Plant Physiol. (1984) 76, 238-2430032-0889/84/76/0238/06/$01.00/0

Activation of NADP-Malate Dehydrogenase, Pyruvate,PiDikinase, and Fructose 1,6-Bisphosphatase in Relation toPhotosynthetic Rate in Maize'

Received for publication February 15, 1984 and in revised form May 21, 1984

HIDEAKI USUDA2, MAURICE S. B. Ku, AND GERALD E. EDWARDS*Botany Department, Washington State University, Pullman, Washington 99164-4230

ABSTRACT

The activity and extent of light activation of three photosyntheticenzymes, pyruvate,Pi dikinase, NADP-malate dehydrogenase (NADP-MDH), and fructose 1,6-bisphosphatase (FBPase), were examined inmaize (Zea mays var Royal Crest) leaves relative to the rate of photosyn-thesis during induction and under varying light intensities. There was astrong light activation of NADP-MDH and pyruvate,Pi dikinase, andlight also activated FBPase 2- to 4-fold. During the induction period forwhole leaf photosynthesis at 30°C under high light, the time required toreach half-maximum activation for all three enzymes was only 1 minuteor less. After 2.5 minutes of illumination the enzymes were fully activated,while the photosynthetic rate was only at half-maximum activity, indi-cating that factors other than enzyme activation limit photosynthesisduring the induction period in C4 plants.

Under steady state conditions, the light intensity required to reachhalf-maximum activation of the three enzymes was similar (300-400microEinsteins per square meter per second), while the light intensityrequired for half-maximum rates of photosynthesis was about 550microEinsteins per square meter per second. The light activated levels ofNADP-MDH and FBPase were well in excess of the in vivo activitieswhich would be required during photosynthesis, while maximum activitiesof pyruvate,Pi dikinase were generally just sufficient to accommodatephotosynthesis, suggesting the latter may be a rate limiting enzyme.

There was a large (5-fold) light activation of FBPase in isolated bundlesheath strands of maize, whereas there was little light activation of theenzyme in isolated mesophyll protoplasts. In mesophyll protoplasts theenzyme was largely located in the cytoplasm, although there was a lowamount of light-activated enzyme in the mesophyll chloroplasts. Theresults suggest the chloroplastic FBPase in maize is primarily located inthe bundle sheath cells.

In C4 plants, light intensity is normally considered a majorlimiting factor for photosynthesis under optimum temperature.Light drives several processes in C4 photosynthesis, including theC4 cycle, provision of energy to the RPP pathway,3 and the light

'Supported by National Science Foundation Grant PCM 82-04625and by the Japan-United States Cooperative Photoconversion and Pho-tosynthesis Research Program.

2 Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, Ohtsuka, Hachioji City, Tokyo, Japan.

3Abbreviations: RPP pathway, reductive pentose phosphate pathway;NADP-MDH, NADP-malate dehydrogenase; FBPase, fructose 1,6-bis-phosphatase; PVP, polyvinylpolypyrrolidone; PEP, phosphoenolpyru-vate.

activation of several enzymes. Under limiting light, it is not clearif all of these factors are co-limiting or if there is one primarylimitation.NADP-MDH and pyruvate,Pi dikinase ofthe C4 cycle are light

activated in C4 plants, and PEP carboxylase is also activated bylight to some extent (8, 11, 21). Several enzymes of the RPPpathway are light activated in C3 plants (fructose 1,6-bisphospha-tase, sedoheptulose 1,7-bisphosphatase, phosphoribulokinaseand NADP-triose-P dehydrogenase) (1, 3) but there is littleevidence that these enzymes are strongly modulated by light invivo in C4 plants. Steiger et al. (23) generally found little lightactivation of NADP-triose-P dehydrogenase and phosphoribu-lokinase in C4 plants compared to C3 plants. Bjorkman andBadger (2) found a several-fold activation of NADP-triose-Pdehydrogenase and phosphoribulokinase in the C4 species Ti-destromia oblongifolia but no light activation of FBPase. Inaddition, activation of photosynthetic enzymes relative to ratesof leaf photosynthesis in C4 plants has not been evaluated. In thepresent study, the activity and extent of activation of NADP-MDH, pyruvate,Pi dikinase, and FBPase in the C4 plant maizewas examined relative to photosynthetic rate during inductionand under varying light intensities.

