Plant Physiol. (1989) 89, 557-5630032-0889/89/89/0557/07/$01 .00/0

Received for publication April 14, 1988and in revised form September 9, 1988

Evidence for a Light Dependent Increase ofPhosphoglucomutase Activity in Isolated, Intact Spinach

Chloroplasts

Richard C. Sicher

U.S. Department of Agriculture, Agricultural Research Service, Plant Photobiology Laboratory, BeltsvilleAgricultural Research Center, Beltsville, Maryland 20705

ABSTRACT

Phosphoglucomutase (PGM) activity was measured in spinach(Spinacia oleracea L.) chloroplasts. Initial enzyme activity in achloroplast lysate was 5 to 10% of total activity measured with 1micromolar glucose 1,6-bisphosphate (Glcl,6-P2) in the assay.Initial PGM activity increased 2- to 3-fold when chloroplasts wereilluminated for 10 minutes prior to enzyme measurement and thendecreased slowly in the dark. Measurements of total enzymeactivity were unchanged by prior light treatment. Inifial PGMactivity from light treated chloroplasts was sufficient to accountfor in vivo rates of starch synthesis. Changes in PGM activitywere affected by stromal pH and orthophosphate concentration.Photosynthetic inhibitors, DL-glyceraldehyde, glycolaldehyde,and glyoxylate, decreased and 3-phosphoglyceric acid increasedlight induced changes of PGM activity. Dark preincubation ofchloroplasts with 10 millimolar dithiothreitol had no effect uponinitial PGM activity, suggesting that light effects did not involve asulfhydryl mechanism. Hexose monophosphate levels increasedin illuminated chloroplasts. Activation of PGM in a chloroplastlysate by Glcl,6-P2 was maximal between pH 7.5 and 8.5. Stromalconcentrations of Glcl,6-P2 were between 20 and 30 micromolarfor both light and dark incubated chloroplasts and these levelsshould saturate PGM activity. Light dependent alterations ofenzyme activity may be due to changes of phosphorylated PGMlevels in the stroma or are the result of changes in residualactivity by the dephosphorylated form of the enzyme. The aboveresults indicate that PGM activity in spinach chloroplasts may beregulated by light, stromal pH, and Glc1,6-P2 concentration.

Stromal and cytosolic isoforms ofPGM' (EC 2.7.1.41) havebeen identified and separated using spinach leaf extracts (18).Both isozymes synthesize Glc 1-P from Glc6-P when function-ing in the gluconeogenic direction. In the stroma, Glc 1-P is asubstrate for ADP-Glc pyrophosphorylase and is an interme-diate of the starch pathway (20). Homogeneous preparationsof PGM have been obtained from animal tissue (5, 14, 21),as well as from potato tuber (24) and yeast (13). The enzymefrom all sources is activated by ,uM amounts of Glc 1,6-P2 (2).

'Abbreviations: PGM, phosphoglucomutase; Glcl-P, glucose 1-phosphate; Glc6-P, glucose 6-phosphate; ADP-Glc, adenosine 5'-diphosphoglucose; Glc1,6-P2, glucose 1,6-bisphosphate; PPFD, pho-tosynthetic photon flux density; Fru6-P, fructose 6-phosphate; Pi,inorganic phosphate; 3-PGA, 3-phosphoglyceric acid; OAA, oxala-cetic acid.

Although the actual enzyme mechanism is uncertain, it isclear that Glc1,6-P2 functions as an obligate cofactor in thePGM reaction (2, 4, 19, 21). Unlike the mammalian enzyme,extractable PGM activity from plant sources is negligible inthe absence of Glc1,6-P2 (4, 9, 18, 24). Beitner (2) reportedthat Glc 1,6-P2 was detectable in plant tissues but did notpresent data supporting this observation. Although PGMcatalyzes a reversible reaction in vitro, there is evidence thatthe enzyme in vivo is slightly displaced from equilibrium (7).Clearly, PGM has properties suggesting that it could be animportant control point in photosynthetic carbon flow.At least six spinach chloroplast enzymes exhibit altered

activity in the light. These chloroplast enzymes are activatedor inactivated either by a thioredoxin-mediated sulfhydrylreduction or by light-driven changes of stromal pH and cationconcentration (3, 13). It is currently accepted that light de-pendent enzymes are important in activating CO2 fixationand other photosynthetic processes (17). At present, there isno information suggesting that any enzyme involved in starchsynthesis is activated by light. This observation may be con-sidered unusual in view of the overall importance of lightdependent enzyme activation to photosynthetic metabolism.Results of the present study suggest that spinach chloroplastPGM activity increases in the light.

