerable attention. Uptake of 02 by Lemna gibba entrained to a short day schedule displays circadian periodicity when transferred to continuous light (20). Also, a CO2 output rhythm has been identified with Lemna perpusilla (14, 15) that is associated with floral induction. The enzyme L-phenylalanine ammonia-lyase (EC 4.3.1.5) catalyzes the deamination reactions in which L-phenylalanine and L-tyrosine are degraded to trans-cinnamic acid and trans-p-coumaric acid plus ammonia, respectively. These reactions represent an important metabolic sequence in plants which results in the diversion of the two amino acids from the pathways of protein synthesis into secondary metabolism with the subsequent production of metabolites such as lignins, flavonoids, tannins, and alkaloids (27). Thus, PAL4 has a pivotal role in the reactions of secondary metabolism and in this manner, is thought to exert its influence on plant growth and development (3). Since oscillations and rhythms have been associated with processes of growth and development (5) and because the activity ofPAL has been reported 'This investigation was supported in part by a fellowship to W. R. G. from the National Fellowships Fund, Atlanta, Georgia 30308. 'This paper is based on a dissertation submitted to fulfill in part the requirements for the degree of Doctor of Philosophy at the University of Minnesota. ' Present address: Department of Biology, Brookhaven National Laboratory, Upton, New York 11973. 4Abbreviations: PAL: phenylalanine ammonia-lyase; TAL: tyrosine ammonia-lyase; DD: continuous darkness; LL: continuous illumination; LD: light-dark. to display fluctuations in response to various environmental conditions (3), an investigation into the nature of fluctuations in PAL activity from members of the Lemnaceae during growth and development seemed appropriate. In this paper, circadian rhythmicity in the activity of PAL is described in L. perpusilla 6746 and Spirodela polyrhiza L. (Schleiden). MATERIALS AND METHODS Cultures of L. perpusilla 6746 and S. polyrhiza L. (Schleiden) were maintained axenically in 125-ml Erlenmeyer flasks containing 50 ml of 0.5-strength Hutner's medium supplemented with 1% sucrose (13). Plants were entrained on a LD 16:8 cycle (16 hr of light followed by 8 hr of darkness) at 22 to 23 C before exposure to LL or DD. The light source used during entrainment for LL was three 15-w cool-white Sylvania fluorescent lamps which provided 235 ft-c (7.4 x 1O' ergs cm-2 sec-') at a distance of 48.4 cm from flasks. PAL was extracted from 3-g fresh weight aliquots of 15-day-old plant tissue in 0.1 M Tris buffer (pH 7.5) containing 28 mM mercaptoethanol. PVP was added in the amount of 10%o (w/w) of the tissue fresh weight during extraction to promote removal of phenols and tannins. A PAL-rich fraction was obtained from crude extracts by treatment of the same with 15% (w/v) (PEG)polyethylene glycol 6000 (7). PAL assays were conducted spectrophotometrically (30). The reaction mixture contained 80 ,umol of borate buffer (pH 8.9), 30 ,umol of L-phenylalanine, and 0.2 ml of enzyme preparation containing 79 to 160 ,ug of protein. Production of trans-cinnamic acid was followed by measuring increases in A at 290 nm against substrate blanks using a Beckman spectrophotometer equipped with sipper-cell accessory. TAL assays were conducted similarly with 3 ,umol of L-tyrosine replacing L-phenylalanine in the reaction mixture and production of pcoumaric acid was followed by measuring changes in A at 333 nm (12). A unit of activity was defined as the amount of enzyme which yields I ,umol/min of product under standard assaying conditions (10). Protein was determined by the method of Potty (23) using BSA as a standard. Enzyme activity measurements at 4 hr intervals were coded onto punch cards for computer analysis by the least squares method. Cosine curves were fitted to the data and rhythm parameters quantified according to methods previously published ( 18).