Light Regulation of Leaf Mitochondrial Pyruvate ... · (pyruvate andsodiumthiamine PPi; refs. 26, 30) and PDH phosphatase (Triton X-100; ref. 6), thereby effectively trap-pingthein

Plant Physiol. (1992) 100, 908-9140032-0889/92/1 00/0908/07/$01 .00/0

Received for publication March 23, 1992Accepted June 3, 1992

Light Regulation of Leaf Mitochondrial PyruvateDehydrogenase Complex'

Role of Photorespiratory Carbon Metabolism

Joanna Gemel* and Douglas D. Randall

Department of Biochemistry, University of Missouri, Columbia, Missouri 65211

ABSTRACT

Light-dependent inactivation of mitochondrial pyruvate dehy-drogenase complex (mtPDC) in pea (Pisum sativum L.) leaves wasfurther characterized, and this phenomenon was extended to sev-eral monocot and dicot species. The light-dependent inactivationof mtPDC in vivo was rapidly reversed in the dark, even afterprolonged illumination. The mtPDC can be efficiently cycledthrough the inactivated-reactivated status by rapid light-dark cy-cling. Light-dependent inactivation of mtPDC was shown to besuppressed by inhibitors of photorespiratory carbon metabolism,including 2-pyridylhydroxymethane sulfonate, isonicotinic acid hy-drazide, and aminoacetonitrile, and by an inhibitor of photosyn-thesis, 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Glycine fed to pealeaf strips in the dark yielded partially inactivated leaf mtPDC, andthis inactivation was blocked by inhibitors of glycine oxidation. Itis concluded that the photorespiratory glycine to serine conversionthat occurs in C3 leaf mitochondria can provide the NADH to driveoxidative phosphorylation and subsequent inactivation of mtPDC.Glycine oxidation also produces ammonium ion, which has beenshown to enhance the inactivation of mtPDC in vitro by stimulatingthe pyruvate dehydrogenase kinase that catalyzes the phosphoryl-ation (inactivation) of the mtPDC. Thus, light-dependent, photo-respiration-stimulated inactivation of the mtPDC can regulate car-bon entry into the Krebs cycle during C3 photosynthesis.

The mtPDC2 links glycolytic carbon metabolism with theKrebs cycle by catalyzing the oxidative decarboxylation ofpyruvate to acetyl-CoA. Pyruvate provides the primary sub-strate for the Krebs cycle, which, in turn, provides the reduc-ing equivalents for ATP production by oxidative phosphoryl-ation. The irreversible nature of the PDC reaction makes it aparticularly valuable site for regulation. Our in vitro and insitu studies have established that there is significant potential

' This research was supported by the Missouri Agricultural Exper-iment Station and National Science Foundation grant No. IBN-9201292. This is journal No. 11,613 of the Missouri AgriculturalExperiment Station. J.G. is the recipient of a Food for the 21st CenturyProgram Visiting Scientist Fellowship.

2Abbreviations: mtPDC, mitochondrial pyruvate dehydrogenasecomplex; PDH, pyruvate dehydrogenase component of the complex;RuBP, ribulose 1,5-bisphosphate; HPMS, 2-pyridylhydroxymethanesulfonic acid; INH, isonicotinic acid hydrazide; AAN, aminoaceto-nitrile.

for regulation of the mitochondrial complex through productfeedback by NADH and acetyl-CoA (23) and inactivation-reactivation by reversible phosphorylation (26, 27). Plantsare unique in having an additional isoform of PDC in theirplastids (10, 11) that is also quite sensitive to product feed-back regulation but does not undergo regulation by reversiblephosphorylation (10, 27). In vitro studies of the PDC kinasehave also shown that the phosphorylation-inactivation re-action is stimulated by micromolar NH4+ (29) and is inhibitedby pyruvate, the substrate for the PDC (26, 30). In situ studieswith purified pea leaf mitochondria have shown that thePDC phosphorylation status is increased when the mitochon-dria are oxidizing substrates other than pyruvate (6), inparticular, glycine, an intermediate of the photorespiratorycarbon oxidation pathway.The controversy of whether or not mitochondrial respira-