MATERIALS AND METHODS

Plant Material

Chloroplasts were isolated from mature leaves of 4- to 5-week-old spinach plants (Spinacia oleracea var. Viroflay) aspreviously described (15). The washed chloroplast pellet (80-90% intact) was resuspended in a small volume of 330 mMsorbitol, 50 mm Mes-NaOH (pH 6.7), 2 mm EDTA, 1 mMMgCl2, and 1 mm MnCl2 and was stored on ice. Plants wereraised from seed in controlled environment chambers provid-ing 700 ,umol m-2 s-', PPFD, with a 9 h photoperiod, a 23°Cday/18°C night cycle and 65% relative humidity. Plants wereraised in large plastic trays containing vermiculite and werewatered daily with complete mineral nutrient solution (22).

Light Treatments and Photosynthesis Measurements

Photosynthetic 02 evolution was measured polarographi-cally with a Clark-type electrode at 25°C in a water-jacketedvessel. Reactions (1.8 mL volume) usually contained 330 mM

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Plant Physiol. Vol. 89, 1989

sorbitol, 50 mM Hepes-NaOH (pH 7.8), 2 mM EDTA, mMMgCl2, mm MnCl2, 0.5 mM K2HPO4 and with other con-ditions as indicated in the text. An irradiance of 600 ,umolm-2 s-1, PPFD, was provided using a 500 W projector lamp.

Enzyme Assays

Enzyme measurements were usually performed at 27°C byinjecting 20 to 50 uL of the chloroplast suspension directlyinto mL reaction mixture containing 50 mM Hepes-NaOH(pH 7.6), 1 mM MgCl2, 0.2 mm NAD+, and unit of Glc6-Pdehydrogenase from Leuconostoc mesenteroides. Assays wereinitiated with 2 mm Glcl-P and increased absorbance was

measured at 340 nm. After measuring initial PGM activity,total enzyme activity for each sample was determined using1 or 2 ,M Glc 1,6-P2. Deviations from the standard assay are

indicated in the text.

Metabolite Measurements

Extraction and analysis of Glcl,6-P2 from whole leaf andisolated spinach chloroplasts was similar to methods describedfor muscle tissue (1). Leaf material (0.2 g fresh weight) andisolated chloroplasts (about 0.3 mg Chl) were extracted with5% (v/v) HC104 at 0°C. Samples were centrifuged at 5OOg for10 min and the supernatants were neutralized with 3 M KOH.Insoluble KCl04 was removed by centrifugation and 1 mL ofthe supernatant was alkalized with 10 uL of 3 M KOH.Inhibitors of PGM activity were destroyed by incubatingsamples at 85°C for 30 to 60 min. After cooling, the extractswere neutralized with 5 ,uL of 4 M HCl and decolorized with5 mg activated charcoal. Samples were either assayed imme-diately or were stored at -20°C before use. The stromalmetabolites, Glc6-P, Glcl-P, and Fru6-P, also were extractedwith HCl04 as described above, except that the final alkali-zation and heating steps were deleted. Hexose monophos-phates were assayed enzymically using fluorimetric detectionofNADPH formation (1 9).

Extractable Glc1,6-P2 was detected as a cofactor of thePGM assay as described by Passoneau et al. (19). Standardcurves were prepared with known amounts of Glc1,6-P2(Sigma Chemical Co., St. Louis, MO).2 Rabbit muscle PGMwas dephosphorylated before use by the method ofLayne andNajjar (16). All Glcl-P preparations used in this study were

freed of contamination by Glc1,6-P2 using ion exchangechromatography (21). Recovery of standard metabolites fromleaf and chloroplast extracts was nearly 100%. Pheophytinand Chl were extracted and measured in 80% (v/v) acetoneby the method of Vernon (25).

RESULTS AND DISCUSSION

Changes of Initial and Total PGM Activity in IsolatedSpinach Chloroplasts

Less than 10% of total PGM activity in the stroma was

detectable in a chloroplast lysate when assays were performedin the absence of added Glc1,6-P2 (Fig. 1). Similar results

2 Names of products are included for the benefit of the reader anddo not imply endorsement by the U.S. Department of Agriculture.