MATERIALS AND METHODS

Material. Seeds of Zea mays (hybrid sweet corn, var RoyalCrest) were obtained from Olds Seed Co., Madison, WI. Plantswere grown in controlled chambers with a 14-h light/10-h darkperiod, with day/night temperatures of 27°C/22°C. The lightintensity was 700 ,E m-2 s-', except in Figure 2 (see legend) inwhich the light intensity during growth was 400 ,E m-2 s-' forsome experiments. Plants were grown in a commercial premix(55% peat moss, 30% pumice, and 15% sand) and watered witha dilute nutrient soution three times a week according to Schmittand Edwards (20). The largest expanded leaves of plants approx-imately 3 weeks of age were used in the experiments.Gas Exchange Measurements. C02 exchange of intact individ-

ual leaves was measured according to Monson et al. (17), withan open IR gas analysis system under 330 1/1 C02 in air. Plantswere incubated in a growth chamber at 30° C. A single leaf wasplaced in a cuvette and maintained at 30 + 0.5C using a Peltier-cooled heat exchanger. Varying levels ofirradiance were achievedby changing the number of cheesecloth screens between the leafchamber and light source. The light source was a l-kw metalhalide lamp which was used both for the gas exchange measure-ments and enzyme analyses (see below). Light intensities weremeasured with a quantum sensor (model 1776 from Li CorInstruments) mounted within the leaf chamber adjacent to theenclosed leaf.

Preparation of Leaf Tissue for Enzyme Assays. Plants were238

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ENZYME ACTIVITY AND PHOTOSYNTHETIC RATE IN MAIZE

incubated in a growth chamber at 30°C. At the same time gasexchange measurements were made on a single leaf, sampleswere taken from other leaves for enzyme assays (see legends fordetails of sampling). A low intensity, green safe light was usedfor samples taken in the dark (18). The leaf samples were frozenand stored in liquid nitrogen until extraction for enzyme assay.For FBPase and NADP-MDH assays, the frozen leaf sampleswere rapidly homogenized using a chilled mortar with 5 mginsoluble PVP and 0.2 g of acid-washed sand plus 0.5 ml of ice-cold medium containing 100 mm Tris-HCI (pH 7.8), 1 mMMgC2, 0.1 mM EDTA, 5 mM DTE, 0.05% (v/v) Triton X-100,and 0.5% (w/v) BSA. Without a reducing agent (e.g. DTE),FBPase (J. Kobza and G. E. Edwards, unpublished data) andNADP-MDH (18) rapidly lose their original in vivo state ofactivation. By extracting at low temperature and assaying withinminutes, no further activation occurs in vitro. For pyruvate,Pidikinase, 5 mg insoluble PVP and 0.2 g of sand, and 0.5 ml ofice-cold medium containing 50 mm Tris-HCI (pH 7.5), 10 mMMgC2, 1 mm EDTA, 2.5 mM pyruvate, 10 mM DTE, and 0.5%(w/v) BSA were used for hotnogenization. After homogenization,1.5 ml of the same medium was added. The homogenates werefiltered through Miracloth. An aliquot of the filtrate was takenfor Chl determination. For the assay of FBPase and pyruvate,Pidikinase, the homogenate was centrifuged at 14,000g for 1 minand the supernatant was used as the crude enzyme extract. Forthe assay of NADP-MDH, a 25 gl aliquot of the homogenatewas added to 100 mm Tris-HCI (pH 8.0), 0.5 mM EDTA, and0.2 mm NADPH (total volume of 1 ml), and centrifuged at14,000g for 1 min. The supernatant was immediately used forenzyme assay.

Isolation of Mesophyll Protoplasts and Bundle SheathStrands. Mesophyll protoplasts and bundle sheath strands wereenzymically isolated according to previous methods using acombination of cellulase and Macerozyme (6, 10). The prepara-tions were suspended in a medium containing 50 mM Hepes-KOH (pH 8.0), 0.4 M sorbitol, and 5mM EDTA.

Isolation of Mesophyll Chloroplasts. Mesophyll protoplastswere suspended in a medium containing 0.4 M sorbitol, 50 mMHepes-KOH (pH 7.6), and 1 mM EDTA, and ruptured by threepasses through a 20-,sm nylon net attached to a l-ml syringe, aspreviously described (6). The protoplast extracts were centrifugedfor 1 min at 650g to pellet the chloroplasts. The pellet wasresuspended in the medium containing 0.4 M sorbitol, 50 mmHepes-KOH (pH 8.0), and 5 mM EDTA.

