Nonaqueous Fractionation Research Articles

Analysis of Cytosolic Heteroglycans from Leaves of Transgenic Potato (Solanum tuberosum L.) Plants that Under- or Overexpress the Pho 2 Phosphorylase Isozyme

During starch degradation, chloroplasts export neutral sugars into the cytosol where they appear to enter a complex glycan metabolism. Interactions between glycans and glucosyl transferases residing in the cytosol were studied by analyzing transgenic potato (Solanum tuberosum L.) plants that possess either decreased or elevated levels of the cytosolic (Pho 2) phosphorylase isoform. Water-soluble heteroglycans (SHGs) were isolated from these plants and were characterized. SHG contains, as major constituents, arabinose, rhamnose, galactose and glucose. Non-aqueous fractionation combined with other separation techniques revealed a distinct pool of the SHG that is located in the cytosol. Under in vitro conditions, the cytosolic heteroglycans act as glucosyl acceptor selectively for Pho 2. Acceptor sites were characterized by a specific hydrolytic degradation following the Pho 2-catalyzed glucosyl transfer. The size distribution of the cytosolic SHG increased during the dark period, indicating a distinct metabolic activity related to net starch degradation. Antisense inhibition of Pho 2 resulted in increased glucosyl and rhamnosyl contents of the glycans. Overexpression of Pho 2 decreased the content of both residues. Compared with the wild type, in both types of transgenic plants the size of the cytosolic glycans was increased.

Identification, subcellular localization and biochemical characterization of water‐soluble heteroglycans (SHG) in leaves of Arabidopsis thaliana L.: distinct SHG reside in the cytosol and in the apoplast

Water-soluble heteroglycans (SHG) were isolated from leaves of wild-type Arabidopsis thaliana L. and from two starch-deficient mutants. Major constituents of the SHG are arabinose, galactose, rhamnose, and glucose. SHG was separated into low (<10 kDa; SHG(S)) and high (>10 kDa; SHG(L)) molecular weight compounds. SHG(S) was resolved into approximately 25 distinct oligoglycans by ion exchange chromatography. SHG(L) was further separated into two subfractions, designated as subfraction I and II, by field flow fractionation. For the intracellular localization of the various SHG compounds several approaches were chosen: first, leaf material was subjected to non-aqueous fractionation. The apolar gradient fractions were characterized by monitoring markers and were used as starting material for the SHG isolation. Subfraction I and SHG(S) exhibited a distribution similar to that of cytosolic markers whereas subfraction II cofractionated with crystalline cellulose. Secondly, intact organelles were isolated and used for SHG isolation. Preparations of intact organelles (mitochondria plus peroxisomes) contained no significant amount of any heteroglycan. In isolated intact microsomes a series of oligoglycans was recovered but neither subfraction I nor II. In in vitro assays using glucose 1-phosphate and recombinant cytosolic (Pho 2) phosphorylase both SHG(S) and subfraction I acted as glucosyl acceptor whereas subfraction II was essentially inactive. Rabbit muscle phosphorylase a did not utilize any of the plant glycans indicating a specific Pho 2-glycan interaction. As revealed by in vivo labeling experiments using 14CO2 carbon fluxes into subfraction I and II differed. Furthermore, in leaves the pool size of subfraction I varied during the light-dark regime.

Mechanical stabilization of desiccated vegetative tissues of the resurrection grass Eragrostis nindensis: does a TIP 3;1 and/or compartmentalization of subcellular components and metabolites play a role?