50

za.-J

201

101

0

0 20 40TIME IN LIGHT OR DARK. min

Figure 1. Time course for light dependent changes of initial PGMactivity from spinach chloroplasts. Intact spinach chloroplasts wereincubated either in the light or dark (indicated with arrows) in 0.33 Msorbitol medium at 250C containing 5 mm NaHC03. Samples wereremoved at indicated times and PGM activity (0) was assayed beforeand after 1 gM Glcl,6-P2 was added to the reaction mixture. TotalPGM activity was about 2.2 ,mol (mg Chl)-' min-' for all samples.

have been reported for PGM preparations from various plantsources (4, 9, 18, 24). However, initial PGM activity increased2- to 3-fold during the first 10 to 15 min of light when intactchloroplasts were illuminated with a saturating irradiance forphotosynthesisjust prior to enzyme measurement. No changein initial PGM activity was observed in dark chloroplastpreparations, suggesting that changes in enzyme activity werelight dependent (see below). Enzyme assays were linear for atleast 15 min, indicating that light effects on PGM activitywere stable. Initial PGM activity decreased slowly during a

subsequent 30 min dark incubation. However, light depend-ent alterations of initial PGM activity were not completelyreversed by this dark treatment. Less than 1% of total PGMactivity in washed chloroplast preparations was attributableto the cytosolic isoform as indicated by chromatographic andelectrophoretic separations (not shown). Total PGM activitywas unaffected by prior light treatment when measured at pH7.6 with 1 uM Glc 1,6-P2. At present, it is not known if a lightdependent change in total PGM activity would be detectedunder different assay conditions.

Photosynthetic 02 evolution by intact spinach chloroplastshas an optimal requirement for 0.3 to 0.5 mm external Pi(10). Photosynthesis is limited by Pi availability when theexternal Pi concentration falls below these levels. Conversely,high concentrations of Pi inhibit CO2 fixation by draining thechloroplast of photosynthetic intermediates via the Pi trans-locator (12). Light activation of several chloroplast enzymesis dependent upon external Pi supply (17). The Pi requirementfor light dependent changes in stromal PGM activity is shownin Figure 2. A small but consistent decrease in initial PGMactivity occurred when isolated spinach chloroplasts were

illuminated in the absence of Pi for 10 min at pH 7.8. Effects

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STROMAL PGM ACTIVITY

> 20-

0.

-j< 1 00

zw0trw

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0.) 2Figure 2. Effects of external Pi concentration on light dependentchanges of initial PGM activity from spinach chloroplasts. Intactspinach chloroplasts were incubated either in the light (0) or dark (0)in 0.33 M sorbitol medium containing various concentrations of Pi.Other experimental conditions were as described under Figure 1.Total PGM activity was about 2.0 umol (mg Chl)-' min-' for allsamples.

of Pi concentration in the dark were minor but initial PGMactivity in illuminated chloroplasts increased rapidly as Pi wasincreased from 0 to 0.5 mm. External Pi concentrations greaterthan 0.5 mm inhibited CO2 fixation (not shown) but did notprevent light dependent changes in initial PGM activity.The pH of the stroma increases from about pH 7.1 in the

dark to about pH 8.0 in the light (1 1). This light dependentalkalization of the stroma is a critical factor in activatingphotosynthetic CO2 fixation (8, 11). In the present study,maximal rates of photosynthetic 02 evolution by isolatedspinach chloroplasts were observed when the pH of the exter-nal medium was between 7.5 and 8.0 (Fig. 3A). This findingwas in agreement with published results (1 1). Light inducedchanges in stromal PGM activity exhibited a similar pHdependence, whereas external pH had little effect on themeasurable activity of PGM in dark incubated chloroplasts(Fig. 3B).

Effects of various additions to the chloroplast medium on

light or dark dependent changes in initial PGM activity areshown in Table I. In the first experiment (Table IA), 3-PGAand OAA were used as terminal electron acceptors in placeof CO2. Initial PGM activity in the light and dark decreasedwhen chloroplasts were exposed to OAA. This chloroplastreaction does not require ATP synthesis. By contrast, lightdependent increases in initial PGM activity were greater when3-PGA rather than CO2 was used to support photosynthesis.In this study, rates of 02 evolution by spinach chloroplastswith CO2, 3-PGA and OAA were 254, 119, and 94 ,umol (mgChl)-' h-', respectively. Note that total PGM activity wassimilar for all three treatments. Effects of various metabolicinhibitors of photosynthetic CO2 fixation on light dependentchanges in initial PGM activity were examined in a secondstudy (Table IB). The inhibitor concentrations used in these