Extraction of Mesophyll and Bundle Sheath Preparations forEnzyme Assays. After light or dark treatment, mesophyll proto-plasts, mesophyll chloroplasts, and bundle sheath strands, sus-pended in plastic tubes in a medium containing 0.4 M sorbitol,50mM Hepes-KOH (pH 8.0), 5 mM EDTA, and 10 mM NaHCO3,were transferred into liquid nitrogen for storage prior to assay.The frozen preparations were thawed with addition of an equalvolume ofmedium containing 200 mM Tris-HCl (pH 7.8), 7 mMMgCl2, 0.2 mM EDTA, 10 mM DTE, 0.2% Triton X-100, and1% (w/v) BSA. In the case of mesophyll protoplasts and meso-phyll chloroplasts, an aliquot was taken from each suspensionfor Chl determination, the remainder was centrifuged at 14,000gfor 10 s and the supernatant was used for enzyme assays. Bundlesheath strands were treated in a similar manner, except they werebroken with an ice-cold Teflon-glass homogenizer after additionof the medium. An aliquot of the homogenized suspensions weresaved for the Chl determination and the rest were centrifuged at14,000g for 10 s and the supernatant used for assay of enzymes.The FBPase assay was initiated within 3 min after thawing ofthe preparations.Assay of Enzyme Activities and Chl Determination. NADP-

MDH was assayed as previously described (18) at 30°C in amedium containing 100 mM Tris-HCl (pH 8.0), 0.5 mM EDTA,

0.2 mm NADPH, and 2 mm oxaloacetate. The assay was initiatedby addition of oxaloacetate.The assay mixture for FBPase contained 100 mm Tris-HCl

(pH 7.8), 5 mM MgCl2, 0.4 mM NADP, 0.5 mM EDTA, 4 unitsofglucose-P isomerase, and 2 units ofglucose 6-P dehydrogenase.The reaction was initiated by addition of FBP to a final concen-tration of 0.25 mm. A relatively low concentration of FBP wasused since higher concentrations of FBP gave less differencebetween the activity from darkened versus illuminated leaves(also, in C3 plants the light-activated form ofFBPase has a lowerKm for the substrate than the dark form). The activity of nonspe-cific phosphatase, which was low or undetected in the prepara-tions, was estimated by omission of MgCl2.

Pyruvate,Pi dikinase and NADP-triose-P dehydrogenase weremeasured as previously described by Nakamoto and Edwards(18) and Spalding et al. (22), respectively.The assay medium for PEP carboxylase contained 50 mM

Hepes-KOH (pH 8.0), 5 mm NaHCO3, 0.2 mm NADH, 10 mmMgC12, and 5 units of malate dehydrogenase. The reaction wasinitiated by adding PEP to a final concentration of 2.5 mm.The assay mixture for NADP-malic enzyme contained 50 mm

Hepes-KOH (pH 8.0), 5 mM EDTA, 5 mm DTE, 0.5 mm NADP,and 5 mm K-malate (pH 7.0). The reaction was initiated byadding MgCl2 to a final concentration of 22.5 mm.Chl was determined according to Wintermans and De Mots

(26).

RESULTSIn maize leaves, FBPase was activated in the light and inacti-

vated in the dark, with the degree of light activation 2- to 4-foldthe activity in darkened leaves (Fig. 1B; Table I). The in vivoactivity of NADP-MDH and pyruvate,Pi dikinase in maize isstrongly dependent on light (Fig. 1, A and C; Hatch [8]). Thetime course of activation of NADP-MDH and pyruvate,Pi diki-nase in the light was similar to that of FBPase (Fig. 1, A-C).Also, the rate of dark inactivation ofNADP-MDH and FBPasewas similar. Dark inactivation of pyruvate,Pi dikinase was notmeasured (the effect of varying environmental conditions on thedark inactivation of pyruvate,Pi dikinase and NADP-MDH hasbeen recently studied [18]). The half-time for the light activationof the three enzymes was about 0.5 to 1 min. The time requiredto reach half-maximum rate ofleafphotosynthesis was about 2.5min, at which time the three enzymes had reached maximumlevel of activation.The influence of light intensity on the degree of activation of

NADP-MDH, pyruvate,Pi dikinase, and FBPase, and on the rateof leaf photosynthesis in maize, is shown in Figure 2. At eachlight intensity, leaves were preilluminated for 20 min to assuresteady state conditions had been reached (preliminary experi-ments at both high and low light intensities indicated steady stateconditionswere obtained within 2 to 3 min forthe light activationof enzymes and within 20 min for photosynthetic rates). A lightintensity of 300 to 400 gE m 2 s-' was required for half-maxi-mum activation of all three enzymes. Approximately 550 MEm-2 s-' of light was required to give half-maximum saturationof the photosynthetic rate and relatively high light intensity wasrequired for photosynthesis to approach a maximum level. Thelight response curve for photosynthesis was similar for plantsgrown at the two light intensities (400 versus 700 ME m 2 s-')and there was no apparent difference in the light required foractivation of the pyruvate,Pi dikinase (plants grown at 400 MEm-2 s-') and NADP-MDH and FBPase (plants grown at 700 MEm-2 s-') (Fig. 2). In addition, outdoor grown plants had a similarlight response curve for photosynthesis (half maximum activityat 600 ME m-2 s-') and activation of pyruvate,Pi dikinase (half-maximum activation at 300 ME m-2 s-') and NADP-MDH (half-maximum activation at 350 ME m-2 s-'), although rates of

