In Vivo Blue-Light Activation of Chiamydomonas reinhardii

CMldmyuOnas rihardii cells, growning photoautotropbical under air, excreted to the culture medium much higher amounts of N02 and NH4 I under ble than under red fight. Under shmilar conditions, but with N02as the only nitrogen source, the cells consumed NO2 and excreted NH4, at shilar rates under blue and red light. In the presence of N03and air with 2% CO2 (v/v), no excretion of NOand NH1+ occurred and, moreover, if the bubbling air of the cells that were currently excreting N02and NH4 + was enrkced with 2% CO2 (v/v), the previously excreted reduced nirogen Ions were rapidly reassimilated. The lels of total nitrate reductase and active nitrate reductase increased several times in the bluelightIrradiated cells growing on NOsunder air. When tugstate replaced molyblate in the medim (conditos that do not allow the formation of functional nitrate reductase), blue light actvated most of the preformed inactive enzyme of the cells. Furthermore, nitrate reductase extracted from the cells in its inactive form was readily activated in vitro by blue ight. It appears that under high irradiance (90 w m-2) and low CO2 tensions, cells growig on NOsor N02may not have sufficient carbon skeletons to incorporate al the pbotogenerated NH4 +. Because these cells should have high levels of reducing power, they might use N03or, in its absence, NO,as terminal electron acceptors. The excretion of the products of N02and NH4, to the medium may provide a mechanism to control reductant level in the cells. Blue lght is suggested as an important regulatory factor of this photorespiratory conmption of N03and possibly of the whole nitrogen metabolism in green algae. Nitrate assimilation in green algae and higher plants is a basic metabolic process because it uses more than 20o of the reducing power generated by their photosynthetic apparatus (11). Among the different steps involved in this metabolic pathway, reduction of NO3 to N02 catalyzed by nitrate reductase has become particularly relevant due to its regulatory features on nitrogen metabolism (5, 12, 36). NADH-nitrate reductase from green algae is a multimeric enzyme of high mol wt with several electron transport components such as flavin adenine dinucleotide, protoheme b557, and molybdenum (8). Recently, it has been reported that molybdenum is held in a special cofactor that contains an unidentified pterin (13). Depending on the growth conditions, nitrate reductase can be extracted from alga cells in two interconvertible forms: active and inactive. In cells optimally grown on NO03 under air enriched in 'Supported in part by grants to P.J.A. and R. Malkin from the USASpain Joint Committee of Cooperative Research III P-7730394/7, and to P.J.A. from the Comisi6n Asesora de Investigaci6n cientifica y Tecnica 1222. This investigation was carried out under Consejo Superior de Investigaciones Cientificas Programme C02, nitrate reductase is usually found in its active form. However, after mid-term (1-2 h) NH4' addition to such cells, most of nitrate reductase appears in its inactive form (I 1, 15, 16). In vitro nitrate reductase can be inactivated by incubation with NAD(P)H or S2042and more readily if CNor C2H2 are also present (14, 18). Both the in vivo inactive form and the in vitro CN-inactivated spinach enzyme can be rapidly reactivated by oxidation with ferricyanide. In vitro blue-light irradiation of CN-or C2H2-inactivated nitrate reductase, as well as the in vivo inactive enzyme from Chlorella fusca, promotes their full activation (1, 18a). Likewise, CN-inactivated nitrate reductase from Neurospora is also fully reactivated by blue light (23). The photoreactivation of spinach nitrate reductase is greatly accelerated by exogenous flavins, especially under anaerobic conditions. Therefore, excited flavins, but not some highly reactive oxygen species, are directly involved in the photoreactivation. Accordingly, it has been proposed that excited flavins that are strong oxidants (38) reactivate nitrate reductase by oxidizing some of its electron transport component(s), probably molybdenum. Alternatively, the excited flavins could act by removing protein-bound CNin a photoaddition reaction that generates cyanoflavins as proposed for the CN--inactivated enzyme (18a). In C. fusca, the assimilation of NO3under nonsaturating irradiation was higher under blue than under red light (7). However, no direct evidence was reported assigning to blue light an in vivo regulatory role of nitrate reductase activity and therefore of nitrogen metabolism. The results presented in this article show that inactive form of nitrate reductase extracted from Chlamydomonas reinhardii can be reactivated by blue light and that cells, growing on N03 under air without supplementary CO2, release NO2and NH4+ to the culture medium at much higer rates under blue than under red light. Furthermore, under these growth conditions, irradiation of the cells with blue light increases the enzyme level and maintains most of the intracellular nitrate reductase in active form. MATERIALS AND METHODS Chlamydomonas reinhardii 11-32b from the Alga Collection of Gottingen University was grown autotrophically in a mineral medium with N03 as the only nitrogen source (31). The medium was adjusted to pH 7.1 with K-phosphate and gassed with air (containing approximately 0.03% C02). Cultures (800 ml) were illuminated with both 160-w model SBR-F, PAR 38, (Eye, Japan) and 100-w tungsten lamps under alternating light-dark periods of 7 and 5 h, respectively. Daily, in the middle of the light period, 300 ml of the growing culture were removed and the remaining culture was refilled up to the initial volume with fresh nutrient solution. The cells were collected by centrifugation and washed three times with the same nutrient solution except that the pH was 7.8, and in some cases N03 was omitted. The resuspended cells were diluted with the pertinent nutrient solution to obtain a final Chl content of 35 ,ug ml-'. Chl concentration did not change