101

7.0 8.0

EXTERNAL pH

9.0

Figure 3. Effects of external pH on rates of photosynthetic 02evolution and on light dependent changes of initial PGM activity fromspinach chloroplasts. A, Photosynthetic 02 evolution (A) by isolatedspinach chloroplasts was measured polarographically in 0.33 M sor-bitol medium at 250C. The pH of the external medium was adjustedusing different buffers, B, Initial PGM activity was determined withchloroplast preparations incubated either in the light (0) or dark (0).Total PGM activity was about 2.0 zmol (mg Chl)-' min-1 for allsamples.

Table I. Effects of Various Electron Transport Acceptors andInhibitors of Photosynthesis on Light Dependent Changes of Initialand Total PGM Activity from Spinach Chloroplasts

Experiment 0 min 10 min 10 min +1 AMExperiment Dark Dark Light Glul,6-P2

pumol (mg Chl) - min-'A. Control 0.18 NDa 0.36 2.0

2 mm 3-PGA 0.18 ND 0.47 1.81 mM OAA 0.04 ND 0.06 1.8

B. Control 0.16 ND 0.38 1.92 mM Glyoxylate 0.14 ND 0.21 1.82 mM Glycolaldehyde 0.19 ND 0.16 1.820 mM DL-Glyceraldehyde 0.19 ND 0.15 1.8

C. Control 0.14 0.16 0.32 2.410 mM DTT 0.14 0.12 ND 2.2

a Not determined.

investigations were sufficient to completely abolish CO2-de-pendent 02 evolution by isolated spinach chloroplasts (notshown). Glyoxylate inhibits photosynthesis by preventing al-kalization of the stroma in the light (8, 23). Initial PGMactivity measured in a spinach chloroplast lysate increasedslightly when 2 mm glyoxylate was present during illumina-

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Plant Physiol. Vol. 89, 1989

tion. Significantly, glycolaldehyde and DL-glyceraldehyde,which inhibit the regenerative phase of the reductive pentosephosphate pathway (23), totally prevented a light dependentincrease in initial PGM activity. It was also not possible toalter initial PGM activity in situ by incubating intact chloro-plasts with 10 mM DTT in the dark (Table IC). This findingsuggests that light effects on stromal PGM activity do notinvolve thioredoxin-dependent sulfhydryl reduction (3). Fur-thermore, it is clear that PGM is not sulfhydryl activatedbased on properties of homogeneous preparations from plantand other sources (5, 13, 14).

Levels of Glcl,6-P2 in Spinach Leaves and ChloroplastsThe light dependent changes of initial PGM activity de-

scribed above may be due to alterations in levels of the active,phosphorylated form of PGM in the stroma. Based upon awell studied series of reactions (13, 14, 21), the phosphoen-zyme intermediate can synthesize Glcl,6-P2 by reacting withGlcl-P and the resultant complex will express PGM activityin the absence of added Glcl,6-P2 in the assay. Based uponresults shown in Table I and using values of molecular weightand specific activity for potato tuber PGM (24), there couldbe up to 5 x 10-8 M phosphorylated PGM in a typicalchloroplast lysate, assuming that there is 1 mole of phosphateper mole ofPGM and that the enzyme is 100% phosphoryl-ated in situ. The chloroplast isozyme ofPGM has an apparentKa for Glc 1,6-P2 of about 50 x 10-8 M (see below). Therefore,the possibility that small, light dependent changes of initialenzyme activity are attributable to fluctuations of phosphor-ylated PGM levels in the stroma, cannot be excluded.At present it is not possible to directly measure the per-