tion is occurring during photosynthesis has been long stand-ing (16), and recent reports by Kromer and colleagues (20,21) working with barley (Hordeum vulgare) leaf protoplastsprovide convincing evidence that mitochondrial ATP pro-duction is required for optimal photosynthesis. However,Budde and Randall (7, 8) recently reported that the pea leafmtPDC is primarily in an inactivated form in illuminatedleaves and that photosynthesis was required for this inacti-vation to occur. Inactivation of the mtPDC, which is theprimary point of entry for carbon into the Krebs cycle, couldcall into question whether the Krebs cycle is providing thereducing equivalents to drive mitochondrial ATP formation.Kromer and colleagues (20, 21) did not establish the sourceof the reducing equivalents for oxidative phosphorylation intheir experiments. Photorespiratory glycine to serine conver-sion in the mitochondria could easily provide sufficientNADH for oxidative phosphorylation, and, in fact, glycinehas been shown to be the preferred substrate for leaf mito-chondria (13). Thus, it would appear that the question is notwhether there is mitochondrial respiratory activity (16) dur-ing photosynthesis but what carbon source(s) is being oxi-dized.The light-dependent inactivation of mtPDC (7) provides a

mechanism to regulate or limit carbon entry into, and thuscarbon oxidation by, the Krebs cycle during photosynthesis.Such curtailment of Krebs cycle activity was suggested morethan 40 years ago by Benson and Calvin (3) when analysesof the Krebs cycle intermediates labeled during 14CO2 fixation

908

https://plantphysiol.orgDownloaded on April 22, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

https://plantphysiol.org

LIGHT REGULATION OF PYRUVATE DEHYDROGENASE COMPLEX

experiments showed that only malate was labeled in thelight, but label quickly moved into other Krebs cycle inter-mediates in the dark. The light-dependent regulation of sev-eral leaf enzymes is known (5, 12), and the light-dependent(photosynthesis-dependent) alteration in protein phosphoryl-ation status has recently been reviewed (8), including suchexamples as Chl a/b light-harvesting complex (2), pyruvate,Pi dikinase (9), phosphoenolpyruvate carboxylase (18, 25),and sucrose phosphate synthase (17). To our knowledge,reports of light-dependent/photosynthesis-dependent regu-lation of mitochondrial enzymes are limited to the pea leafmtPDC (7, 8). An observation of phytochrome-dependentenhancement of three other mitochondrial enzymes, Cytoxidase, succinate dehydrogenase, and fumarase, in cotyle-dons of Sinapis seedlings (28) was linked to increased denovo synthesis of these enzymes.

In this report, we describe further characterization of theconditions affecting the light-dependent inactivation of theleaf mtPDC and evidence linking this phenomenon to pho-torespiratory glycine oxidation.

MATERIALS AND METHODS

Plant Material

Pea (Pisum sativum L., cv Little Marvel), Brassica campestris,Brassica napus, and zucchini (Cucurbita pepo) seedlings weregrown in a growth chamber (10 h photoperiod, 250 ,E m-2s-', 180C) for 12 to 14 d before harvesting. For some exper-iments, zucchini seedlings were grown in the greenhouse forthe same period. Before experimental use, plants were rou-tinely dark adapted overnight.

Biochemicals

Biochemicals were purchased from Sigma (St. Louis, MO).Amersham (Arlington Heights, IL) supplied radiochemicals.Solutions of [1_14C]pyruvate were prepared by dissolving 50,uCi of [1-_4C]pyruvate in 6 mL of 20 mm sodium pyruvatecontaining 3 mm HCl and stored at -200C.