During dehydration, numerous metabolites accumulate in vegetative desiccation-tolerant tissues. This is thought to be important in mechanically stabilizing the cells and membranes in the desiccated state. Non-aqueous fractionation of desiccated leaf tissues of the resurrection grass Eragrostis nindensis (Ficalho and Hiern) provided an insight into the subcellular localization of the metabolites (because of the assumptions necessary in the calculations the data must be treated with some caution). During dehydration of the desiccant-tolerant leaves, abundant small vacuoles are formed in the bundle sheath cells, while cell wall folding occurs in the thin-walled mesophyll and epidermal cells, leading to a considerable reduction in the cross-sectional area of these cells. During dehydration, proline, protein, and sucrose accumulate in similar proportions in the small vacuoles in the bundle sheath cells. In the mesophyll cells high amounts of sucrose accumulate in the cytoplasm, with proline and proteins being present in both the cytoplasm and the large central vacuole. In addition to the replacement of water by compatible solutes, high permeability of membranes to water may be critical to reduce the mechanical strain associated with the influx of water on rehydration. The immunolocalization of a possible TIP 3;1 to the small vacuoles in the bundle sheath cells may be important in both increased water permeability as well as in the mobilization of solutes from the small vacuoles on rehydration. This is the first report of a possible TIP 3;1 in vegetative tissues (previously only reported in orthodox seeds).

Maltose is the major form of carbon exported from the chloroplast at night.

Transitory starch is formed in chloroplasts during the day and broken down at night. We investigated carbon export from chloroplasts resulting from transitory-starch breakdown. Starch-filled chloroplasts from spinach ( Spinacia oleracea L. cv. Nordic IV) were isolated 1 h after the beginning of the dark period and incubated for 2.5 h, followed by centrifugation through silicone oil. Exported products were measured in the incubation medium to avoid measuring compounds retained inside the chloroplasts. Maltose and glucose made up 85% of the total exported products and were exported at rates of 626 and 309 nmol C mg(-1) chlorophyll h(-1), respectively. Net export of phosphorylated products was less than 5% and higher maltodextrins were not detected. Maltose levels in leaves of bean ( Phaseolus vulgaris L. cv. Linden), spinach, and Arabidopsis thaliana (L.) Heynh. were low in the light and high in the dark. Maltose levels remained low and unchanged during the light/dark cycle in two starch-deficient Arabidopsis mutants, stf1, deficient in plastid phosphoglucomutase, and pgi, deficient in plastid phosphoglucoisomerase. Through the use of nonaqueous fractionation, we determined that maltose was distributed equally between the chloroplast and cytosolic fractions during darkness. In the light there was approximately 24% more maltose in the cytosol than the chloroplast. Taken together these data indicate that maltose is the major form of carbon exported from the chloroplast at night as a result of starch breakdown. We hypothesize that the hydrolytic pathway for transitory-starch degradation is the primary pathway used when starch is being converted to sucrose and that the phosphorolytic pathway provides carbon for other purposes.

Differential accumulation of dimethylallyl diphosphate in leaves and needles of isoprene- and methylbutenol-emitting and nonemitting species.

The biosynthesis and emission of volatile plant terpenoids, such as isoprene and methylbutenol (MBO), depend on the chloroplastic production of dimethylallyl diphosphate (DMAPP). To date, it has been difficult to study the relationship of cellular DMAPP levels to emission of these volatiles because of the lack of a sensitive assay for DMAPP in plant tissues. Using a recent DMAPP assay developed in our laboratories, we report that species with the highest potential for isoprene and MBO production also exhibit elevated light-dependent DMAPP production, ranging from 110% to 1,063%. Even species that do not produce significant amounts of volatile terpenoids, however, exhibit some potential for light-dependent production of DMAPP. We used a nonaqueous fractionation technique to determine the intracellular distribution of DMAPP in isoprene-emitting cottonwood (Populus deltoides) leaves; approximately 65% to 70% of the DMAPP recovered at midday occurred in the chloroplasts, indicating that most of the light-dependent production of DMAPP was chloroplastic in origin. The midday concentration of chloroplastic DMAPP in cottonwood leaves is estimated to be 0.13 to 3.0 mM, which is consistent with the relatively high K(m)s that have been reported for isoprene synthases (0.5-8 mM). The results provide support for the hypothesis that the light dependence of isoprene and MBO emissions is in part due to controls over DMAPP production.

Probing the circadian control of phosphoenolpyruvate carboxylase kinase expression in Kalanchoë fedtschenkoi.

