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 ofBiochemistry and Biophysics, University ofCalifornia, Davis, California 95616 ABSTRACI leaves. The sucrose is either degraded in the apoplast or in the cytoplasm of the storage cell. Sucrose, or its degradation prod- The lliztion ofenzymes involved in the flow of carbon into and out ucts, can be further metabolized to the triose-P or 3-PGA level. of starch was determined in guard cells of Commelina communis. The These compounds may then move into the amyloplast via the guard cell chloroplasts were separated from the rest of the cellular triose-P/Pi translocator and are converted into starch. However, components by a modification of published microfuge methods. The at present, the presence of the triose-P/Pi translocator in amy- enzymes of interest were then assayed in the supernatant and chloroplast loplasts has not been demonstrated. Assuming that the triose-P/ fractions. The chloroplast yield averaged 75% with 10% cytoplasmic Pi translocator is present, the movement of carbon into starch contamination. The enzymes involved in starch biosynthesis, ADPglucose would be a reversal of the enzymic steps occurring in the cyto- pyrophosphorylase, starch synthase, and branching enzyme, are located plasm with the last several steps resulting in the direct incorpo- exclusively in the chloroplast fraction. The enzymes involved in starch ration of carbon into starch. The above process will be reversed degradation show a more complex distribution. Phosphorylase is located with starch degradation occurring and the products moving into in both the supernatant and chloroplast fraction, 50% in each fraction. the cytoplasm presumably via the triose-P/Pi translocator. Most of the amylase and debranching enzyme activity is present in the Triose-P and 3-PGA are then further metabolized, as needed, in supernatant (70%) fraction. The majority of the rest of the enzymes the cytoplasm. involved in the degradation of starch to malate and synthesis of starch Using the amyloplast system as a model for guard cells, it was from a hexose precursor were also investigated. All of the enzymes were ofinterest to determine the enzyme activity ofthe starch biosyn- present in the chloroplast except for hexokinase and phosphofructoki- thetic and degradative pathways and their localization. A chlo- nase. The inability to assay these enzymes could possibly have been due roplast and cytoplasmic (supernatant) fraction were obtained to the lack of or low activity of the enzymes or to nonoptimal assay from guard cell protoplasts using a microfuge technique. En- conditions. zymes involved in the flow ofcarbon 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 ofthe consequences ofstomatal opening is the breakdown epidermis ofthe 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 contami- for 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 ofthe 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 ofCalifornia, 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, phos- Isolation of Guard Cell Chloroplasts. The pellet was resus- phofructokinase; 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 361 both Refs. 15 and 26). This was centrifuged for 15 s in a overnight in ethanol: 1 M ammonium acetate (pH 3.8, 5:2) after microfuge. The supernatant was removed, containing most of adding sufficient EDTA to make the concentration 20 Mmol. the cell contents except the chloroplasts, and the pellet, mostly This was done to improve the resolution. Standards of frutose, chloroplasts, resuspended in 1 ml of the rinsing solution. The Fru 6-P, and Fru 1,6-bisP were run at 0.1 smol and the location enzyme assays were done on these two fractions. determined with silver nitrate dip (1). The second reaction did Enzyme Assays. Assay of Starch Biosynthetic Enzymes. not go to completion so the label was present in both Fru 1,6- ADPGlc pyrophosphorylase was assayed only in the pyrophos- bisP and Fru 6-P. Both radioactive spots were eluted after soaking phorylase direction (24, 25). The reaction mixture contained 20 the chromatograms in 100% ethanol for 3 h. ,gmol ofglycylglycine (pH 8.0), 1.5 JUmol ofMgCl2, 0.25 umol of Protein was determined using the BCA protein reaction from ADPGlc, 0.5 gmol 32PPi (1000-6000 cpm/nmol), 0.1 mg BSA, Pierce with crystalline BSA as the standard. The cellulase used 0.2 ,mol 3-PGA, and extract in a final volume of 0.25 ml. The in the digestion was obtained from Cooper Biomedical and the reaction mixture was incubated for 30 min at 37°C. Starch macerase was obtained from Calbiochem. The 5 and 20 Mm synthase was assayed as described by Hawker et al. (6). The meshes were obtained from Spectrum Medical Industries, Inc. specific activity of ADP-['4C]Glc was increased to 2000 cpm/ The ['4CJfrutose and ["4C]Glc 1-P were both obtained from nmol and the incubation time was increased to 30 min. Branch- Amersham. The ADP['4C]glc was produced as described by ing enzyme activity was determined as described previously (6). Hawker et al. (6). The coupling enzymes that were used in the In some experiments it was necessary to add a known amount spectrophotometric and fluorometric assays and the Percoll were of rabbit liver glycogen as primer. The reaction was terminated obtained from Sigma. Those enzymes suspended in ammonium after 2 h at 30C. sulfate that were used in the same reaction mixture as the extracts Assay ofStarch Degradative Enzymes. Both amylase and R- were dialyzed overnight in 50 mM Hepes (pH 7.5), 1 mM EDTA, enzyme were assayed by determining the concentration ofreduc- and 1 mM GSH or DTE. Rabbit muscle phosphorylase a, used ing sugars present at the completion of the reaction (18). Both in the branching enzyme assay, was obtained from Boehringer- reactions were terminated after 2 h at 37°C and the concentration Mannheim. ofreducing sugars determined using Nelson's (16) reducing sugar assay. Hexokinase was assayed using two different methods (27, RESULTS AND DISCUSSION The assay was converted to a 29). phosphorylase (18) two-step Several methods were tried before the microfuge method was fluorometric assay (14) because ofthe low activity ofthe enzyme. found to provide the highest chloroplast yields and lowest cyto- In the first step of the reaction, Glc 1-P was formed while in the plasmic contamination in the chloroplast fraction ofa guard cell second step the concentration of Glc 1-P was determined. The communis first was incubated at for 15 protoplast C. preparation. The chloroplast yield and step 37C min.