(Hordeum vulgare L.) Etioplasts'

Plant Physiol. (1992) 100, 184-190 0032-0889/92/100/01 84/07/$01 .00/0

Received for publication October 22, 1991 Accepted April 20, 1992

Transport Processes and Corresponding Changes in Metabolite Levels in Relation to Starch Synthesis in Barley (Hordeum vulgare L.) Etioplasts' Olaf Batz, Renate Scheibe, and H. Ekkehard Neuhaus* Lehrstuhl fur Pflanzenphysiologie, Universitat Osnabruck, Barbarastrasse 11, D-4500 Osnabruck, Federal Republic of Germany ABSTRACT Intact etioplasts with an intactness of 85% and with a cytosolic and a mitochondrial contamination of less than 10% were isolated from 8-d-old dark-grown barley (Hordeum vulgare) leaves. These plastids contained starch equivalent to 21.5 Amol of glucose per mg protein. From various likely precursors applied to isolated etioplasts, only dihydroxyacetone phosphate (DHAP) had significant effects on metabolite levels and on the internal ATP/ADP ratio. The concentration dependence of DHAP uptake exhibited saturation characteristics with half saturation at 0.36 mM DHAP and a maximal velocity of 6.6 Mmol mg-1 of protein h-1. The transport was significantly inhibited by inorganic phosphate, pyridoxal-5'-phosphate, and 4,4'-diisothiocyano-2,2'-stilbenedisulfonate. The rate of glucose-6-phosphate uptake was much lower and not saturable up to a concentration of 10 mm. Exogenously applied [14C]DHAP was incorporated into starch at a rate of 0.14 ,gmol of DHAP mg-1 of protein h-1. Enzyme activities required to convert DHAP into starch were found to be present in etioplasts. Furthermore, enzymes generating ATP from DHAP for ADPglucose synthesis were also detected. Finally, a scheme is presented suggesting DHAP uptake to serve both as carbon skeleton and as energy source for starch synthesis, mediated by a translocator with properties similar to those of the triose phosphate translocator from chloroplasts.

phosphates are possible precursors for starch synthesis in heterotrophic tissues. In addition, it is not yet clear how ATP required for the synthesis of ADPGlc2 is generated inside the plastids. The methods of examination used so far are isolation of plastids and estimation of their enzymic content (6), randomization experiments using position-labeled precursor and subsequent analysis of newly synthesised starch (10), and direct uptake experiments using isolated plastids (13). Until now no attempt has been made to analyze the formation of etioplastic starch, which are functionally heterotrophic because carbon as well as energy need to be imported for starch synthesis (29). Here, an attempt is made to characterize the processes involved in starch synthesis in etiolated barley (Hordeum vulgare) leaves comprising the following aspects: (a) transport of precursor metabolites across the intact membrane, (b) changes of metabolite levels within the plastids, (c) estimation of the rate of starch synthesis, and (d) detection and estimation of the relevant enzyme equipment. MATERIALS AND METHODS

Plant Material

Barley (Hordeum vulgare L. cv Roland) was purchased from a local distributor (Kobe and Hopfer, Osnabrick, FRG). The grains (approximately 25 g, previously soaked in water for 14 h) were transferred to 20- x 40-cm plastic boxes containing a layer of vermiculite and saturated with water. The boxes were kept dark at 80 to 90% RH at 210C. After 8 d, the plants were harvested by cutting 4 cm above the ground.

Compartmentation of the complex metabolic pathways and controlled transport processes across organelle membranes are required to maintain regulation. The transport of carbohydrate across the inner envelope membrane of isolated chloroplasts is well known. Export of carbohydrate occurs via the chloroplastic phosphate-triose phosphate translocator (9). In addition, there is evidence for a glucose transport system with low substrate affinity (25). Furthermore, a hexose phosphate transport system in the chloroplasts of Sedum (20) and Codium (24) has been described. Information about transport processes involved in starch synthesis in plastids of heterotrophic tissue is scarce due to the fact that the isolation of these starch-containing organelles is extremely difficult (6). Various attempts have been made to determine which carbohydrate is transported across the envelope of nongreen plastids, showing that hexoses, hexose phosphates, and triose '

Isolation of Intact Etioplasts Approximately 200 g of barley leaves were cut into pieces 5 mm long and transferred to 600 mL of ice-cold isolation

