Apr 8, 1987 - pelleting material) by a piece of 5 ,m mesh (modification of. 360 ..... the appropriate compartments with sufficient activities to ac- count for starch ...
Plant Physiol. (1987) 85, 360-364
0032-0889/87/85/0360/05/$01.00/0
Localization of Carbohydrate Metabolizing Enzymes in Guard Cells of Commelina communis1 Received for publication April 8, 1987 and in revised form June 13, 1987
NINA L. ROBINSON2 AND JACK PREISS*3 Department of Biochemistry and Biophysics, University ofCalifornia, Davis, California 95616 ABSTRACI The lliztion of enzymes involved in the flow of carbon into and out of starch was determined in guard cells of Commelina communis. The guard cell chloroplasts were separated from the rest of the cellular components by a modification of published microfuge methods. The enzymes of interest were then assayed in the supernatant and chloroplast fractions. The chloroplast yield averaged 75% with 10% cytoplasmic contamination. The enzymes involved in starch biosynthesis, ADPglucose pyrophosphorylase, starch synthase, and branching enzyme, are located exclusively in the chloroplast fraction. The enzymes involved in starch degradation show a more complex distribution. Phosphorylase is located in both the supernatant and chloroplast fraction, 50% in each fraction. Most of the amylase and debranching enzyme activity is present in the supernatant (70%) fraction. The majority of the rest of the enzymes involved in the degradation of starch to malate and synthesis of starch from a hexose precursor were also investigated. All of the enzymes were present in the chloroplast except for hexokinase and phosphofructokinase. The inability to assay these enzymes could possibly have been due to the lack of or low activity of the enzymes or to nonoptimal assay conditions.
leaves. The sucrose is either degraded in the apoplast or in the cytoplasm of the storage cell. Sucrose, or its degradation products, can be further metabolized to the triose-P or 3-PGA level. These compounds may then move into the amyloplast via the triose-P/Pi translocator and are converted into starch. However, at present, the presence of the triose-P/Pi translocator in amyloplasts has not been demonstrated. Assuming that the triose-P/ Pi translocator is present, the movement of carbon into starch would be a reversal of the enzymic steps occurring in the cytoplasm with the last several steps resulting in the direct incorporation of carbon into starch. The above process will be reversed with starch degradation occurring and the products moving into the cytoplasm presumably via the triose-P/Pi translocator. Triose-P and 3-PGA are then further metabolized, as needed, in the cytoplasm. Using the amyloplast system as a model for guard cells, it was of interest to determine the enzyme activity of the starch biosynthetic and degradative pathways and their localization. A chloroplast and cytoplasmic (supernatant) fraction were obtained from guard cell protoplasts using a microfuge technique. Enzymes involved in the flow of carbon into and out of starch were then assayed in the two fractions.