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. In crassulacean acid metabolism (CAM) plants, phosphoenolpyruvate carboxylase (PEPC) kinase is expressed at night under the control of a circadian oscillator. We have proposed that this is an indirect effect, secondary to circadian fluctuations in the level of a metabolite, possibly cytosolic malate, resulting from a primary effect on the permeability of the tonoplast (Nimmo 2000, Trends in Plant Science 5, 75-80). Here we show that the nocturnal accumulation of PEPC kinase translatable mRNA and phosphorylation of PEPC in Kalanchoë fedtschenkoi is blocked by the protein phosphatase inhibitor cantharidin. This implicates protein dephosphorylation in the circadian pathway that regulates expression of PEPC kinase. We also show that the effect of reducing the temperature from 30 to 15 °C on CO2 fixation by detached leaves held in constant darkness and normal air is 'gated' by the circadian clock. This strongly supports the view that the effect of the clock on the expression of PEPC kinase is secondary rather than direct. We have developed a non-aqueous fractionation protocol that separates the cytosolic material in mature leaves from vacuolar material. The cytosolic malate in mature leaves represents a very small part of the total malate, and its concentration cannot be measured precisely by this method.

Analysis of the Compartmentation of Glycolytic Intermediates, Nucleotides, Sugars, Organic Acids, Amino Acids, and Sugar Alcohols in Potato Tubers Using a Nonaqueous Fractionation Method

Analysis of the Compartmentation of Glycolytic Intermediates, Nucleotides, Sugars, Organic Acids, Amino Acids, and Sugar Alcohols in Potato Tubers Using a Nonaqueous Fractionation Method

Analysis of the compartmentation of glycolytic intermediates, nucleotides, sugars, organic acids, amino acids, and sugar alcohols in potato tubers using a nonaqueous fractionation method.

The compartmentation of metabolism in heterotrophic plant tissues is poorly understood due to the lack of data on metabolite distributions and fluxes between subcellular organelles. The main reason for this is the lack of suitable experimental methods with which intracellular metabolism can be measured. Here, we describe a nonaqueous fractionation method that allows the subcellular distributions of metabolites in developing potato (Solanum tuberosum L. cv Desiree) tubers to be calculated. In addition, we have coupled this fractionation method to a recently described gas chromatography-mass spectrometry procedure that allows the measurement of a wide range of small metabolites. To calculate the subcellular metabolite concentrations, we have analyzed organelle volumes in growing potato tubers using electron microscopy. The relative volume distributions in tubers are very similar to the ones for source leaves. More than 60% of most sugars, sugar alcohols, organic acids, and amino acids were found in the vacuole, although the concentrations of these metabolites is often higher in the cytosol. Significant amounts of the substrates for starch biosynthesis, hexose phosphates, and ATP were found in the plastid. However, pyrophosphate was located almost exclusively in the cytosol. Calculation of the mass action ratios of sucrose synthase, UDP-glucose pyrophosphorylase, phosphoglucosisomerase, and phosphoglucomutase indicate that these enzymes are close to equilibrium in developing potato tubers. However, due to the low plastidic pyrophosphate concentration, the reaction catalyzed by ADP-glucose pyrophosphorylase was estimated to be far removed from equilibrium.

Metabolite transport in apoplastic and symplastic phloem loaders

If leaves are able to fix more CO2 during the light period than they need for their metabolism, the surplus of carbon is exported to support sink tissues. Long distance transport of metabolites from the source leaves to the sink tissues takes place in the phloem. The production and export of assimilates involves a cooperation of various cell types and requires several transfer steps across membranes. To understand these complex interactions, we have analyzed the concentrations of metabolites in the subcellular compartments of mesophyll cells and in the phloem sap by the methods of non-aqueous fractionation and laser-aphid-stylet-technique. Metabolites can enter the phloem via apoplastic or symplastic loading steps, depending on the number of plasmodesmata between the phloem and the surrounding cells. In apoplastic phloem loaders sucrose is the only form of carbohydrate which is exported, whereas the export of amino acids is not specific. All the amino acids found in the cytosol of mesophyll cells also occur in the phloem sap. To compare symplastic phloem loaders with apoplastic phloem loaders we analyzed several members of the Scrophulariaceae. The main carbon transport form of Asarina is sucrose and in Alonsoa raffinose and stachyose (ca. 600 mM). Independent of the kind of the translocated sugar, we identified full length cDNAs of sucrose transporters in the leaves of both plant species. SUT mRNA could also be detected in pure phloem sap. The apoplastic and symplastic phloem loading mechanism will be discussed.

