Amino acid and sucrose contents were analyzed in the chloroplastic, cytosolic, and vacuolar compartments and in the phloem sap of illuminated spinach leaves (Spinacia oleracea L.). The determination of subcellular metabolite distribution was carried out by nonaqueous fractionation of frozen and lyophilized leaf material using a novel three-compartment calculation method. The phloem sap was collected by aphid stylets which had been severed by a laser beam. Subcellular analysis revealed that the amino acids found in leaves are located mainly in the chloroplast stroma and in the cytosol, the sum of their concentrations amounting to 151 and 121 millimolar, respectively, whereas the amino acid concentrations in the vacuole are one order of magnitude lower. The amino acid concentrations in the phloem sap are found to be not very different from the cytosolic concentrations, whereas the sieve tube concentration of sucrose is found to be one order of magnitude higher than in the cytosol. It is concluded that the phloem loading results in a preferential extraction of sucrose from the source cells.

In leaves of spinach plants (Spinacia oleracea L.) grown in ambient CO2 the subcellular contents of adenylates, pyridine nucleotides, 3-phosphoglycerate, dihydroxyacetone phosphate, malate, glutamate, 2-oxoglutarate, and aspartate were assayed in the light and in the dark by nonaqueous fractionation technique. From the concentrations of NADP and NADPH determined in the chloroplast fraction of illuminated leaves the stromal NADPH to NADP ratio is calculated to be 0.5. For the cytosol a NADH to NAD ratio of 10-3 is calculated from the assay of the concentrations of NAD, malate, glutamate, aspartate, and 2-oxoglutarate on the assumption that the reactions catalyzed by the cytosolic glutamate oxaloacetate transaminase and malate dehydrogenase are not far away from equilibrium. For the transfer of redox equivalents from the chloroplastic NADPH to the cytosolic NAD two metabolite shuttles are operating across the inner envelope membrane: the triosephosphate-3-phosphoglycerate shuttle and the malate-oxaloacetate shuttle. Although both shuttles would have the capacity to level the redox state of the stromal and cytosolic compartment, this apparently does not occur. To gain an insight into the regulatory processes we calculated the free energy of the enzymic reactions and of the translocation steps involved.From the results it is concluded that the triosephosphate-3-phosphoglycerate shuttle is mainly controlled by the chloroplastic reaction of 3-phosphoglycerate reduction and of the cytosolic reaction of triosephosphate oxidation. The malate-oxaloacetate shuttle is found to be regulated by the chloroplastic NADP-malate dehydrogenase and also by the translocating step across the envelope membrane.The metabolism of a leaf cell is distributed between various compartments, e.g. the cytosol, chloroplast stroma, and mitochondrial matrix. Each of the metabolic compartments has its specific function and hence also its special milieu. Specific translocators catalyze the transfer of metabolites between these compartments (16 transferred from the chloroplast stroma to the cytosol by two different metabolite shuttles, the triosephosphate-3-phosphoglycerate shuttle ( 14) catalyzed by the Pi-trioseP-3-PGA3 translocator (6) and the malate-OAA shuttle (1) facilitated by specific transport of malate and OAA ( 12). As both metabolite shuttles would have the capacity to level the redox state of the stromal and cytosolic compartment, a regulation of these processes is required to maintain the specific redox states of the two metabolic compartments.To gain an insight into such regulatory processes, we attempted in the present publication to analyze the redox state of pyridine nucleotides by the measurement of their concentrations and the concentrations of substrates of pyridine nucleotide-linked reactions in subcellular compartments of spinach leaves. These measurements were carried out mainly by nonaqueous fractionation of frozen leaves carried out by Heber (13) earlier and later refined in our laboratory (8). It will be shown that ...

The concentrations of sucrose, amino acids, nitrate and malate in the apoplastic compartment of illuminated leaves of barley and spinach were determined and compared with the corresponding concentrations in the cytosolic compartment of mesophyll cells and in the phloem sap, as measured previously with plants grown under identical conditions. The concentrations of sucrose and amino acids in the apoplast are found to be much lower than in the cytosol and in the phloem sap, indicating that not only the uptake into the phloem of sucrose, but also of amino acids, requires transport against a concentration gradient. The gradient of sucrose and amino acids between the cytosol and the apoplast was maintained when phloem transport had been blocked by cold girdling. Apparently, the efflux of sucrose and amino acids from the source cells to the apoplast is regulated in such a way that it meets the requirements of phloem transport. The percentages of the single amino acids as part of the total amino acids are quite similar in the cytosol, apoplast and phloem sap. The ratio of sucrose to the total amino acids in the cytosol is similar to that in the apoplast but about five times higher in the phloem sap. It appears from these results that the preferential extraction of sucrose over amino acids from the source cells to the phloem is due to the uptake from the apoplast into the phloem.