Enzyme Extraction and Assay

The in vivo steady-state activity of PDC was determinedusing the rapid sampling technique of Budde and Randall(7). Pea leaves (1-2 g fresh weight) were harvested and placedin the well of an ice-cold stainless steel cylinder with a 0.5-mm hole in the bottom covered by cheesecloth. The cell sapfrom the leaves was rapidly expressed by applying pressurevia a stainless steel piston attached to a drill press. The sapwas collected in a microfuge tube (no buffer or additives) andan aliquot immediately used to initiate the radiometric assayfor PDC activity. The pH of the sap was 6.8 ± 0.1. It tookapproximately 8 to 10 s to prepare the crude cell sap and toinitiate the reaction. A 50- to 100-,L aliquot of the expressedsap was injected into a serum stoppered 20-mL glass scintil-lation vial. The vial was equipped with a hanging center well(Kontes) containing a 2.5-cm2 piece of filter paper impreg-nated with 70 ,L of 5 M ethanolamine to trap CO2. The vialcontained the following assay reagents: 80 mm Tes-NaOH(pH 7.6), 0.1% (v/v) Triton X-100, 0.5 mM MgCl2, 0.2 mM

thiamine PPi, 2 mM fl-NAD, 0.12 mm Li-CoA, 2 mM cysteine,and 1 mm [1-14C]sodium pyruvate (750-1000 dpm/nmol) in1.0 mL. The assays were performed in a shaking water bath(300C). The basis for the rapid sampling of the tissue is tominimize any changes in the phosphorylation status ofmtPDC between the intact tissue state and the in vitro assaystate. For each analysis, a control assay was performed in theabsence of added extract or with boiled extract. After 2 min,the reaction was stopped by the addition of 50 uL of 6 MHCl. After 20 min, the paper wick used to trap "4CO2 wasremoved and assayed by liquid scintillation counting. Eachdatum point represents the mean of duplicate assays. Eachexperiment was repeated at least three times.The trapping of '4CO2 from the enzymic decarboxylation

of pyruvate was linear with increasing amount of expressedsap (up to 0.1 mL) and with time (up to 2 min). Deviationsfrom linearity with extended time or greater amounts ofexpressed sap were due to product inhibition (23). Whenisolated mitochondria were used, the activity of the mtPDCassayed by this radiometric procedure was 90 to 95% of therate determined by the standard spectrophotometric assay(6). Components of the reaction medium inhibit PDH kinase(pyruvate and sodium thiamine PPi; refs. 26, 30) and PDHphosphatase (Triton X-100; ref. 6), thereby effectively trap-ping the in vivo steady-state level of phosphorylation of themtPDC.

Inhibition of Photorespiration or Photosynthesis

In all experiments with inhibitors, detached leaves werefed an appropriate solution through the transpiration streamfor 1 h in the dark before illumination and assay. This enabledthe compounds tested to penetrate into cells at reasonableconcentration. INH and AAN solutions were neutralizedbefore use, but the pH of the HPMS solution was not adjustedbecause at neutral pH it is negatively charged and does notreadily enter cells (31). DCMU was dissolved in 0.1% (v/v)ethanol-water.

Mitochondria Isolation

Intact mitochondria were isolated and purified using a two-consecutive discontinuous Percoll gradient procedure as de-scribed by Fang et al. (14). Protein was determined by themethod of Bradford (4) using BSA as the standard.

Partial Purification of PDC

For the experiment described in Table I, mitochondria weretreated as follows: isolated mitochondria (20-40 mg of pro-tein mL-1) were frozen, thawed, diluted to 3 mg of proteinmL-1 in 50 mM Tes-KOH (pH 7.2), 0.1 M KCl, and 2 mm DTT,and homogenized with a Polytron (two 10-s bursts). Themitochondrial homogenate was centrifuged at 27,000g (max-imum) for 20 min to remove membranes. PDC was collectedfrom the supematant fraction by centrifugation at 200,000g(maximum) for 4 h and the pellet resuspended in a minimalvolume of 50 mm Tes-KOH (pH 7.2), 0.5 mM MgCl2, and 2mM DTT.