NADPH Supply and Mannitol Biosynthesis. Characterization, Cloning, and Regulation of the Non-Reversible Glyceraldehyde-3-Phosphate Dehydrogenase in Celery Leaves

Mannitol, a sugar alcohol, is a major primary photosynthetic product in celery (Apium graveolens L. cv Giant Pascal). We report here on purification, characterization, and cDNA cloning of cytosolic non-reversible glyceraldehyde-3-P dehydrogenase (nr-G3PDH, EC 1.2.1. 9), the apparent key contributor of the NADPH required for mannitol biosynthesis in celery leaves. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, purified nr-G3PDH showed a molecular mass of 53 kD. A 1,734-bp full-length cDNA clone (accession no. AF196292) encoding nr-G3PDH was identified using polymerase chain reaction and rapid amplification of cDNA ends techniques. The cDNA clone has an open reading frame of 1,491 bp encoding 496 amino acid residues with a calculated molecular weight of 53,172. K(m) values for the celery nr-G3PDH were low (6.8 microM for NADP(+) and 29 microM for D-glyceraldehyde-3-P). NADPH, 3-phosphoglycerate, and ATP were competitive inhibitors, and cytosolic levels of these three metabolites (as determined by nonaqueous fractionation) were all above the concentrations necessary to inhibit activity in vitro, suggesting that nr-G3PDH may be regulated through feedback inhibition by one or more metabolites. We also determined a tight association between activities of nr-G3PDH and mannose-6-P reductase and mRNA expression levels in response to both leaf development and salt treatment. Collectively, our data clearly show metabolic, developmental, and environmental regulation of nr-G3PDH, and also suggest that the supply of NADPH necessary for mannitol biosynthesis is under tight metabolic control.

Disturbance of myocardial energy metabolism in experimental virus myocarditis by antibodies against the adenine nucleotide translocator.

The adenine nucleotide translocator (ANT) of the inner mitochondrial membrane is an autoantigen in myocarditis and in dilated cardiomyopathy. Clinical and experimental studies showed that specific autoantibodies inhibit the transmembrane nucleotide transport. In isolated hearts of guinea pigs immunized with the ANT, energy metabolism is disturbed. This metabolic disorder is related to functionally active specific antibodies and to a reduced heart function. This study tests whether similar immunological, metabolical and functional responses also occur in experimental virus myocarditis. Experimental virus myocarditis was induced in A.SW/SnJ-mice by Coxsackie B3 virus infection. Specific antibodies against the ANT were detected by Western Blot in 14 out of 19 infected animals. In the isolated perfused hearts of five of these 14 mice cytosolic and mitochondrial ATP/ADP-ratios, determined by nonaqueous fractionation, were significantly altered, signalling a reduced ANT function [cytosolic ATP/ADP: 59 +/- 18 vs. 136 +/- 20 (controls), mitochondrial ATP/ADP: 4.2 +/- 1.0 vs. 1.1 +/- 0.3], all P < 0.05. Also, left ventricular pressure [43 +/- 9 vs. 78 +/- 6 mmHg (noninfected controls)], rate-pressure product (15.8 +/- 3.2 vs. 30.5 +/- 3.0 mmHg/min/1000), dp/dt (2410 +/- 222 vs. 3250 +/- 118 mmHg/s), and oxygen consumption (4.7 +/- 0.9 vs. 7.3 +/- 0.7 mumol/g/min), all P < 0.05, were lowered. The data support the hypothesis that a virus infection alters cardiac energy metabolism and function by an antibody-mediated modulation of the function of the ANT.

