The assimilation of nitrogen by N-limited microalgae has profound effects on respiratory and photosynthetic metabolism. The addition of inorganic nitrogen causes a rapid increase in the rate of amino acid synthesis, which increases the requirements for keto-acids. This results in a large increase in the demand for tricarboxylic acid cycle intermediates. To meet this demand, tricarboxylic acid cycle activity increases, resulting in high rates of respiratory CO2 release during photosynthesis. Tricarboxylic acid cycle reductant, produced during ammonium assimilation, is oxidized via the mitochondrial electron-transport chain, resulting in a substantial increase in the rate of O2 consumption during photosynthesis. When [Formula: see text] is assimilated, tricarboxylic acid cycle activity increases, but there is little effect on mitochondrial O2 consumption. This implies that the tricarboxylic acid cycle reductant produced during [Formula: see text] assimilation is oxidized by some mechanism other than the mitochondrial electron-transport chain, possibly through the reduction of [Formula: see text].These results show that both the tricarboxylic acid cycle and the mitochondrial electron-transport chain are capable of operation during photosynthesis and that a major role of mitochondrial respiration during photosynthesis is the provision of carbon skeletons for biosynthetic reactions. The increase in tricarboxylic acid cycle activity during nitrogen assimilation is supported by anaplerotic reactions. The requirement for substrates by these reactions causes a redirection of recent photosynthate from the synthesis of starch to glycolysis and the tricarboxylic acid cycle. This corresponds with a decrease in the concentration of ribulose bisphosphate in the chloroplast. Under some conditions the concentration of ribulose bisphosphate drops below the ribulose bisphosphate binding site density of ribulose bisphosphate carboxylase:oxygenase resulting in ribulose bisphosphate limitation of photosynthetic carbon fixation. When ammonium is the added N source, there is a corresponding decrease in gross photosynthetic oxygen evolution. When [Formula: see text] is added, the decreased demand for photogenerated reductant brought about by a decrease in Calvin cycle activity is offset by an increase in electron flow to [Formula: see text].

A model which predicts total photosynthetic electron flow from a linear regression of the relationship between corrected steadystate quantum yield and nonphotochemical quenching (E Weis, JA Berry [1987] Biochem Biophys Acta 894: 198-208) was formulated for N-limited cells of the green alga Selenastrum minutum. Unlike other models based on net CO2 fixation, our model is based on total photosynthetic electron flow measured as gross 02 evolution. This allowed for the prediction of total photosyn- Chlorophyll a fluorescence provides a sensitive indicator of photochemical processes (12,14). Qualitative relations between fluorescence emission and photochemistry have frequently been demonstrated, but these relations have been obscured by the fact that there are two major mechanisms of fluorescence quenching (10, 13). Differentiation of these mechanisms via the light doubling procedure of Bradbury and Baker (2, 3) and subsequent development of the 'saturation pulse' method (22) and modulation techniques (9,15,20,22) have allowed the formulation of models predicting rates of photosynthetic electron transport based upon quenching analysis of fluorescence emission (22,31,32 (photochemical quenching; Qq). Oxidized QA allows excitation energy to be used for photochemistry, preventing the reemission of light energy as fluorescence (i.e. 'quenching' the fluorescence). Quenching may also result from nonphotochemical processes (nonphotochemical quenching; QNP). Although there are potentially several mechanisms accounting for QNP, it is generally believed that increases in the transthylakoid pH gradient may result in structural changes which are thought to increase thermal dissipation ofabsorbed light energy. While Qq is a measure of the oxidation state of QA, and therefore one indicator of the ability of PSII to utilize excitation energy to perform work, it has been repeatedly shown that QNP quenching is negatively correlated with quantum yield of PSII (10,11,17,18,31,32). This suggests an important role for thylakoid membrane energization in the regulation of PSII activity. Thus, models for the estimation of photosynthetic electron transport from fluorescence must take into account both of these quenching mechanisms.Weis and Berry (31) have shown that quenching analysis can be used to estimate total electron transport rates (J) from fluorescence emission according to the equation:where I is the incident PAR, and m and b are empirically derived constants. Rates of steady-state photosynthetic electron transport calculated using these fluorescence-derived parameters were highly correlated to electron transport rates calculated from net CO2 exchange (23,31,32).In this report we utilize gross photosynthetic 02 evolution, measured via mass spectrometry, to derive the constants m and b, and to confirm fluorescence-based estimates the total photosynthetic electron transport chain activity.

The effects of ammonium assimilation on photosynthetic carbon fixation and 02 exchange were examined in two species of N-limited green algae, Chlorella pyrenoidosa and Selenastrum minutum. Under light-saturating conditions, ammonium assimilation resulted in a suppression of photosynthetic carbon fixation by S. minutum but not by C. pyrenoidosa. These different responses are due to different relationships between cellular ribulose bisphosphate (RuBP) concentration and the RuBP binding site density of ribulose bisphosphate carboxylase/oxygenase (Rubisco). In both species, ammonium assimilation resulted in a decrease in RuBP concentration. In S. minutum the concentration fell below the RuBP binding site density of Rubisco, indicating RuBP limitation of carboxylation. In contrast, RuBP concentration remained above the binding site density in C. pyrenoidosa. Compromising RuBP regeneration in C. pyrenoidosa with low light resulted in an ammonium-induced decrease in RuBP concentration below the RuBP binding site density of Rubisco. This resulted in a decrease in photosynthetic carbon fixation. In both species, ammonium assimilation resulted in a larger decrease in net 02 evolution than in carbon fixation. Mass spectrometric analysis shows this to be a result of an increase in the rate of mitochondrial respiration in the light.The assimilation of ammonium by photosynthetic organisms results in changes to both photosynthetic and respiratory carbon metabolism. This is illustrated by the diversion of recent photosynthate from the synthesis of starch to the production of TCA cycle2 intermediates. The keto-acids thus produced are then available for assimilation of ammonium into amino acids (1, 9, 31). There is evidence that in natural environments algal growth may be limited by inorganic nitrogen (7). A key adaptation under these conditions is an increase in the saturated rate of nitrogen uptake and assimilation (29). It is therefore not surprising that the supply of nitrogen to N-limited algae affects photosynthetic and respiratory metabolism to a much greater extent than in Nsufficient algae (8,16,25).In some species of N-limited microalgae, the rapid assimilation (5, 6, 17-20, 22, 26, 27). In a study with the N-limited green alga Selenastrum minutum (8), it was shown that the suppression of photosynthetic carbon fixation during the assimilation of inorganic nitrogen coincided with a decrease in RuBP concentration. This implied that RuBP levels may be responsible for limiting carbon fixation. In Chlorella pyrenoidosa, however, nitrogen assimilation results in large decreases in RuBP with little or no effect on photosynthetic carbon fixation (12). Given this apparent discrepancy, the role of RuBP in determining the rate of photosynthetic carbon fixation during N-assimilation requires further clarification.Net photosynthetic 02 evolution also exhibits a wide range of responses to N assimilation. In some N-limited algae, N assimilation results in only a slight suppression of net 02 evolution (10), while in others it ...

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