Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. Alfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose and Reactive Blue agarose chromatography, and led to 60-fold and 150-fold purification for the two enzymes, respectively. The Glc6PDH 1 isoform accounts for 17% of total activity of the enzyme in roots, and is very sensitive to the effects of NADP+/NADPH ratio and dithiothreitol; the Glc6PDH 2 isoform is less affected by reducing power and represents 83% of the total activity. The isoforms showed distinct pH optima, isoelectric points, Km for glucose-6-phosphate and a different electrophoretic mobility. The kinetic properties for the two enzymes were affected by ATP and metabolites. Both enzymes are inhibited to different extents by ATP when magnesium is omitted from the assay mixture, whereas the addition of ATP-Mg2+ had no effect on Glc6PDH activities. The Glc6PDH isoforms are usually present in the plastids and cytosol of plant cells. To verify the intracellular locations of the enzymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location; the enzyme purified from the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots.

In barley (Hordeum vulgare L. var. Nure), glutamate synthesis and the production of reducing power by the oxidative pentose phosphate pathway (OPPP) are strictly correlated biochemical processes. NADH-GOGAT was the major root isoform, whose activity increased on a medium supplied with NH4+ or NO3-; by contrast, no noticeable variations could be observed in the leaves of plants supplied with nitrogen. In the leaves, the major isoform is Fd-GOGAT, whose activity increased under nitrogen feeding. G6PDH activity increased in the roots supplied with nitrogen; no variations were observed in the leaves. Moreover, an increase of the P2 isoform in the roots was measured, giving 13.6% G6PDH activity localized in the plastids under ammonium, and 25.2% under nitrate feeding conditions. Western blots confirmed that P2-G6PDH protein was induced in the roots by nitrogen. P1-G6PDH protein was absent in the roots and increased in the leaves by nitrogen supply to the plants. The changes measured in cytosolic G6PDH seem correlated to more general cell growth processes, and do not appear to be directly involved in glutamate synthesis. The effects of light on Fd-GOGAT is discussed, together with the possibility for P2-G6PDH to sustain nitrogen assimilation upon illumination.

Summary• Temperature effects on growth, photosynthesis, respiration and nitrate reductase (NR) were studied in the cryophilic algae Koliella antarctica and ' Chlorella ' saccharophila , and in the mesophilic Chlorella sorokiniana .• Growth rate was measured as increase in optical density. Photosynthesis at saturating light and respiration in darkness were measured as O 2 exchange. NADH : NR was assayed in crude extracts.• The two cryophilic algae grew below 15 ° C, and C. sorokiniana above 20 ° C. Photosynthetic and respiration rates of K. antarctica and ' C .' saccharophila were elevated at 5 ° C, and peaked at 30 ° C. Arrhenius plots from 5 to 25 ° C were linear in K. antarctica , whereas in ' C .' saccharophila and C. sorokiniana they exhibited breaks at 15 and 20 ° C, respectively. Values for activation energy ( E a ) and the factor by which the rate increases with raising the temperature 10 ° C ( Q 10 ) differed. Nitrate reductase had its optimum at 25 ° C in cryophilic algae and at 35 ° C in C. sorokiniana .• We conclude that growth of cryophilic algae at low temperature is favoured by elevated photosynthesis and respiration rates, but that it could be limited by a high respiration : photosynthesis ratio.