Subcellular and tissue Mn compartmentation in bean leaves under Mn toxicity stress

Non-aqueous fractionation was used to characterize subcellular and tissue Mn compartmentation of mature and immature leaves of two common bean (Phaseolus vulgaris L.) cultivars contrasting in their response to Mn toxicity. Excess Mn decreases leaf CO2 assimilation through a reduction of chlorophyll content in immature leaves with no effect detected on mature leaves. We hypothesized that differential accumulation of Mn in chloroplasts occurs at different leaf developmental stages. Chloroplasts of immature leaves accumulated at least three times as much Mn as those of mature leaves at equivalent total foliar Mn. Chlorosis was positively correlated with Mn concentration in chloroplasts from high-Mn plants (r2 = 0.96; P = 0.003) but was not correlated with Mn in unfractionated tissue (r2 = 0.026; P = 0.793) nor with Mn in the epidermis-enriched fraction (r2 = 0.33; P = 0.314). Both cultivars showed high accumulation of Mn in the vacuoles as determined by the co-localization of α-mannosidase and Mn content on a continuous density gradient. Cultivars differed significantly in Mn concentration in an epidermis-enriched fraction, with the tolerant cultivar Calima accumulating more Mn in this fraction than the sensitive cultivar ZPV-292. In both cultivars, Mn was accumulated up to 2400 µg g–1 dry weight in crystal-type structures whereas the unfractionated leaf tissue contained about 500 µg g–1 dry weight. The results demonstrate that Mn compartmentation occurs at both the tissue and the organelle level and that Mn accumulation in the epidermis-enriched fraction could contribute to Mn tolerance in common bean. The role of Mn accumulation in structures resembling oxalate crystals is discussed.

The Use of Nonaqueous Fractionation to Assess the Ionic Composition of the Apoplast during Fruit Ripening.

We have examined the possibility that pectin solubilization and cell separation in fruit may be due to organic acids disrupting calcium bridges between pectic polysaccharides. With fruit from a wild tomato (Lycopersicon pimpinellifolium [Dunal]) we demonstrated the validity of a nonaqueous fractionation method to obtain reliable estimates of the ionic content of the apoplast. In unripe fruit no organic acids were associated with the cell wall, which contained 67% of the total calcium and 47% of the magnesium. In ripe fruit 4% of the malate, 10% of the citrate, and 15% of the oxalate were estimated to be in the cell wall, together with 84% of the calcium and 52% of the magnesium. In contrast to the cultivated tomato, we did not find a consistent decrease in the degree of methyl esterification between unripe and ripe fruit, and an overall average of 75% was observed. In the cell walls of ripe fruit the ratio of calcium:magnesium:organic acid:unesterified uronic acid, on the basis of charge, was 15:4:4:16. The use of a computer program to predict the proportions of different ionic species in complex mixtures suggested that in ripe fruit 70% of the unesterified uronic acid would be complexed with calcium. Our results show that organic acids do not accumulate in the cell wall sufficiently to disrupt calcium cross-linking, nor is the calcium removed from the wall into the cell. We therefore conclude that organic acids do not contribute to cell separation during the ripening of tomato fruit.