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0)

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31 4142

TIME (min)FIG. 1. Time course of the light activation of FBPase, NADP-MDH,

and pyruvate,Pi dikinase (PPDK) relative to induction of photosynthesis(APS) in maize. Experiments were initiated in the morning after approx-imately 2 h into the light period. Following a preconditioning for 21 minin the dark (see figure) the plants were illuminated with a light intensityof 1500 gE m-2 s-'. Photosynthesis was measured continuously on a

single leaf. A separate leaf of similar age was used for taking samples forthe assay ofFBPase and NADP-MDH. At each time point a leaf segment,approximately I x 1 cm, was cut from along the middle portion of theleaf (segments were taken from the leaf margin up to the mid-rib, andsuccessively toward the base of the leaf). In each sample taken, bothNADP-MDH and FBPase were assayed. For the assay of PPDK, seg-ments were removed from a separate leaf at the time points indicated.Immediately upon removal from the plant the samples were frozen andstored in liquid nitrogen prior to extraction and enzyme assay. The arrow

on each curve indicates the time required to reach half the maximumactivity.

photosynthesis and the pyruvate,Pi dikinase activity were slightlyhigher than in the growth chamber plants (data not shown).

It is known from previous work that pyruvate,Pi dikinase andNADP-MDH are located in the mesophyll chloroplasts of C4plants (5, 8). To determine the intercellular location of the light-activated FBPase, mesophyll protoplasts and bundle sheathstrands were isolated from maize and preincubated in the darkor light for 20 min at 30°C prior to enzyme extraction and assay.Both mesophyll and bundle sheath preparations treated in thedark had FBPase activity (TableI). Light-treated bundle sheathstrands showed a large increase in FBPase activity (about 5-fold),while light-treated mesophyll protoplasts showed only a slightincrease in activity of the enzyme. DCMU, an inhibitor ofnoncyclic electron flow, at 10 gM levels prevented the light-dependent increase in activity in the preparations. Assay of PEPcarboxylase (marker for mesophyll protoplasts) and NADP-malicenzyme (marker for bundle sheath cells) showed that good sep-aration of the cell types was obtained.Mesophyll protoplasts were fractionated and the chloroplast

and extrachloroplastic fractions separated to determine the intra-

cellular location of FBPase (Table II). Based on the distributionof the chloroplastic NADP-triose-P dehydrogenase among twopreparations, the chloroplasts were 93 to 95% intact. The distri-bution of the cytosolic enzyme PEP carboxylase between thechloroplast pellet and supernatant indicated that only about 4%of the cytosolic fraction contaminated the chloroplast pellet. Inthese dark-treated preparations, the FBPase was totally locatedin the extrachloroplastic fraction (the low percentage of activityin the chloroplast pellet of 1 to 3% can be accounted for bycytosolic contamination). The activity of NADP-malic enzymein the mesophyll preparations (Table II) was only 1% of that inbundle sheath cells (see Table I). Thus, the contribution ofFBPase activity from bundle sheath contamination is insignifi-cant (1% of the activity of FBPase from dark-treated cells, TableI, is only 0.3 jmol mg-' Chl h-'). The low activity of NADP-malic enzyme in the mesophyll preparation was extrachloroplas-tic, which suggests any contaminating bundle sheath chloroplastswere broken since NADP-malic enzyme is located in the bundlesheath chloroplasts in C4 plants (5).When mesophyll chloroplasts isolated from protoplasts were

illuminated, the activity of FBPase increased to 8 to 11 ,umolmg'I Chl h-', compared to activity of less than 1 ,umol mg' Chlh-' in the dark-treated chloroplasts. DCMU strongly inhibitedthe light-dependent activation. Also, PGA, which uses ATP andreductive power during its reduction to glyceraldehyde-3-P, in-hibited the light activation of FBPase in the mesophyll chloro-plasts (Table III).

DISCUSSION

Induction of Photosynthesis and Light Activation of Enzymesin Maize, a C4 Plant. Although light is known to be required foractivation of NADP-MDH and pyruvate,Pi dikinase of the C4cycle in C4 plants, measurements on enzyme activation andphotosynthesis under varying conditions have not been studied.In the present study, when maize leaves were illuminated at30C, within 2.5 min the enzymes NADP-MDH, pyruvate,Pidikinase, and FBPase had reached a maximum degree of acti-vation, although photosynthesis had reached only half its maxi-mum rate (Fig. 1). This finding indicates that, during induction,factors in addition to enzyme activation are critical for photo-synthesis in C4 plants to reach maximum capacity. Followingillumination, photosynthesis may be limited until certain metab-olites of the C4 cycle and RPP pathway build up to a steady statelevel. Leegood (13) found that substantial changes occur in thelevels of several metabolites of carbon assimilation over severalminutes following a dark/light transition with maize. In partic-ular, there was a large buildup of pyruvate, 3-P glycerate anddihydroxyacetone-P over the first 10 to 40 min of illumination.In C4 plants enzyme activation may occur well before buildupof metabolites and increases in concentrations of metabolites ofthe C4 cycle and RPP pathway may be important during induc-tion. In addition, it takes about 15 min for full opening ofstomata of maize leaves when plants are illuminated (1500,uEm-2 s-') following a 30-min period of darkness. However, cal-culations of the intercellular [CO2] during induction indicateCO2 is not limiting to photosynthesis (H. Usuda, unpublisheddata).