centage of phosphorylated PGM in isolated spinach chloro-plasts. However, a rapid, light dependent increase of Glc 1,6-P2 in illuminated chloroplasts would support the suggestionthat the phoshorylated PGM concentration goes up in thelight. The Glc 1,6-P2 level of spinach leaves sampled in thedark was 1.6 ± 0.1 1 nmol (mg Chl)- . Results are the averageof six measurements from two experiments. Spinach leavescontained about 1.3 mg Chl (g fresh weight)-'. If it is assumedthat the vacuole occupies 90% of the leaf cell by volume, thenthe concentration of Glc 1,6-P2 in the extra-vacuolar com-partment of spinach leaves is comparable to that of animaltissues [10 to 80 nmol (g fresh weight)-'] (19). The aboveresults are based upon an enzyme bioassay that has been usedextensively by animal investigators. In the future, it will benecessary to positively demonstrate that the factor activatingPGM in plant extracts is Glcl ,6-P2. The Glcl ,6-P2 concentra-tion of isolated spinach chloroplasts incubated in sorbitolmedium in the dark at 25°C for 10 min was 0.7 ± 0.04 nmol(mg Chl)-'. Although chloroplasts contained about one-halfof the total cellular concentration of this metabolite, photo-synthetic intermediate levels can be underestimated by usingmeasurements of aqueously prepared chloroplasts (26). Toour knowledge, Glc 1,6-P2 measurements previously have notbeen reported for leaf or chloroplast extracts. It is not surpris-ing that Glc 1,6-P2 is found in chloroplast preparations becausestromal PGM is strongly activated by the presence of coen-zyme (21) (Table I). Chloroplast Glc 1,6-P2 levels decreasedslightly to 0.5 ± 0.05 nmol (mg Chl)-' after 10 min of

illumination in sorbitol medium containing 5 mM NaHCO3.Therefore, it is not likely that the light dependent changes ofstromal PGM activity shown in Figures 1 through 3 were theresult of increased Glc 1,6-P2 levels.The internal volume of spinach chloroplasts is about 25 gL

(mg Chl)-' when determined with 3H20 (11). The stromalGlcl,6-P2 concentration for these chloroplast preparationswas between 20 and 30 gM. These cofactor levels are oneorder of magnitude higher than necessary to fully saturatePGM activity (4, 9). The concentration ofGlc 1,6-P2 in animaltissue is also present in vastly excessive quantities (2), sug-gesting that measurements of total cofactor concentration donot accurately reflect the effective concentration in vivo. Theamount of Glcl,6-P2 released upon chloroplast lysis duringthe measurement of initial PGM activity as in Figure 1 wouldproduce a final coenzyme concentration of only 1 or 2 nM,which is far below levels required for PGM activity. BecauseGlcl,6-P2 levels in the stroma are so low, it is not possible atthe present time to test for the presence of enzyme-boundforms of Glcl,6-P2 in chloroplast extracts. It will be necessaryin the future to directly measure changes in the activatedform of PGM, in order to show that the light dependentchanges in initial PGM activity described above are attribut-able to altered levels of phosphorylated PGM in thechloroplast.

Dependence of PGM Activity in a Chloroplast Lysate onpH and Glcl,6-P2 Levels

Results presented in Figure 3 and Table I make it clear thatincreased PGM activity in the light is dependent upon stromalalkalization. The effects of pH on PGM activity in a chloro-plast lysate were examined in an effort to better understandthe regulation of this enzyme in situ. In the first experiment,intact spinach chloroplasts were diluted 50-fold into reactionmedia containing 5 gM Glc1,6-P2. Assays were initiated witha final concentration of 2 mm Glc 1-P after chloroplast lysis.The activity profile for stromal PGM was strongly alkaline(Fig. 4). Maximal enzyme activity between pH 8.0 and 8.5was 2.5 times greater than similar assays performed at pH7.0. Muhlbach and Schnarrenberger (18) previously reportedthat the plastid and cytosolic isoforms of spinach leaf PGMexhibit maximal activity between pH 7.8 and 7.9.The effects of preincubating stromal PGM with Glcl,6-P2

were examined in a related experiment. In this study, 0.5 mLof a chloroplast suspension was collected by centrifugationand the sorbitol medium was removed. The chloroplast pelletwas osmotically shocked by resuspension in 1.0 mL of buffercontaining 50 mM Hepes-NaOH (pH 8.0), and 1 mM MgC12and the chloroplast membranes were removed by centrifuga-tion. The supernatant was made 2 ,M with Glc1,6-P2 and wasincubated at room temperature for 5 min. Assays of PGMactivity were performed by diluting the lysate preparation 20-fold into assay media that contained only Glc1,6-P2 that wasintroduced with the enzyme extract. Enzyme rates measuredbetween pH 6.5 and 9.0 were about 0.2 gmol (mg Chl)-'min-' or less (i.e., in the presence of 0.1 AM Glcl,6-P2). Thisactivity was less than 10% of the rate observed with 5 umGlcl,6-P2 included in the assay. A 3- to 4-fold increase inPGM activity occurred when the volume of lysate added to