Antibody-Mediated Imbalance of Myocardial Energy Metabolism

The ADP-ATP carrier of the inner mitochondrial membrane is an autoantigen in myocarditis and dilated cardiomyopathy. Sera of patients with these diseases contain carrier-specific autoantibodies that inhibit the transmembrane nucleotide transport on isolated mitochondria. Guinea pigs immunized with the isolated ADP-ATP carrier protein also generate specific carrier-inactivating antibodies. In this study, we measured the cardiac function of guinea pigs immunized with the ADP-ATP carrier by determining the external heart work (EHW) of their isolated perfused spontaneously beating hearts stimulated by 4.0 mmol/L calcium and aortic ligature. Further, the electrogenic transport activity of the ADP-ATP carrier was estimated by calculating the cytosolic-mitochondrial difference of the phosphorylation potential of ATP [delta G(cyt-mit)] in the freeze-clamped isolated hearts by nonaqueous fractionation. The EHW of immunized guinea pigs was seen to be reduced by 54% (P < .005) compared with nonimmunized control guinea pigs, and delta G(cyt-mit) declined from 4.9 kJ/mol ATP in nonimmunized control hearts to 2.3 kJ/mol ATP in the hearts of the immunized guinea pigs (P < .005). The decisive result of this study, however, is the close relation observed between the magnitude of reduction of delta G(cyt-mit) and the size of the decrease in EHW (r = .87). Therefore, it seems plausible that antibody-mediated carrier dysfunction (creating the observed imbalance in myocardial energy metabolism) is responsible for the impairment of cardiac function. Our data support the hypothesis that immunopathic mechanisms in myocarditis and dilated cardiomyopathy can trigger subsequent heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of hexoses in leaf vacuoles: Studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol, vacuole or apoplasm

The subcellular distribution of hexoses, sucrose and amino acids among the stromal, cytosolic and vacuolar compartments was analysed by a nonaqueous fractionation technique in leaves of tobacco (Nicotiana tabaccum L.) wild-type and transgenic plants expressing a yeast-derived invertase in the cytosolic, vacuolar or apoplasmic compartment. In the wild-type plants the amino acids were found to be located in the stroma and in the cytosol, sucrose mainly in the cytosol and up to 98% of the hexoses in the vacuole. In the leaves of the various transformants, where the contents of hexoses were greater than in wild-type plants, again 97–98% of these hexoses were found in the vacuoles. It is concluded that leaf vacuoles contain transporters for the active uptake of glucose and fructose against a high concentration gradient. A comparison of estimated metabolite concentrations in the subcellular compartments of wild-type and transformant plants indicated that the decreased photosynthetic capacity of the transformants is not due to an osmotic effect on photosynthesis, as was shown earlier to be the case in transformed potato leaves, but is the result of a long-term dedifferentiation of tobacco leaf cells to heterotrophic cells.

Subcellular volumes and metabolite concentrations in barley leaves

Metabolite concentrations in subcellular compartments from mature barley (Hordeum vulgare L. cv. Apex) leaves after 9 h of illumination and 5 h of darkness were determined by nonaqueous fractionation and by the stereological evaluation of cellular and subcellular volumes from light and electron micrographs. Twenty one-day-old primary leaves of barley with a total leaf volume of 902 μL per mg chlorophyll were found to be composed of 27% epidermis, 42% mesophyll cells, 6% veins, 4.5% apoplast and 23% gas space. While in epidermal cells 99% of the volume was occupied by the vacuole, mesophyll cells with an average volume of 31.3 pL consisted of 23 pL (73%) vacuole, 4.6 pL (19%) chloroplasts, 2.06 pL (6,7%) cytosol (including smaller organelles and vesicles), 0.34 pL (1%) mitochondria and 107 fL (0.34%) nucleus. The differences between leaves harvested after 9 h of illumination and after 5 h of darkness were in the size of the stromal compartment and the starch grains therein. Subcellular metabolite concentrations were calculated from the compartmental volumes and metabolite contents of the compartments as determined by nonaqueous fractionation. The amino-acid concentrations in stroma and cytosol were rather similar after 9 h of illumination and 5 h of darkness. In contrast, the vacuolar amino-acid concentrations were about one order of magnitude lower than the stroma and cytosol values, and there was a slight increase in concentration after 5 h of darkness.