During induction of photosynthesis in C3 plants, it has longbeen considered that light activation of enzymes and/or buildupof metabolites may be important factors (7). A current view oninduction of photosynthesis in C3 species links enzyme activa-tion, specifically activation of FBPase, and changes in metabolitelevels. Based on studies with isolated chloroplasts and the enzymeFBPase, it is suggested that an increase in the ratio of organic-P/Pi in the chloroplast may be required for the enzyme to reachmaximum level of activation (9, 12, 15). Maximum degree ofactivation ofFBPase appears to require both reductive conditions

240 USUDA ET AL.

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ENZYME ACTIVITY AND PHOTOSYNTHETIC RATE IN MAIZE

Table I. Activity ofFnrtose 1,6-Bisphosphatase in Whole Leaf, Mesophyll Protoplasts, and Bundle SheathStrands ofMaize

Three separate extracts were made on the whole leaf, while two separate replications were made on themesophyll and bundle sheath preparations. Whole plants were illuminated (1500 gE m-2 s-') for 20 min orkept in the dark at room temperature prior to extraction. Mesophyll and bundle sheath preparations werepreilluminated (1500 uE m-2 s-') or kept in the dark at 30°C for 20 min prior to extraction. DCMU was addedas ethanol solution (0.9% final concentration). Ethanol at this concentration does not affect the rate ofphotosynthetic "C02 fixation by isolated bundle sheath cells (25).

Fructose 1,6-

Material Light bisphosphatase Dark PEP NADP-malicLight + 10 M Carboxylase EnzymeDCMU

'tmol/mg Chl hLeaf 292 63.5

201 102201 78

Mesophyll protoplasts 58.6 51.1 54.4 947 11.355.9 41.5 1140 19.5

Bundle sheath 156 32.5 29.0 0 1172strands 203 42.2 31.8 7.3 1413

and the presence of the substrate fructose 1,6-bisP. Buildup oforganic-P outside the chloroplast can be important during in-duction of photosynthesis with isolated chloroplasts ofC3 plants.However, from studies with protoplasts and leaves of the C3species spinach, no substantial difference was found in darkversus light in the total pool oforganic-P (24), so it was concludedthat buildup ofthe total pool of organic-P may not occur in vivo.Rather, in leaves ofC3 plants, activation ofenzymes and changesin the relative concentrations of certain organic-P within thechloroplast (e.g. ribulose-1 ,5-bisP and fructose- 1,6-bisP) may beimportant during induction. In both C3 and C4 plants there isstill insufficient data with intact leaves on activation of variousenzymes, changes in metabolite pools, and rates of photosyn-thesis to make complete conclusions about the induction process.

Influence of Light Intensity on Photosynthesis and Light Ac-tivation of Enzymes in Maize. The light-response curves foractivation of NADP-MDH, pyruvate,Pi dikinase and FBPasewere similar to the light-response curve for photosynthesis, al-though higher light intensity was required to reach maximumrates ofphotosynthesis. While light activation ofenzymes in boththe C4 cycle and RPP pathway is required for C4 photosynthesis,light activation ofenzymes in C4 plants may serve as more thanan off/on switch for certain enzymes between dark/light. Ifserving only as an on/off switch, full activation of the enzymesmight occur at very low light intensities, well below that requiredto reach maximum rates of photosynthesis. In C4 plants, lightactivation of enzymes may regulate the flux of carbon throughvarious phases of carbon assimilation, or the degree of lightactivation of enzymes may limit the rate of photosynthesis at agiven light intensity. The activity of NADP-malate dehydroge-nase is well in excess ofphotosynthetic rate, so the enzyme wouldnot appear rate-limiting. The activity of FBPase due to lightactivation is equivalent to the rate of leaf photosynthesis (Figs.1, 2; Table I). In vivo, the chloroplastic FBPase needs to functionat one-third the rate of CO2 fixation during the regeneration ofRuBP. Additional activity of FBPase would be required in thechloroplast ifthe product ofphotosynthesis is converted to starch(maximum of one-sixth of the rate of CO2 fixation). The chlo-roplastic FBPase would therefore need to function, at maximum,up to one-half the rate of CO2 fixation. Since the activity ofFBPase due to light activation was approximately equivalent tothe rate of photosynthesis under a given condition (Figs. 1, 2;Table I), the chloroplastic FBPase would appear more thansufficient to accommodate CO2 assimilation. However, pyru-