Evidence is provided for a close link between glutamate (Glu) synthesis and the production of reducing power by the oxidative pentose phosphate pathway (OPPP) in barley (Hordeum vulgare L. var. Alfeo) root plastids. A rapid procedure for isolating organelles gave yields of plastids of over 30%, 60% of which were intact. The formation of Glu by intact plastids fed with glutamine and 2-oxoglutarate, both substrates of glutamate synthase (GOGAT), depends on glucose-6-phosphate (Glc-6-P) supply. The whole process exhibited an apparent K m Glc-6-P of 0.45 mM and is abolished by azaserine, a specific inhibitor of GOGAT; ATP caused a decrease in the rate of Glu formation. Glucose and other sugar phosphates were not as effective in supporting Glu synthesis with respect to Glc-6-P; only ribose-5-phosphate, an intermediate of OPPP, supported rates equivalent to Glc-6-P. Glucose-6-phosphate dehydrogenase (Glc6PDH) rapidly purified from root plastids showed an apparent K m Glc-6-P of 0.96 mM and an apparent K m NADP + of 9 lM. The enzyme demonstrated high tolerance to NADPH, exhibiting a K i NADPH of 58.6 lM and selectively reacted with antibodies against potato plastidic, but not chloroplastic, Glc6PDH isoform. The data support the hypothesis that plastidic OPPP is the main site of reducing power supply for GOGAT within the plastids, and suggest that the plastidic OPPP would be able to sustain Glu synthesis under high NADPH:NADP + ratios even if the plastidic Glc6PDH may not be functioning at its highest rates.

Cyanidium caldarium (Tilden) Geitler, a non‐vacuolate unicellular alga, resuspended in medium flushed with air enriched with 5% CO2, assimilated NH4+ at high rates both in the light and in the dark. The assimilation of NO3−, by contrast, was inhibited by 63% in the dark. In cell suspensions flushed with CO2‐free air, NH4+ assimilation decreased with time both in the light and in the dark and ceased almost completely after 90 min. The addition of CO2 completely restored the capacity of the alga to assimilate NH4+. NO3− assimilation, by contrast, was 33% higher in the absence of CO2 and was linear with time. It is suggested that NO3− and NH4+ metabolism in C. caldarium are differently controlled in response to the light and carbon conditions of the cell.

The effects of ammonium and glutamine supply on amino acid levels and the activity of glucose‐6P dehydrogenase (G6PDH EC 1.1.1.49), the main regulated enzyme of the oxidative pentose phosphate pathway, were investigated in barley roots (Hordeum vulgare cv. Alfeo). Feeding ammonium to barley plants increased the contents of glutamine, asparagine and G6PDH in roots. These effects were abolished by using inhibitors of glutamine synthetase. Glutamine‐fed barley roots showed a similar increase in G6PDH activities to ammonium‐fed plants. Two G6PDH enzymes (G6PDH 1 and 2) were partially purified and characterized from ammonium‐fed and glutamine‐fed roots. The isozymes had different pH optima and apparent Km values for glucose‐6P. G6PDH 2 showed similar kinetic parameters to the G6PDH present in root extracts of barley grown without any nitrogen source, while G6PDH 1 exhibited different kinetic parameters, suggesting the appearance of a second G6PDH isoform in response to ammonium. Western blot analysis demonstrated the existence of two G6PDH subunits of different molecular mass in barley roots grown in the presence of ammonium or glutamine, while only one isoform could be detected in roots grown without any nitrogen source. The results suggest a primary role of ammonium and/or glutamine in the appearance of a novel G6PDH isoform; this enzyme (G6PDH 1) shows kinetic parameters similar to those measured previously for chloroplastic and plastidic isoforms and seems to be induced by changes in glutamine content or a related compound(s) in the roots.