MATERIALS AND METHODS Isolation of Guard Cell Protoplasts. The plant material was Commelina communis, 4- to 6-week-old plants. The abaxial One of the consequences ofstomatal opening is the breakdown epidermis of the leaves was peeled and placed in 0.25 M mannitol of starch during the day into malate and citrate, for use as an with 0.5 mMCaCl2 for at least 30 min. The peels were collected osmoticum. In the late afternoon or evening starch is resynthe- and digested for approximately 1 h in 2% cellulase, 10 mm Mes sized. This is the reverse of the situation in mesophyll cells. At (pH 5.3), 0.25 M mannitol, and 0.5 mm CaCl2 at 25°C on an the present time there is no evidence as to the source of carbon orbital shaker at 100 rpm. During this digestion any contamifor the resynthesis of starch due to the lack of Rubisco4 activity nating epidermal and mesophyll cells form protoplasts and burst (22) and the malate formed during opening is not converted due to the low osmoticum. The peels were collected on Miracloth back into starch (19). Based on this, what is the pathway for and transferred to a solution containing 2% cellulase, 0.5% starch synthesis in guard cells and in which of the compartments macerase, 10 mm Mes (pH 5.3), 0.4 M mannitol, and 0.5mm are the enzymes localized? These same questions can also be CaCl2. This digestion lasted for approximately 4 h at 25°C on an asked in reference to the starch degradation pathway. orbital shaker at 60 rpm. In storage tissue, which contains starch in amyloplasts, the The guard cell protoplasts were harvested by passing them source of carbon for starch synthesis is sucrose imported from through 20 ,um nylon mesh followed by centrifugation at 200g for 100 min. The supernatant was discarded and the pellet 'Supported in part by a National Science Foundation grant PCM82- resuspended in 2 ml of 0.4 M mannitol with 0.5 mm CaCl2. This 0570. was layered on a 6 ml discontinuous 22/67/90% Percoll gradient 2McKnight Foundation trainee. Present address: Mann Laboratory, (5) and centrifuged for 5 min at 400g. The guard cell protoplasts Department of Vegetable Crops, University of California, Davis, CA. were recovered from the 22/67% interface and rinsed with 0.4 3Present address: Department of Biochemistry, Michigan State Uni- mannitol, 10 mm Mes (pH 6.5), 1 mm EDTA, and 0.5 mM CaCl2 versity, East Lansing, MI 48824. (rinsing solution) and collected by a 10 min centrifugation at 4Abbreviations: Rubisco, ribulose 1,5-bisphosphate carboxylase/oxy- 200g. genase; ADPGlc, ADPglucose; RuBP, ribulose bisphosphate; PFK, phosIsolation of Guard Cell Chloroplasts. The pellet was resusphofructokinase; PFP, pyrophosphate:fructose-6-P phosphotransferase; pended in 0.3 ml of fresh rinsing solution. This solution was 3-PGA, glyceric acid 3-phosphate; PEP, phosphoenolpyruvate; Glc I-P, placed in an Eppendorf tube separated from 1 ml of 0.7 M glucose 1-phosphate; Fru 6-P, fructose 6-phosphate; Fru 1,6-bisP, fruc- sucrose, 50 mm Tricine (pH 7.9), and 0.5 mm CaCl2 (sucrose tose 1,6-bisphosphate; Fru 2,6-bisP, fructose 2,6-bisphosphate. pelleting material) by a piece of 5 ,m mesh (modification of 360
CARBOHYDRATE METABOLISM IN GUARD CELLS
both Refs. 15 and 26). This was centrifuged for 15 s in a microfuge. The supernatant was removed, containing most of the cell contents except the chloroplasts, and the pellet, mostly chloroplasts, resuspended in 1 ml of the rinsing solution. The enzyme assays were done on these two fractions. Enzyme Assays. Assay of Starch Biosynthetic Enzymes. ADPGlc pyrophosphorylase was assayed only in the pyrophosphorylase direction (24, 25). The reaction mixture contained 20 ,gmol of glycylglycine (pH 8.0), 1.5 JUmol of MgCl2, 0.25 umol of ADPGlc, 0.5 gmol 32PPi (1000-6000 cpm/nmol), 0.1 mg BSA, 0.2 ,mol 3-PGA, and extract in a final volume of 0.25 ml. The reaction mixture was incubated for 30 min at 37°C. Starch synthase was assayed as described by Hawker et al. (6). The specific activity of ADP-['4C]Glc was increased to 2000 cpm/ nmol and the incubation time was increased to 30 min. Branching enzyme activity was determined as described previously (6). In some experiments it was necessary to add a known amount of rabbit liver glycogen as primer. The reaction was terminated after 2 h at 30C. Assay of Starch Degradative Enzymes. Both amylase and Renzyme were assayed by determining the concentration of reducing sugars present at the completion of the reaction (18). Both reactions were terminated after 2 h at 37°C and the concentration of reducing sugars determined using Nelson's (16) reducing sugar assay. Hexokinase was assayed using two different methods (27, 29). The phosphorylase assay (18) was converted to a two-step fluorometric assay (14) because of the low activity of the enzyme. In the first step of the reaction, Glc 1-P was formed while in the second step the concentration of Glc 1-P was determined. The first step was incubated at 37C for 15 min. The second step went to completion at room temperature. Phosphoglucomutase was assayed spectrophotometrically (3). Assays of Glycolytic Enzymes. Aldolase was assayed spectrophotometrically over a 10 min period (10). Fructose bisphosphatase was assayed in two ways, fluorometrically and radioactively, using a modification of Kelly et al. (12). The fluorometric assay was converted to a two-step reaction. In the first step Fru 6-P was formed while in the second step the Fru 6-P concentration was fluorometrically determined. The radioactive assay used ['4C]Fru-1,6-bisP. After 10 min at room temperature the products of the reaction were determined using descending paper chromatography in ethanol: 1 M ammonium acetate (5:2, pH 3.8) solvent system and rechromatographed in butanol: propanol:acetone:80% formic acid:30% TCA (40:20:25:25:1.5) with 0.5 g of EDTA (100 ml solvent)-'. The location of the standards were determined (1). The location of the radioactivity was determined by counting 1 cm sections in a toluene based cocktail. PEP carboxylase was determined using a modification of Bahr and Jensen's (2) Rubisco assay. The reaction mixture contained 1 gmol of PEP in place of RuBP and the reaction was terminated after 10 min at room temperature. PFK was assayed spectrophotometrically (4, 11) with 25 gmol of Pi (pH 7.5) included when the cytoplasmic isozyme was assayed. Phosphoglucose isomerase was assayed spectrophotometrically ( 17). PFP was assayed as described previously (13). Glyceraldehyde 3-P dehydrogenase was assayed spectrophotometrically (8) as was triose-P isomerase (28). Production of/4JFru-1,6-BisP. The ['4C]Fru-1,6-bisP used in the radioactive fructose bisphosphatase assay was made from ['4C]Fru using yeast hexokinase (Sigma) and rabbit muscle Fru 6-P kinase (Sigma) in a two-step procedure. The reaction mixture for the hexokinase reaction contained 50 smol of Tris-HCl (pH 8.0), 1 ,umol of frutose, 10 Mmol of ATP, 10 ,umol of MgCl2, 1.7 units of hexokinase, and 120 uCi of ['4C]frutose in 1 ml. After 15 min at room temperature 0.02 unit of Fru 6-P kinase was added and the reaction proceeded for an additional 30 min at room temperature. The reaction mixture was chromatographed
361
overnight in ethanol: 1 M ammonium acetate (pH 3.8, 5:2) after adding sufficient EDTA to make the concentration 20 Mmol. This was done to improve the resolution. Standards of frutose, Fru 6-P, and Fru 1,6-bisP were run at 0.1 smol and the location determined with silver nitrate dip (1). The second reaction did not go to completion so the label was present in both Fru 1,6bisP and Fru 6-P. Both radioactive spots were eluted after soaking the chromatograms in 100% ethanol for 3 h. Protein was determined using the BCA protein reaction from Pierce with crystalline BSA as the standard. The cellulase used in the digestion was obtained from Cooper Biomedical and the macerase was obtained from Calbiochem. The 5 and 20 Mm meshes were obtained from Spectrum Medical Industries, Inc. The ['4CJfrutose and ["4C]Glc 1-P were both obtained from Amersham. The ADP['4C]glc was