vate,Pi dikinase activity was similar to the photosynthetic rate ata given light intensity (Fig. 2; H. Usuda, M. S. B. Ku, and G. E.Edwards, unpublished data with plants grown at different lightintensities). Therefore, pyruvate,Pi dikinase may be a rate limit-ing enzyme for C4 photosynthesis in vivo. Other enzymes, likechloroplastic FBPase and NADP-MDH, can apparently functionin vivo below substrate saturation and accommodate rates ofcarbon assimilation.

In leaves of wheat, a C3 plant, FBPase reached one-half max-imum degree of activation at about 18 w m-2 (4% of fullsunlight), and full saturation at 60 w m-2 (about 12% of fullsunlight) (14). As FBPase reached one-half maximum degree ofactivation at 300 ;&E m 2 sg- (about 15% full sunlight) and fullactivation at 700 tE m-2 sg' (about 35% of full sunlight) inmaize (Fig. 2), it appears much higher light intensity may berequired to activate the enzyme in C4 plants than in C3 plants.

Characteristics of FBPase in laize. In the C4 plant maize,the present study shows a several-fold activation of FBPase inthe light and inactivation in the dark, which is similar to thatreported in the C3 plant wheat (14). This is in contrast to resultsof Bjorkman and Badger (2) in which no light activation ofFBPase was found in leaves ofthe C4 plant T. oblongifolia. Eitherconditions used during extraction may mask the light activationof the enzyme or there may be species differences. In C3 plants,FBPase is located in the chloroplast and cytosol of mesophyllcells, with the FBPase of the chloroplast being light activated (1,2). The activity of FBPase obtained from darkened leaves ofmaize in the present study is similar to that found in darkenedleaves ofC3 plants (14; J. Kobza and G. E. Edwards, unpublisheddata) which may be predominantly that of the cytosolic enzyme.In darkened mesophyll protoplasts of maize the enzyme activitywas only found in the extrachloroplastic fraction following dis-ruption of the protoplasts.Maize leaves have a thioredoxin-linked FBPase and the en-

zyme can be activated in vitro by a reducing agent (DTT orphotochemically from spinach thylakoids) and thioredoxinf( 19).The present study indicates FBPase is light activated in vivo inmaize, apparently in both the mesophyll and bundle sheathchloroplast by a mechanism similar to that in C3 plants. Withthe isolated mesophyll and bundle sheath preparations it wasfound that both cell types had substantial activity of FBPase inthe dark, but the main part of the light-activated FBPase was inthe bundle sheath cells. It appears that both cell types may havesignificant amounts of cytosolic FBPase and that the chloroplas-

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0 400 800 1200 1600LIGHT INTENSITY (MuE m-2 sec')

FIG. 2. Influence oflight intensity on the activities ofFBPase, NADP-MDH, pyruvate,Pi dikinase, and photosynthesis under steady state con-ditions in leaves of maize. See legend of Figure I for an explanation ofthe abbreviations in the figure. 0, plants were grown at 700 uE m-2 s5';0, plants were grown at 400 PE ml2 s'. Initially, plants were given highlight intensity, 20 min dark, and then APS was measured on a single leafby increasing increments fiom low to high light (similar results wereobtained in a preliminary experiment if photosynthesis rates were meas-ured when changing increments fiom high to low light intensities).Between each light intensity a period of 20 min elapsed to allow a newsteady state to be reached. Likewise, for enzyme assays leafsamples weretaken at 20-min intervals, killed in liquid nitrogen, and stored in liquidnitrogen until assayed. For FBPase and NADP-MDH segments wereremoved from a single leaf during successive increases in the lightintensity (both enzymes assayed in each sample as in Fig. 1). For thePPDK, each sample (about I x I cm) was taken from a separate, similaraged leaf. The activity of the FBPase obtained from dark-treated leaves(at least part ofwhich is cytosolic) was subtracted from that of the light-treated leaves in order to compare photosynthetic rates with the light-dependent activation ofthe enzyme. The arrow on each curve indicatesthe light intensity required to reach the half-maximum light-dependentactivity.

tic FBPase is mainly in the bundle sheath chloroplast Due tothe difficulty in isolating intact, functional chloroplasts frommaize bundle sheath strands, the association ofthe light-activatedFBPase with bundle sheath chloroplasts of maize is yet to be

demonstrated. In C4 plants, the regenerative phase of photosyn-thesis is located in bundle sheath chloroplasts, and in somespecies, such as maize, starch is normally only synthesized inthese chloroplasts. Thus, as already mentioned, in these chloro-plasts FBPase may be involved in both the RPP pathway andstarch synthesis. Considering the distribution of these two func-

