ELSEVIER Biochimica et Biophysica Acta 1249 (1995) 72-78 BB Biochi~ic~a et Biophysica A~ta Regulation of the expression of ferredoxin-nitrite reductase in synchronous cultures of Chlamydomonas reinhardtii Elofsa Pajuelo, Purificaci6n Pajuelo, Ma6a Teresa Clemente, Antonio J. Mfirquez Departamento de BioqMmica, Facultadde QMmica, Universidadde Sevilla, Apartado 553, 41080 Sevilla, Spain Received 18 October 1994; accepted 7 December 1994 Abstract The regulation of ferredoxin-nitrite reductase - the second enzyme involved in the nitrate assimilatory pathway - in synchronous cultures of C. reinhardtii has been studied both at the activity and protein levels using specific antibodies. During a cycle of 12 h light/12 h dark (12L:12D), ferredoxin-nitrite reductase activity shows a 24-h fluctuation with a maximum in the middle of the light period. The increase of activity during the first few hours of the light phase is due to de novo synthesis of the enzyme. This synthesis occurs in the absence of NH~- and it is highly induced by either nitrate or nitrite, but it does not require light so long as carbon skeletons are available. The decrease of ferredoxin-nitrite reductase activity during the last hours of the light period and during the dark phase is suggested to be due to protein degradation, although this process is slow because of the high stability of the enzyme. The changes in the level of ferredoxin-nitrite reductase seem to be related to events in the cell cycle under the illumination conditions used. Thus, synthesis of the enzyme correlates to growth periods within the cell cycle, and it does not seem to be under the control of a circadian rhythm. Keywords: Nitrogen assimilation regulation; Nitrite reductase; Synchronous culture; Light/dark cycle; (Green alga) 1. Introduction Ferredoxin-nitrite reductase is the second enzyme in the assimilatory reduction of nitrate and catalyzes the reduc- tion of NO~- produced by nitrate reductase to NH~-. The regulation of ferredoxin-nitrite reductase is poorly under- stood compared, for instance, to that of nitrate reductase [1]. In Chlamydomonas reinhardtii the enzyme is NH~--re- pressible [2]. Although removal of NH~- from the medium allows some extent of ferredoxin-nitrite reductase activity derepression, maximal activity levels are achieved in the presence of NO 2 and particularly NO 3 [3,4]. Light is another factor shown to be required for the induction of ferredoxin-nitrite reductase in several plants (see Refs. [5-8] for reviews), although little is known with regard to C. reinhardtii enzyme. The ability to grow C. reinhardtii cells synchronously offers an interesting alternative approach to the study of the regulation of ferredoxin-nitrite reductase. On the one Abbreviations: 12L:12D, 12 h light/12 h dark cycle; Ln, hour n of the light phase; Dn, hour n of the dark phase. * Correspondingauthor. E-mail: cabeza@cica.es. Fax: + 34 5 4626853. Elsevier Science B.V. SSDI 0167-4838(95)00066-6 hand, the environmental laboratory conditions used as synchronizing procedure (12 h light/12 h dark (12L: 12D) cycle) mimic the conditions in which cells are found in nature. Also, the use of synchronous cultures makes that all the cells can be in the same phase of the cell cycle and therefore are physiologically behaving as a single cell. Thus, the changes observed in the culture are more closely related to what is happening in a single cell, and therefore a process can be studied with regard to the cell cycle which takes place under these conditions. Recent work in our laboratory [9] has established that the nitrate-reducing and ammonium-assimilatory systems are regulated differentially in synchronous cultures of C. reinhardtii. Nitrate and nitrite reductases reached a maxi- mum level of enzyme activity around the middle of the light period (L6) and then decreased to a minimum and constant value in the dark period. On the contrary, glu- tamine synthetase and glutamate synthases increased dur- ing the light phase and remained at high level in the dark. The availability of anti-C, reinhardtii-ferredoxin-nitrite reductase antibodies [10] has allowed us now to investigate the molecular mechanism involved in the fluctuation of the ferredoxin-nitrite reductase in synchronous cultures of C. reinhardtii grown under a 12L:12D cycle. We have stud-