produced as described by Hawker et al. (6). The coupling enzymes that were used in the spectrophotometric and fluorometric assays and the Percoll were obtained from Sigma. Those enzymes suspended in ammonium sulfate that were used in the same reaction mixture as the extracts were dialyzed overnight in 50 mM Hepes (pH 7.5), 1 mM EDTA, and 1 mM GSH or DTE. Rabbit muscle phosphorylase a, used in the branching enzyme assay, was obtained from BoehringerMannheim. RESULTS AND DISCUSSION Several methods were tried before the microfuge method was found to provide the highest chloroplast yields and lowest cytoplasmic contamination in the chloroplast fraction of a guard cell protoplast C. communis preparation. The chloroplast yield and cytoplasmic contamination were determined by assaying the activity of a cytoplasmic, PEP carboxylase, and chloroplastic, ADPGlc pyrophosphorylase, marker in both fractions. ADPglc pyrophosphorylase was used as the chloroplast marker due to the lack of Rubisco activity in guard cells (22). The chloroplast yield is the percentage ofthe ADPGlc pyrophosphorylase activity present in the chloroplast fraction. The cytoplasmic contamination is the percent of PEP carboxylase activity present in the chloroplast fraction. The microfuge method described above resulted in 74% chloroplast yield (n = 48; SD = 6.2) and 10% cytoplasmic contamination (n = 36; SD = 3.6) of the chloroplast fraction. Starch Biosynthetic Pathway. Results. The enzymes involved in the conversion of Glc 6-P to triose-P, and 3-PGA in the cytoplasm were assayed (Table I) and found to be present in the cytoplasm. PFK and PFP catalyze the conversion of Fru 6-P to Fru 1,6-bisP. The highest activities for PFP were obtained only in the presence of the activator, Fru 2,6-bisP. In the absence of Fru 2,6-bisP there was a 75% decrease in activity. The triose-P/Pi translocator found in the chloroplast membrane of mesophyll cells exchanges triose-P or 3-PGA for phosphate into or out of the chloroplasts (7). Presumably this translocator also functions in guard cells. Once the three-carbon units are inside the chloroplast they could be converted into starch by a partial reversal of the steps outlined above in the cytoplasm. The activity of the chloroplastic isozymes of NADH triose-P dehydrogenase, triose-P isomerase, and aldolase are lower than for their cytoplasmic isozymes. The conversion of Fm 1,6-bisP to Fru 6-P is catalyzed by fructose bisphosphatase. Fructose bisphosphatase was difficult to assay because of its low activity. Two methods were used as assays. A two-step fluorometric assay monitoring the production of NADH and a radioactive assay. When the radioactive assay was used the product of the reaction was shown to be Fru 6-P. The enzymes necessary for the conversion of Fru 6-P to Glc I-P, the substrate for ADPGlc pyrophosphorylase, were present in the chloroplast. The last steps in the starch biosynthetic pathway result in the direct incorporation of carbon into starch. The first enzyme in
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ROBINSON AND PREISS
Plant Physiol. Vol. 85, 1987
Table I. Distribution and Activity of Enzymes Involved in the Flow of Carbon into and out of Starch in Guard Cells of C. communis The protoplasts and chloroplasts were prepared by modification and combination of Miills and Joy (15) and Shimazaki and Zeiger (26).
Enzyme
Supernatant
Chloroplast
Percent in
nmol (mg protein. min)-'
Marker Enzymes PEP carboxylase ADPglucose pyrophosphorylase Starch Degradative Enzymes Amylase Phosphorylase R-enzyme Starch Biosynthetic Enzymes ADPglucose pyrophosphorylase Starch synthase Branching enzyme 1 mgprimer 0.01 mg primer Glycolytic Enzymes Aldolase Fructose-1,6-bisphosphatase Phosphoglucose isomerase Phosphoglucomutase PFK PFP
96.5 11.3
10.0 42.8
10.4 (36)a 73.7 (48)
29.5 41.8 22.1
16.3 (2) 34.0 (2) 6.2 (3)
34.5 (6)b 42.8 (6)b 24.7 (6)b
11.3 0.4
42.8 1.8 (2)
73.7 (48) 76.8 (4)b
78.5 42.3
324.4(1) 136.5 (1)
69.0 ( I)b
27.0 7.6 283 192 8.9 9.5
9.1 10.4 46.0 52.5