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STROMAL PGM ACTIVITY

3

E

E

I-

E

v-

I

0

0~

1

0

6.5 7.0 7.5 8.0 8.5ASSAY pH

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Figure 4. Effect of pH and Glc ,6-P2 on PGM activity in a chloroplastlysate. The pH dependence of PGM activity was measured in reactionmedia containing 5 Mm Glc ,6-P2 (0) and 2 mm GIcl -P using a solubleextract prepared from osmotically ruptured spinach chloroplasts.Similar samples (A) were preincubated for 5 min with 2 Mm Glcl,6-P2and then PGM activity was measured after diluting the chloroplastextract 20 fold into assay media prepared without additionalcoenzyme.

the reaction mixture was doubled (data not shown). Thesefindings indicated that PGM activity was strictly dependentupon the presence of Glc 1,6-P2 in the assay. If the lightinduced changes in stromal PGM activity shown in Figures 1

through 3 were due to a pH and Glc 1,6-P2-dependent enzymeactivation in situ, then it should have been possible to phos-phorylate PGM in a chloroplast lysate at pH 8.0 with addedGlc1,6-P2. In addition to control by Glcl,6-P2, it is possiblethat light dependent changes of stromal PGM activity in thechloroplast are mediated by changes in the residual enzymeactivity possessed by the dephosphorylated form of PGM(21).

Effects of pH on the stimulation of stromal PGM activityby Glc 1,6-P2 was examined in Figure 5. In this study, spinachchloroplasts were injected into reaction media containingdifferent levels of Glc 1,6-P2. Stromal PGM activity was satu-rated with 1 gM Glc 1,6-P2 when assayed at pH 7.2. In similarexperiments performed at pH 8.2, stromal PGM was activatedby up to 5 ,M Glc 1,6-P2 and the enzyme rate increased about45% over that seen at lower pH. Replots of the data in Figure5 indicated that the apparent Ka for Glc 1,6-P2 increased from0.14 to 0.54 gM when the pH was increased from 7.2 to 8.2.Similar half-maximal activation constants have been reportedfor PGM from pea and mungbean seedlings (9). The Km forGlcl-P in this study was about 40 gM at both pH 7.2 and 8.2(data not shown).

Effects of Illumination on Hexose Monophosphate Levelsin Isolated Spinach ChloroplastsA thorough description of hexose monophosphate levels in

the chloroplast compartment of intact spinach leaves has

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stromal PGM activity. The Glcl,6-P2 dependence of PGM activityfrom osmotically ruptured chloroplasts (10.7 Ag Chl) was measuredwith 2 mm GIcl -P at either pH 7.2 (A) or pH 8.2 (0).

Table II. Hexose Monophosphate Levels in Isolated SpinachChloroplasts Incubated for up to 8 Min in the Light or Dark

Sample Glc6-P GIcl-P Fru6-P

nmol (mg Chl)'A. Light0min 43±2.6a 2±0.5(21)b 11±2.4(4)2min 38±2.4 2±0.5(19) 10±1.5(4)4min 44±4.9 3±0.8(15) 14±2.4(3)8min 58±7.8 3±0.8(19) 25±5.2(2)

B. Dark8min 14±2.4 1±0.2(14) 4±1.1(4)

a Results are given as means ± SE for n = 4 from two experi-ments. b Values in parentheses are Glc6-P/Glc1-P and Glc6-P/Fru6-P ratios, respectively.

appeared previously (6, 7). Starch synthesis in the stroma isentirely dependent upon the synthesis of Glcl-P from Glc6-P, which in turn, is synthesized from Fru6-P. Hexose phos-phate measurements using aqueously prepared spinach chlo-roplasts are shown in Table II. After a brief lag period, bothGlc6-P and Fru6-P increased when intact chloroplasts were

illuminated for 8 min. There was also an apparent increasein stromal Glcl-P concentration in the light, although levelsof this metabolite were quite low and approached the lowerlimit of detection. All three photosynthetic intermediatesdecreased substantially in the dark samples. Although metab-olite levels in the current study were slightly lower than innonaqueously prepared chloroplast samples, there was stillexcellent agreement with published findings. The results inTable II support earlier suggestions (6, 7) that PGM andphosphoglucoisomerase are near or slightly displaced fromequilibrium and that the low levels of Glcl-P in the stromamay impose a rate-limitation on starch synthesis.