Table II. Activity and Intracellular Localization ofSeveral Enzymes inMesophyll Protoplast Extracts ofMaize

Values in parentheses are results from a second experiment.Enzyme Total 650-g Pellet Supernatant

pmol/mg Chl-h % distributionPEP carboxylase 792 (815) 4.6 (3.9) 95.4 (96.1)NADP-triose-P 1470 (1432) 92.9 (94.7) 7.1 (5.3)dehydrogenase

NADP-malic 10.8 9 (0) 100 (100)enzyme (11.4)

Fructose 1,6-bis- 30.6 2.9a (1.4r 97.1(98.6)phosphatase (28.3)' Activities measured using darkened chloroplasts.

Table III. Effect ofLight on Fructose l,6-BisphosphataseActivity ofMesohyll Chloroplasts ofMaize

Mesophyll chloroplasts were incubated for 20 min at 30C under light(1500 uE m2 s-') or dark prior to extraction. Two replications were runon chloroplasts isolated from mesophyll protoplasts.

LightExperiment Light +3 mM iDark

PGA + 10 ;sm DCMU Drj.mol/mg Chlh

1 8.2 3.0 2.4 0.92 10.9 3.6 3.6 0.4

tions in the maize leaf, it is reasonable that the chloroplasticform of FBPase would be primarily located in the bundle sheathcell. Low activity of a light-activable FBPase was found in themesophyll chloroplast of maize. Under continuous illuminationstarch is syntheized in mesophyll as well as bundle sheathchloroplasts of maize (4). Therefore, under some circumstancesthe low activity FBPase in the mesophyll chloroplasts may func-tion in starch synthesis, utilizing organic-P synthesized in bundlesheath chloroplasts. Presumably, the cytosolic FBPase in maizewould be involved in metabolism of triose-P to sucrose.Reducing equivalents are required for activating the thiore-

doxin-linked FBPase in maize (19). Lack of complete inhibitionof activation by PGA or DCMU in the mesophyll chloroplasts(Table III) may be related to the fact that the capacity for theHill reaction in these chloroplasts is quite high (7) while theFBPase activity in these chloroplasts is relatively low. On theother hand, in maize bundle sheath chloroplasts the activity ofthe Hill reaction is quite low in comparison to maize mesophylland C3 plant mesophyll chloroplasts (7, 16, 25). However, itappears there is sufficient reductant generated from the Hillreaction in bundle sheath chloroplasts of maize to activateFBPase (Table II), and reductant from other sources may not benecessary (i.e. from malate decarboxylation through NADP-malic enzyme).Acknowidgment-The authors apprecate the valuable dions with John

Kobza and Hitoshi Nakamoto and the help by Hitoshi Nakamoto in growing theplant material.

LITERATURE CITED

1. ANDEON LE, AR ASHTON, AH MOHAMED, R SCHEmE 1982 Light/darkmodulation ofenzyme activity in photosynthesis. Boscence 32: 103-107

2. BORKMAN 0, M BADGER 1977 Thermal stabilHty of photosynthetic enzymain heat- and cool-adaed C4 species. Carnegie Inst Wash Year Book 76:346-354

3. BUCHANAN BB 1980 Role of light in the regulation of chloroplas enzymes.Annu Rev Plant Physiol 31: 341-374

4. DOWNTON WJS, JS HAWKER 1974 Enzymes ofstarch and sucrowe meabolismin mesophyil and bundle sheath cells of maize leaves. In RL Biesi, ARFerguson, MM Creswell, eds, MecLanisms of Regulation of Plant Growth,Bulletin 12. The Royal Society ofNew Zealand, Wellington, pp 229-233.

242 USUDA ET AL.

www.plantphysiol.orgon January 23, 2020 - Published by Downloaded from Copyright © 1984 American Society of Plant Biologists. All rights reserved.