2Abbreviations:

ADPGlc, ADPglucose; ADPGlc-PPiase, ADPgl-

ucose pyrophosphorylase; DHAP, dihydroxyacetone phosphate;

DIDS, 4,4'-diisothiocyano-2,2'-stilbenedisulfonate; FBPase, fructose-1,6-bisphosphatase; Fru6P, fructose-6-phosphate; Frul,6P, fructose-1,6-bisphosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GlclP, glucose-i-phosphate; Glc6P, glucose-6phosphate; PFP, pyrophosphate: fructose-6-phosphate phosphotransferase; 3PGA, 3-phosphoglycerate; PLP, pyridoxal-5'-phosphate; UDPGlc-PPiase, UDPglucose pyrophosphorylase.

Supported by the Deutsche Forschungsgemeinschaft, SFB 171,

C16.

184

METABOLITE LEVELS AND STARCH SYNTHESIS IN BARLEY

medium consisting of 330 mm sorbitol, 5 mM MgCl2, 10 mM sodium PPi, 2 mm ascorbate, adjusted with HCl to pH 6.5 (buffer A). The material was homogenized in a Waring Blendor with three strokes for 3 s each. The preparation was gently squeezed through four layers of cheesecloth and one layer of nylon gauze (30-,um mesh). The filtrate was centrifuged for 1 min at 600g in a Sorvall SS34 rotor at 40C. The supematant was discarded, and the pellet was resuspended in 20 mL of medium containing 330 mm sorbitol, 50 mM Hepes-KOH (pH 7.6), 2 mm EDTA, 1 mM MgC12, 1 mm MnCl2 (buffer B). For further purification, the etioplast suspension was centrifuged a second time (1 min, 600g, 40C), and the resulting pellet was suspended in 1 to 2 mL of buffer B to yield a protein concentration of 1 to 2 mg-ml-'. Protein was measured using a dye-binding assay with Coomassie brilliant blue (Serva). To check the purity of intact etioplast preparations, enzyme activities specific for different cell compartments were measured in crude extracts and in purified etioplasts (UDPGlcPPiase and PFP for cytosol, citrate synthase for mitochondria, and ADPGlc-PPiase for etioplasts). Crude extracts were prepared by grinding 0.1 g of etiolated barley leaves in a mortar containing 1 mL of a medium that consisted of 50 mm HepesKOH (pH 7.4), 5 mM MgCl2, 2 mm MnCl2, 1 mm EDTA, 1 mM EGTA, 0.1% Triton X-100 (40C). The resulting suspension was centrifuged (2 min, 16,000g), and aliquot amounts of the supematant were stored in liquid nitrogen until measurements were carried out. The influence of allosteric effectors on ADPGlc-PPiase activity was measured using a crude enzyme extract. This extract was obtained using an NAP-5 column (Pharmacia, Sweden) used according to the supplier's instruction. Intactness of Isolated Etioplasts

Intactness of isolated plastids was estimated by performing latency experiments according to the method of Entwistle and ap Rees (6). ADPGlc-PPiase activity was determined under isoosmotic conditions (addition of 330 mm sorbitol to the assay) and in ruptured etioplasts. For complete lysis, etioplasts were treated with the small tip of a sonicator with three strokes for 5 s each before the measurement.

185

subsequent analysis (27). Recoveries were: Glc6P, 109.6 ± 0.63%; Glc1P, 78.1 ± 0.55%; Fru6P, 97.6 ± 3.17%; ATP, 97.6 ± 0.78%; ADP, 94.7 ± 1.48%; 3PGA, 106.0 ± 3.5% (mean ± SE, n = 4). Metabolite measurements were carried out on a spectrophotometer (Aminco DW 2a) operating in double-wavelength mode according to the method of Lowry and Passonneau (16).