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Plant Physiol. Vol. 89, 1989

Physiological Significance of Stromal PGM Activity

The above findings suggest that the activity of PGM in thechloroplast can effectively be controlled by stromal pH andby Glc 1,6-P2 concentration. The expected activity ofPGM inthe dark would be low because the pH of the stroma is low.Illumination increases the pH of the stroma by about 1.0 pHunit and this alkalization is capable of increasing PGM activ-ity in the chloroplast by two mechanisms. Activation ofPGMby Glcl,6-P2 increases substantially at higher pH, and sec-

ondly, the rate of catalysis in the presence of saturatingcoenzyme is maximal at pH 8.0 or above.

In addition to pH mediated effects of light on PGM activity,a small, light induced change in initial PGM activity can bemeasured immediately after chloroplast lysis. Light dependentincreases of initial PGM activity as shown in Figure 1, maybe due to the increased formation of phosphoenzyme inilluminated chloroplasts. Indeed, comparisons of initial andtotal PGM activity have been used previously to calculate thepercentage of phosphoenzyme in plant extracts (9). AlthoughPGM in mammalian tissue extracts is almost entirely phos-phorylated, results reported above and previously (4, 9, 24)suggest that PGM from plant sources is mostly dephospho-rylated. Difficulty in phosphorylating PGM, both in situ (Fig.1) and in vitro (Fig. 4), is potentially significant and raises thepossibility that the activation ofPGM by Glcl ,6-P2 may havean important, metabolic control function in the plant cell.Alternatively, light effects on initial PGM activity may reflectchanges in the residual activity of the inactive form of theenzyme.The experiments reported above permit the following, ad-

ditional conclusions. Light dependent alterations in PGMactivity are affected by chloroplast pH and require that Pi besupplied to the stroma. Studies with OAA, 3-PGA, glycolal-dehyde and DL-glyceraldehyde show that carbon flow isnecessary to obtain light induced changes of PGM activity.One possible explanation of these collective results is thatlight dependent alterations of PGM activity require turnoverof the ATP pool. Further research is necessary to elucidatethis matter.

Light mediated changes in PGM activity are considerablysmaller than activity increases observed for other light de-pendent stromal enzymes. The initial activity ofPGM in thelight was about 0.4 ,mol (mg Chl)-' min-'. This enzyme

activity is sufficient to process about 50% ofcarbon fixed intostarch when the leaf is performing at maximal rates of pho-tosynthesis [i.e., 5 ,umol CO2 (mg Chl)-' min-']. The actualactivity ofPGM in the stroma will depend upon several factorsincluding pH, Glc6-P and Glc 1,6-P2 levels, and Pi availability.Additional factors, such as Mg2+ concentration, temperatureand enzyme effectors, may also combine to influence theactivity expressed in vivo. Total PGM activity was 5- to 10-fold higher than initial activity. This result suggests that thechloroplast may have a large reserve capacity forPGM activitythat can be accessed when there is a need for additional starchsynthesis. It was also interesting to note that the highest initialPGM activities observed in this study were when 3-PGA was

included in the chloroplast medium. It has been reported

previously that 3-PGA is a powerful, positive effector of starchsynthesis ( 12, 20).

ACKNOWLEDGMENT

Expert technical assistance was provided by D. F. Kremer.

LITERATURE CITED

1. Bassols AM, Carreras J, Cusso R (1986) Changes in glucose 1,6-bisphosphate content in rat skeletal muscle during contraction.Biochem J 240: 747-751

2. Beitner R (1985) Glucose-1,6-bisphosphate- the regulator of car-bohydrate metabolism. In R Beitner, ed, Regulation of Car-bohydrate Metabolism, Vol 1. CRC Press, Boca Raton, FL, pp1-27

3. Buchanan BB (1980) Role of light in the regulation ofchloroplastenzymes. Annu Rev of Plant Physiol 31: 341-374

4. Carreras M, Bartrons R, Climent F, Carreras J (1986) Effects offructose 2,6-bisphosphate on phosphoglucomutase fromplants. Plant Physiol 82: 619-621

5. Chiba H, Ueda M, Hirose M (1976) Purification and propertiesof beef liver phosphoglucomutase. Agric Biol Chem 40: 2423-2431

6. Dietz K-J (1985) A possible rate-limiting function of chloroplasthexosemonophosphate isomerase in starch synthesis in leaves.Biochim Biophys Acta 839: 240-248