In leaves of spinach plants (Spinacia oleracea L.) performing CO2 and N03-assimilation, at the time of sudden darkening, which eliminates photosystem I-dependent nitrite reduction, only a minor temporary increase of the leaf nitrite content is observed. Because nitrate reduction does not depend on redox equivalents generated by photosystem I activity, a continuation of nitrate reduction after darkening would result in a large accumulation of nitrite in the leaves within a very short time, which is not observed. Measurements of the extractable nitrate reductase activity from spinach leaves assayed under standard conditions showed that in these leaves the nitrate reductase activity decreased during darkening to 15% of the control value with a half-time of only 2 minutes. Apparently, in these leaves nitrate reductase is very rapidly inactivated at sudden darkness avoiding an accumulation of the toxic nitrite in the cells.In a green plant cell, photosynthetic nitrate assimilation involves two reductive steps, located in different subcellular compartments. In the cytosol, nitrate is reduced to nitrite via NADH-dependent NR2 (27), and after transport into the chloroplasts (3), nitrite is further reduced to ammonia by nitrite reductase located in the stroma (2). Because this reaction needs reduced ferredoxin, which is provided by PSI, it is essentially light dependent, although a minor amount of nitrite reduction may also occur during darkness at the expense of reducing equivalents generated by the oxidative pentose phosphate pathway (1, 24) and starch breakdown (13 (27) are too slow to account for a rapid decrease of enzyme activity during darkening. Although a number of metabolites such as ADP and ATP (5, 17), Pi (20), NADH and cyanide (7,26), and amino acids (18,19) were reported to affect NR activity, a systematic study of the putative regulatory role of these and other metabolites showed that none of these substances, when added to partially purified NR at those concentrations occurring in the cytosol of illuminated and darkened spinach leaves, were able to change NR activity to such an extent that the enzyme could be switched off during sudden darkening (25).Recently, Kaiser and Forster ( 11) presented evidence that NR may be altered in its activity by interconversion of the enzyme protein. These authors found that in illuminated spinach leaves the extractable NR activity assayed under standard conditions largely decreased when leaves kept under ambient CO2 were flushed with C02-free air, and the deactivation was reversed when the leaves were brought back to air. Moreover, the extractable NR activity was found to increase when darkened leaves were illuminated (1 1). The deactivation of NR during the exposure of leaves to C02-free air occurred with a half-time of about 20 min (10, 11). To avoid an accumulation of nitrite when a leaf suddenly becomes shaded, a much higher rate of NR deactivation would be required. In the present report, from measurements of nitrite contents and of NR activity in spinach le...

Abstract.Rates of CO2 fixation during the light period and the rates of CO 2 release during the night period were measured using mature leaves from 39-to 49-d-old spinach (Spinacia oleracea L., US Hybrid 424; grown in 9 h light, 15 h darkness, daily) and mature leaves from 21-d-old barley (Hordeum vulgare L., cv. Apex; grown in 14 h light, 10 h darkness, daily). At certain times during the light and dark periods leaves were harvested for assay of their contents of soluble carbohydrates, starch, malate and the various amino acids. Evaluation of the results of these measurements shows that in spinach and barley leaves 46% and 26%, respectively, of the carbon assimilated during the light period is deposited in the leaves for export during the night period. Taking into account the carbon consumption in the source leaves by dark respiration, it is evaluated that rates of assimilate export during the light period from spinach and barley leaves [38 and 42 laatom C.(mg Chl) -~. la 1] are reduced in the dark period to 16 patom C. (mg Chl) 1. h-i in both species. The calculated C/N ratios of the photoassimilates exported during the dark period were 0.029 and 0.015 for spinach and barley leaves, respectively.

Acetyl-CoA and Fatty Acid Synthesis, Chloroplasts The present investigation indicates that photosynthetically active chloroplasts can synthesize acetyl-CoA either from acetate via acetyl-CoA synthetase (ACS) or from pyruvate via the pyru­ vate dehydrogenase complex (PDC). Both enzyme systems have been assayed in rapidly prepared extracts of chloroplasts isolated from spinach, peas and maize mesophyll. Their kinetic properties showed few species-specific differences. The differing pyruvate and acetate concentrations within the corresponding leaf tissues have been interpreted, therefore, as constituting a major factor determining the relative involvement of both acetyl-CoA synthesizing systems within the different types of chloroplasts. The idea that acetate originates from mitochondria and pyruvate from the cytosol has been supported by nonaqueous fractionation studies. Diffusion-mediated faster up­ take of acetate may indicate a predominant role of the ACS in spinach chloroplasts. Higher cellular pyruvate/acetate-ratios (2-5) in pea and maize leaves may enhance pyruvate uptake into chloroplasts and thus PDC-driven acetyl-CoA synthesis in pea and maize mesophyll chloroplasts. Maize mesophyll chloroplasts even show a light-driven pyruvate uptake accompanied by a stimulated acetyl-CoA and fatty acid formation. Assuming light-dependent increasing parameters in the stroma space, like Mg2+-concentrations, pH and ATP, as further control criteria in chloroplast acetyl-CoA formation, the ACS appears better adapted to the circumstances in illuminated chloroplasts because of the fact that 1. the ACS requires these cofactors altogether; 2. the PDC is stimulated by increasing pH (up to 8) and Mg-levels (up to 5 mᴍ) alone.

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