Pyrophosphate:Fructose-6-Phosphate 1-Phosphotransferase and Fructose 2,6-Bisphosphate in the Bundle Sheath of Maize Leaves

The aim of this work was to discover whether the cells of the bundle sheath of the leaves of maize ( Zea mays) contained pyrophosphate:fructose-6-phosphate 1-phos-photransferase (PFP) and fructose 2,6-bisphosphate (Fru-2,6-P 2). Physiologically active preparations of bundle sheath cells from leaves of 4- to 6-week-old plants showed activities of PFP, 6-phosphofructo-2-kinase (6-PF-2-K), and fructose-2,6-bisphosphatase (Fru-2,6-Pase) of 38, 1.8, and 15 nmol min −1 mg −1 chlorophyll, respectively, and contained 75 pmol mg −1 chlorphyll Fru-2,6-P 2. For the above enzymes, and marker enzymes for the bundle sheath and for mesophyll cells, the ratios of the activities in leaf extracts to those in bundle sheath extracts were determined. The ratios for PFP, 6-PF-2-K, Fru-2,6-Pase, and Fru-2,6-P 2 were intermediate between those found for the mesophyll markers and bundle sheath markers. The distribution of PFP activity after nonaqueous fractionation of leaves differed from that of the bundle sheath and mesophyll marker enzymes. It is argued that maize bundle sheaths can contain significant activity of PFP and amounts of Fru-2 ,6-P 2.

Nucleotide Sequence of a cDNA Clone Encoding a β-Amylase from Arabidopsis thaliana

,B-Amylase (EC 3.2.1.2) hydrolyzes a1,4-glucosidic linkages from the nonreducing ends of starch or glycogen, releasing maltose with the 3-anameric configuration. This enzyme occurs in plants and in certain bacteria; however, its physiological function in some plants is not clear because the enzyme and its substrates are spatially separated. In the leaves of a variety of plants including Arabidopsis thaliana (L.) Heynh. (3), 3-amylase is located outside the chloroplast (5, 7). In pea and wheat, 3-amylase appears to be confined to the vacuole (1 1). The A. thaliana 3-amylase is also located in the vacuole as determined by nonaqueous fractionation (our unpublished results). However, the only known substrates for ,-amylase, soluble and insoluble starch, are confined to the chloroplast (8). The A. thaliana 3-amylase is the most abundant of the three major leaf amylases, making up about 80% of the total crude amylolytic activity (3). In the course of studying starch metabolism in A. thaliana, leaves of several starchless and starch-overproducing mutants were found to contain 10to 40-fold more 3-amylase activity than wild-type leaves when the plants were grown under a 12-h/ 1 2-h light/dark cycle (1). Under continuous light, mutant and wild-type plants were indistinguishable. In addition, ,3-amylase activity was increased in both the wild type and mutants by growing plants under a higher light intensity. Using polyclonal antibodies raised to the purified A. thaliana 3-amylase, we determined that the ,3-amylase protein accumulated in the mutants and in plants grown under high light intensity (5). To investigate how 3-amylase expression is regulated in A. thaliana, we isolated five cDNA clones from a X-Zap expression library using immunoaffinity-purified anti-3-amylase IgG. Restriction mapping and partial sequence analysis indicated that the coding regions of all five cDNAs were identical (data not shown). Here, we present the sequence of the longest complete cDNA which contains 2469 bases and a single open reading frame encoding a protein of 498 amino acids (Table I, Fig. 1). The mol wt of the protein from the deduced amino acid sequence is 56,069, and the apparent molecular mass of the purified protein as determined from SDS-PAGE was 55,000 D (5). 3-Amylase sequences are known from three other plants: soybean (4), sweet potato (9), and barley (2). The A. thaliana 3-amylase sequence is 69% identical with the soybean sequence at the amino acid level. When amino acids with similar side groups are included in the comparison, the A. thaliana and soybean sequences are 79% similar. The A. thaliana sequence is 64 and 63% identical with, and 75 and 73% similar to, the sweet potato and barley sequences, respectively. The active sites of the soybean (6) and sweet potato (9) f-amylases were identified by modification of the proteins

Amino Acid and Sucrose Content Determined in the Cytosolic, Chloroplastic, and Vacuolar Compartments and in the Phloem Sap of Spinach Leaves

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.

Redox Transfer across the Inner Chloroplast Envelope Membrane

In leaves of spinach plants (Spinacia oleracea L.) grown in ambient CO(2) 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.