SUMMARYBarley plants {Hordeum vulgare L.) grown for 10 d in nitrogen-free hydroponic culture, after a rapid initial phase ahsorbed supplied NH,' ai a constant rate of 131 +1'2//mol h"^ g"'f. wt in the light, and at a rate of 13-81 ± 1-6/imol h"'g"'f. wt in darkness. Atnmonium-grown plants assimilated NH^' at a raie of 7-5 ±0'33//.mo! h"'g"'f. wt and at a 5O'\, lower rate in darkness. Nitrogen-free grown plants showed low concentrations of free amino acids in both root and shoot tissues. Supplying NH^" caused an imtnediate increase in the concentration of glutamine in the root tissues of both illuminated and darkened plants over a 120 tnm period. The increase in concentration of glutaminc then exhibited a lap period of 120 min, after wbich it resumed, but to a very small extent. Glutamine also accumulated in shoot tissue of illuminated plants at increasing rates, attaining a concentration which, 8 h after XH^* supply, was 1'61-foId greater than that attained in the roots. In shoots of darkened plants, by contrast, the concentration of glutamine increased slowly and was always smaller than that in the root tissue. Overall formation of glutamine (in sboots and roots) occurred at decreasing rates during the first 4 h, and then at increasing rates. The increase was more pronounced in illuminated plants than in darkened plants. Even 24 h after ^^H^' was supplied, glutamine content in root tissue was lower than that in sboot tissue. However, 48 h later, tbe concentrations of glutamine in root and shoot were similar, attaining values that were almost 47-fold (in root) and 134-fold (in shoot) greater than initial values. Significant levels of asparagine were detected in the root and in the shoot 24 h after adding NH/, These increased further during the succeeding period. Ammonium supply caused a transitory' drop in the concentration of ATP in root tissue, along with noticeable transitory \'ariations in glucose-6-P concentration. A permanent decrease in free glucose concentration was also detected. Addition of NH/ caused 2-and ]-43-fold increases in respiratory oxygen consumption by roots of ijluminated and darkened plants, respectively. Both in the light and in the dark, the root tissue accumulated methylammonium up to a concentration of 55-67 //mol h'' g"^ f. wt. Methylammonium was never found in shoot tissue of either illuminated or darkened plants. Methylammonium stimulated respiration of root barley plants by a factor of 1-2, Regulatory aspects of NHj' metabolism are discussed.

Temperature responses of nitrate reductase (NR) were studied in the psychrophilic unicellular alga, Koliella antarctica , and in the mesophilic species, Chlorella sorokiniana . Enzymes from both species were purified to near homogeneity by Blue Sepharose (Pharmacia, Uppsala, Sweden) affinity chromatography and high-resolution anionexchange chromatography (MonoQ; Pharmacia; Uppsala, Sweden). Both enzymes have a subunit molecular mass of 100 kDa, and K. antarctica NR has a native molecular mass of 367 kDa. NR from K. antarctica used both NADPH and NADH, whereas NR from C. sorokiniana used NADH only. Both NRs used reduced methyl viologen (MVH) or benzyl viologen (BVH). In crude extracts, maximal NADH and MVH-dependent activities of cryophilic NR were found at 15 and 35 ∞ C, respectively, and retained 77 and 62% of maximal activity, respectively, at 10 ∞ C. Maximal NADH and MVH-dependent activities of mesophilic NR, however, were found at 25 and 45 ∞ C, respectively, with only 33 and 23% of maximal activities being retained at 10 ∞ C. In presence of 2 m M flavin adenine dinucleotide (FAD), activities of cryophilic NADH:NR and mesophilic NADH:NR were stable up to 25 and 35 ∞ C, respectively. Arrhenius plots constructed with cryophilic and mesophilic MVH:NR rate constants, in both presence or absence of FAD, showed break points at 15 and 25 ∞ C, respectively. Essentially, similar results were obtained for purified enzymes and for activities measured in crude extracts. Factors by which the rate increases by raising temperature 10 ∞ C ( Q 10 ) and apparent activation energy ( E a ) values for NADH and MVH activities measured in enzyme preparations without added FAD differed slightly from those measured with FAD. Overall thermal features of the NADH and MVH activities of the cryophilic NR, including optimal temperatures, heat inactivation (with/without added FAD) and break-point temperature in Arrhenius plots, are all shifted by about 10 ∞ C towards lower temperatures than those of the mesophilic enzyme. Transfer of electrons from NADH to nitrate occurs via all three redox centres within NR molecule, whereas transfer from MVH requires Mo-pterin prosthetic group only; therefore, our results strongly suggest that structural modification(s) for cold adaptation affect thermodynamic properties of each of the functional domains within NR holoenzyme in equal measure.