ENZYME ACTIVITY AND PHOTOSYNTHETIC RATE IN MAIZE5. EDWARDs GE, SC HUBER 1981 TheC4pathway. InMD Hatch, NK Boardman,

eds, The Biochemistry of Plants. A Comprehensive Treatise, Vol 8, Photo-synthesis. Academic Press, New York, pp 237-281

6. EDWARDs GE, SP ROBINSON, NJC TYLER, DA WALKER 1978 Photosynthesisby isolated protoplasts, protoplat extracts, and chloroplasts of wheat. PlantPhysiol 62: 313-317

7. EDWARDs GE, DA WALKER 1983 C3, C4: Mechanisms, and Cellular andEnvironmental Regulation of Photosynthesis. Blackwell Scientific, Oxford

8. HATCH MD 1978 Regulation ofenzymes in C4photosynthesis. In BL Horecker,ER Stadtman, eds, Current Topics in Cellular Regulation, Vol 14. AcademicPress, New York, pp 1-27

9. HUBER SC 1978 Substrates and inorganic phosphate control: The light activa-tion of NADP-glyceraldehyde-3-phosphate dehydrogenase and phosphori-bulokinase in barley (Hordeum vulgare) chloroplas FEBS Lett 92: 12-16

10. KANAI R, GE EDWARDS 1973 Separation of mesophyll protoplasts and bundlesheath cells from maize leaves for photosynthetic studies. Plant Physiol 51:1133-1137

1 1. KARABOURNIoTI G, Y MANETAS, NA GAVALAS 1983 Photoregulation ofphosphoenolpyruvate carboxylase in Salsola soda L. and other C4 plants.Plant Physiol 73: 735-739

12. LAING WA, M STITr, HW HELDT 1981 Control of CO2 fixation. Changes inthe activity of ribulosephosphate kinase and fructose- and sedoheptuose-bisphosphatase in chloroplasts. Biochim Biophys Acta 637: 348-359

13. LEEGOOD RC 1984 Rapid fractionation of leaves of Zea mays: Contents ofmetabolites in mesophy II and bundle sheath compartments. In C Sybesma,ed, Advances in Photosynthesis Research, Vol 3. Martinus Nijhoff/Dr WJunk, The Hague, pp 441-444

14. LEEGEOD RC, DA WALKER 1982 Regulation of fructose-1,6-bisphosphataseactivity in leaves. Planta 156: 449-456

15. LEEGOOD RC, Y KOBAYASHI, S NEIMANIs, DA WALKER, U HEBER 1982 Co-operative activation ofchloroplast fructose-1,6-bisphosphatase by reductant,pH and substrate. Biochim Biophys Acta 682: 168-178

16. LEEGOOD RC, D CROWTHER, DA WALKER, G HIND 1983 Energetics ofphotosynthesis in Zea mays. 1. Studies of the flash-induced eectrochromicshift and fluorescence induction in bundle sheath cells Biochim Biophys

Acta 722: 116-12617. MONSON RK, MA STIDHAM, GJ WILLIAMS III, GE EDWARDs, EG URIBE 1982

Temperature dependence of photosynthesis in Agropyron smithii Rydb. I.Factors affecting net C02 uptake in intact leaves and contribution fromribulose-l,5bisphosphate carboxylase measured in vivo and in vitro. PlantPhysiol 69: 921-928

18. NAKAMOTo H, GE EDWARDS 1983 Influence of oxygen and temperature onthe dark inactivation of pyruvate,Pi dikinase and NADP-malate dehydro-nase in maize. Plant Physiol 71: 568-573

19. NISHAZAWA AN, BB BUCHANAN 1981 Enzyme regulation in (4 photosynthesis.Purification and ppties of thioredoxin-linked fiuctose iphataseand sedoheptulose bisphosphatase from corn leaves. J Biol Chem 256: 6119-6126

20. ScHmTrT MR, GE EDWARDS 1981 Photosynthetic capacity and nitrogen useefficiency of maize, wheat and rice: A comparison between C3 and C4photosynthesis. J Exp Bot 32: 459-466

21. SLACK CR 1968 The photoactivation of a phosphopyruvate synthase in leavesofAmaranthus palmeri. Biochem Biophys Res Commun 30: 483-488

22. SPALDING MH, MR SCHurr, SB Ku, GE EDWARDS 1979 Intrcellular local-ization of some key enzymes of Crassulacean acid metabolism in Sedwnpraealtum. Plant Physiol 63: 738-743

23. STEIGER E, I ZIEGLE, H ZIEGLE 1971 Differences in the light activation ofNADP dependent glyceraldehyde-3-phosphate dehydrogenase and of ribu-lose-5-phosphate kinase between plants containing the Calvin and thosecontaining the C4-dicarboxylic acid pathway of photosynthetic carbon re-duction. Planta 96: 109-18

24. S-rr M, W WIRT, HW HELDT 1980 Metabolite levels during induction inthe chooplas and extrachloroplast compartments of spinach protoplass.Biochim Biophys Acta 593: 85-102

25. USUDA H, R KANAI, S MIYACHI 1975 Carbon dioxide assimilation andphotosystem II deficiency in bundle sheath stands isolated from (4 plants.Plant Cell Physiol 16: 485-494

26. WNTEMANS JFGM, A DE MoTS 1965 Spectrphotometric char ofchlorophyll and their pheophytins in ethanol. Biochim Biophys Acta 109;448-453

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