7. Dietz K-J (1987) Control function of hexosemonophosphateisomerase and phosphoglucomutase in starch synthesis inleaves. In J Biggins, ed, Proceedings of the 7th InternationalCongress on Photosynthesis, Vol 3. Martinius/Nijhoff/Dr.Junk Publishers, The Hague, pp 329-332

8. Flugge UI, Freisl M, Heldt HW (1980) The mechanism of thecontrol of carbon fixation by the pH in the chloroplast stroma.Studies with acid mediated proton transfer across the envelope.Planta 149: 48-51

9. Galloway C, Dugger,WM Jr, BlackCC (1985) In vitro activationof phosphoglucomutase by fructose 2,6-bisphosphate. PlantPhysiol 79: 920-922

10. Heldt HW, Chon CJ, Maronde D, Herold A, Stankovic ZS,Walker DA, Kraminer A, Kirk MR, Heber U (1977) Role oforthophosphate and other factors in the regulation of starchformation in leaves. Plant Physiol 59: 1146-1155

11. Heldt HW, Werdan K, Milovancev M, Geller G (1973) Alkali-zation of the chloroplast stroma caused by light-dependentproton flux into the thylakoid space. Biochim Biophys Acta314: 224-241

12. Heldt HW, Rapley L (1970) Specific transport of inorganicphosphate, 3-phosphoglycerate and dihydroxyacetone-phos-phate and of dicarboxylates across the inner membrane ofspinach chloroplasts. FEBS Lett 10: 143-148

13. Hirose M, Sugimoto E, Sasaki R, Chiba H (1970) Crystallizationand reaction mechanism of yeast phosphoglucomutase. JBiochem 68: 449457

14. Jamil H, Clarke JB (1985) Mechanism of action of rabbit liverphosphoglucomutase. Biochem J 230: 791-795

15. Jensen RG, Bassham JA (1966) Photosynthesis by isolated chlo-roplasts. Proc Natl Acad Sci USA 56: 1095-1101

16. Layne PP, Najar VA (1975) The dephosphorylation ofphospho-glucomutase by nucleophilic reagents. J Biol Chem 250: 966-972

17. Leegood RC (1985) Regulation ofC02 pathway enzymes by lightand other factors. Photosynth Res 6: 247-259

18. Muhibach H, Schnarrenberger C (1978) Properties and intracel-lular distribution of two phosphoglucomutases from spinachleaves. Planta 141: 65-70

19. Passoneau JV, Lowry OH, Schulz DW, Brown JG (1969) Glu-cose 1,6-diphosphate formation by phosphoglucomutase inmammalian tissues. J Biol Chem 244: 902-909

20. Preiss J (1982) Biosynthesis of starch and its regulation. In FALoewus, W Tanner, eds. Encyclopedia of Plant Physiology(New Series), Vol 13A. Springer-Verlag, Berlin, pp 397-417

562 SICHER

www.plantphysiol.orgon February 2, 2019 - Published by Downloaded from Copyright © 1989 American Society of Plant Biologists. All rights reserved.

STROMAL PGM ACTIVITY

21. Ray WJ Jr, Roscelli G (1964) A kinetic study ofthe phosphoglu-comutase pathway. J Biol Chem 239: 1228-1236

22. Sicher RC (1984) Characteristics of light-dependent inorganiccarbon uptake by isolated spinach chloroplasts. Plant Physiol74: 962-966

23. Sicher RC (1984) Glycolaldehyde inhibition of photosyntheticcarbon assimilation by isolated chloroplasts and protoplasts.In C Sybsema, ed, Advances in Photosynthesis Research, Vol3. Martinius Nijhoff/Dr. Junk Publishers, The Hague, pp 413-416

24. Takamiya S, Fukui T (1978) Purification of multiple forms ofphosphoglucomutase from potato tubers. Plant Cell Physiol19: 759-768

25. Vernon LP (1960) Spectrophotometric determination of chloro-phylls and pheophytins in plant extracts. Anal Chem 32: 1144-1150

26. Wirtz W, Stitt M, Heldt HW (1980) Enzymic determination ofmetabolites in the subcellular compartments of spinach pro-toplasts. Plant Physiol 66: 187-193

563

www.plantphysiol.orgon February 2, 2019 - Published by Downloaded from Copyright © 1989 American Society of Plant Biologists. All rights reserved.