Starch Measurement Starch was measured spectrophotometrically as glucose units after hydrolysis. Etioplasts corresponding to 50 jig of protein were diluted to a final volume of 500 ,uL with a 50 mm sodium acetate-HCl buffer, pH 4.7. After the material was autoclaved for 3 h at 1400C and then cooled, a-amylase and amyloglucosidase (3 units each) were added. After the material was incubated for 3 h at 370C, the reaction was stopped by transfer of the reaction vessels to a boiling water bath for 3 min. After the vessels were cooled on ice, 50 to 100 ,uL of the solution were added to a cuvette containing 100 mM Hepes-KOH (pH 7.8), 5 mM MgCl2, 1 mm NADP+, 1 mM ATP, Glc6P dehydrogenase (1 unit/mL), hexokinase (1 unit/mL). Samples treated similarly but in the absence of hydrolyzing enzymes and controls without etioplasts were found to contain no glucose.

Synthesis of [14CJDHAP

[I4C]DHAP was synthesized according to the method of Tyson and ap Rees (28). ["4C]Glycerol-3-P (Amersham CFB 171, 0.139 MBq corresponding to 75 ,uL) was diluted to 300 ,uL in a buffer medium consisting of 50 mm Hepes-KOH (pH 8.0) and incubated for 10 min with 10 units of glycerol-3-P oxidase and 2600 units of catalase. The reaction was stopped by transfer to a boiling water bath for 3 min. Assays of glycerol-3-P and DHAP (according to procedure in ref. 16) revealed that 98.1% (±0.6%, n = 4) of the glycerol-3-P was oxidized to DHAP. The solution was diluted with unlabeled DHAP to the final specific activities as given below. Transport Experiments

Metabolite Measurements Etioplasts (approximately 1 mg of protein) were incubated in a reaction vessel for 10 min (200C) in buffer B in the presence or absence of 10 mm DHAP. Metabolism was stopped by addition of a mixture of HCl04 (final concentration, 7%) and EDTA (final concentration, 10 mm). After incubation on ice for 10 min, the precipitated protein was removed by centrifugation, and the supematant was neutralized by successive addition of 1 M triethanolamine, 5 M KOH according to the method of Stitt et al. (27). The resulting KCl04 precipitate was removed by centrifugation for 5 min at 16,000g. The clear supematant was transferred to a new reaction vessel and stored in liquid nitrogen until measurements were carried out. Recoveries for all measured metabolites were estimated after addition of a mixture containing a 2- to 3-fold excess over the endogenous metabolites and

For short-term transport experiments using isolated etioplasts, the silicon oil filtration technique according to the method of Heldt and Sauer (12) was applied. Eppendorf reaction vessels (400 uL) were filled with three layers. The lower layer was 50 ,uL of 700 mM sucrose containing 7% HCl04; the middle layer was 100 uL of silicon oil AR 200 (Wacker-Chemie, Munich, FRG); the upper one consisted of etioplasts equivalent to 50 yg of protein diluted with buffer B up to a final volume of 200 ,L containing different concentrations of ['4C]DHAP (specific activity, 25.9 MBq/mmol) or ['4C]Glc6P (specific activity, 30 MBq/mmol). The incubation was carried out at room temperature for 20 s and was terminated by centrifugation for 20 s in a Beckman Microfuge E equipped with a horizontal rotor. The transport rates were corrected for the radioactivity trapped in the sorbitol-permeable (intermembrane) space.

BATZ ET AL.

186

SDS-PAGE, Western Blotting, and Immunoprinting

Estimation of Sorbitol-Permeable Space Etioplasts

were

incubated with ["4C]sorbitol and tritiated

water with specific activities of 0.112 and of 0.067 MBq/ mmol, respectively. The assay was carried out as described

by Heldt (11).

Synthesis of Starch from ['4C]DHAP Isolated etioplasts (about 50 ,ug of protein) were incubated (250C, 30 min) in buffer B containing 10 mm ['4C]DHAP (specific radioactivity, 4.6 MBq/mmol). The reaction was stopped by transfer of the reaction vessels to boiling water for 5 min. To remove labeled soluble compounds, the insoluble fraction was sedimented by centrifugation at 16,000g for 5 min and subsequently washed three times with doubledistilled water until no radioactivity was detectable in the supematant. Finally, the pellet was resuspended with 200 gL of 50 mm sodium acetate-HCl, pH 4.7, and autoclaved for 3 h (1400C) to solubilize starch. This solution was incubated for at least 3 h (370C) with 3 units of a-amylase and 3 units of amyloglucosidase to hydrolize starch into glucose units. The reaction was stopped by transfer to a boiling water bath (3 min). After cooling, the solution was incubated for 15 min with 10 units of hexokinase, 10 mm ATP in 50 mm HepesKOH (pH 7.8), 5 mM MgCl2 to activate hexokinase (final volume, 1.4 mL). The resulting ['4C]Glc6P was separated from other radioactively labeled material by anion exchange chromatography (15). The radioactivity in the anionic fraction containing [14C]Glc6P was determined, and the rate of starch synthesis was calculated with respect to the supplied specific radioactivity.

Enzyme Activity Measurements Enzyme activity measurements were carried out at 250C an Eppendorf spectrophotometer. ADPGlc-PPiase measurements were carried out as described by Sowokinos (26). FBPase was measured according to the method of Entwistle and ap Rees (6) with 80 mm ,B-mercaptoethanol in the assay mixture. NADP-GAPDH was measured according to the procedure of Winter et al. (30). PFP was assayed as described by Neuhaus et al. (19). Citrate synthase was measured as described by Stitt et al. (27). UDPGlc-PPiase, aldolase, triose phosphate isomerase, pyruvate kinase, phosphoglucose isomerase, phosphoglucose mutase, phosphoglycerate mutase, and phosphoglycerate kinase were measured according to the method of Bergmeyer (1). in

The protein samples were subjected to SDS-PAGE, blotted a polyclonal serum against spinach chloroplast FBPase raised in a rabbit (dilution 1:400). For detection, the blot was incubated with a goat antirabbit immunoglobulin G alkaline phosphatase conjugate. The color reaction was obtained with naphtole-AS-E-phosphate/Fast Violet B as substrate. onto nitrocellulose, and incubated with

RESULTS AND DISCUSSION

Properties of the Isolated Etioplasts

Etioplasts of high intactness and purity were isolated routinely from dark-grown barley leaves. Using ADPGlc-PPiase latency as a marker for intactness of the plastids revealed that the preparations contained at least 85% (SE 1.8%, n = 8) intact etioplasts. Measurement of marker enzymes for other cell compartments resulted in the finding that cytosolic contamination was 6.4% (SE 0.7%, n = 5) or 9.2% (SE 0.8%, n = 4) using UDPGlc-PPiase and PFP, respectively, as marker enzymes. Mitochondrial contamination was not detectable (values detected were below 1.2% of the citrate synthase activity in crude extracts) in the etioplast preparations as determined by measuring citrate synthase activity (Table IB). The sorbitol-impermeable space of the isolated barley etioplasts was determined to be 3.26 ,uL (SE 0.09 ,L, n = 9) per mg of protein. This was 38% of the total etioplast volume (tritiated water-permeable space). The ratio of stroma to

Table I. Enzyme Activities in Crude Extracts and Isolated Etioplasts from Dark-Grown Barley Leaves A, Enzyme activities of the glycolytic and the gluconeogenetic pathway in etioplasts. Percentage of activity was calculated according to the formula given in "Materials and Methods." B, Marker enzyme activities were used to determine the cytosolic (UDPGlcPPiase, PFP) and mitochondrial (citrate synthase) contamination. ADPGlc-PPiase was measured under Vmax conditions according to the method of Sowokinos (26). NADP-GAPDH is used as the plastidic marker enzyme activity (28). Activity Enzyme

Crude extract Etioplast preparation units -mg-' of protein % of total

A Triose phosphate isomerase NADP-GAPDH

3PGA-kinase 3PGA-mutase Pyruvate kinase Aldolase

Estimation of Plastidic Enzyme Localization Enzyme activities were measured in the crude extract (CE) and in the etioplast preparation (EP). The percentage of the total activity localized in the plastidic fraction was calculated according to the following equation: NADP-GAPDH (CE) Enzyme (EP) NADP-GAPDH (EP) Enzyme (CE) NADP-GAPDH activity (28).

was

used

as

Plant Physiol. Vol. 100, 1992

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the plastidic marker

100

FBPase

Phosphoglucoisomerase Phosphoglucomutase ADPGlc-PPiase

6.63 0.26 3.03 0.15 0.02 0.16 0.04 0.21 0.51 0.14

11.5 100 69.0 20.1 9.1 44.0 35.8 15.8 82.6 97.9

2.17 0.21 0.02

0.18 0.017

6.4 9.2