909



Plant Physiol. Vol. 100, 1992

9A8 Dark [=l Light Light l7 - night 15 min 30 min 45 min

6-5-4 -

3 -2 -

-

0100 500 1000

987654

32

87

654

3

2

0

100 250 1000

c

~~Dark = Light M Dark Mnight 45 min 5 min

100 1000Ught intensity, uE m2 s-'

DarkI0 mir

attributable to the chloroplast isoform of PDC that does notundergo reversible phosphorylation (10, 11) and is not inac-tivated in the light (data not shown). Details of this calcula-tion were described by Budde and Randall (7).To establish that this inactivation was not simply inhibition

of PDC activity by some low mol wt metabolite(s) producedduring photosynthesis, cell sap from illuminated leaves wasmixed with PDC partially purified from purified and lysedmitochondria. In addition, the cell sap from illuminatedleaves was desalted on Sephadex G-25 to remove any metab-olites that were present. Table I shows that the sap fromilluminated tissues had no effect on the activity of the par-tially purified PDC and that Sephadex G-25 treatment didnot reactivate or relieve inhibition of PDC activity in sapfrom illuminated tissues. The addition of pyruvate, whichinhibits the PDH kinase reaction (30), and NaF, which inhib-its the PDH-P phosphatase (24), also did not affect theactivation status of PDC in the cell sap (Table I). Thus, itwould appear that the greatly reduced PDC activity in illu-minated tissue is not due to any photosynthetically producedlow mol wt inhibitors. Because neither pyruvate nor fluorideappear to alter the activation status of the complex, we haveconcluded that the assay reflects the in vivo phosphorylationstatus of the mtPDC.To determine whether the light-dependent inactivation of

PDC activity was a more general phenomenon and notunique to pea leaves, we examined several other species, bothmonocots and dicots. Although total PDC activity from dark-ened leaf tissues tends to be lower in zucchini, soybean(Glycine max), bean (Phaseolus vulgaris), barley, tobacco (Ni-cotiana tabacum), red clover (Trifolium pratense), and maize,significant light-dependent inactivation was observed, andthis inactivation is illustrated for zucchini for which bothgrowth chamber- and greenhouse-grown tissue was used(Fig. 2, B and C). Arabidopsis thaliana (Columbia wild type)

Figure 1. A, Light-dependent PDC inactivation. Pea seedlings were

dark adapted overnight and then illuminated at the designated lightintensities for 15 to 45 min. B, Reversibility of light/dark effect on

PDC activity. Pea seedlings, dark adapted overnight, were illumi-nated for 45 min at the designated light intensities. Subsequently,plants were transferred to darkness for 10 min and then reillumi-nated at the same light intensity for 15 min. C, Reversibility of light-dependent PDC inactivation. Pea seedlings, dark adapted over-

night, were illuminated for 45 min at either 100 or 1000 ,uE m-2S-2.Subsequently, they were returned to darkness for 5 or 10 min. Thedashed lines show the level of estimated chloroplast PDC activity(see ref. 7).

RESULTS

We confirmed that leaf mtPDC activity was suppressed ina light-dependent manner (Fig. 1A) as reported by Buddeand Randall (7). This inactivation was readily reversible inthe dark (Fig. 1B). Time as short as 5 min (shortest timetested) was sufficient to restore PDC activity to 100% or more

of the overnight, dark-adapted level (Fig. 1C). To evaluatethe effect of light on mtPDC activity, it should be kept inmind that the radiometric assay using the crude cell sapmeasures total PDC activity. We used 1.4 nmol min-' mg ofprotein-' as that portion of PDC activity calculated to be

Table I. Effect of Plant Sap on mtPDC Activity

Experimental Conditions

PDC (partially purified)Sap (dark-adapted control)'PDC + sap (dark-adapted control)PDC (partially purified)Sap (illuminated)bPDC + sap (illuminated)

Sap (dark adapted)Sap (dark adapted) + Sephadex G-25Sap (illuminated)Sap (illuminated) + Sephadex G-25Sap (dark adapted)Sap (dark adapted) + 0.2 mM pyruvate +20 mm NaF

Sap (illuminated)Sap (illuminated) + 0.2 mm pyruvate +

20 mm NaF

cpm (percent)

182241685961 (100)182213123178 (101)

cpm (min mgof protein)

39743969 (100)23532294 (97)42994276 (100)

20491907 (93)

d Leaf tissue was dark adapted overnight. i Leaf tissue wasilluminated at 250 AE m-2 s-1, 45 min.

c

0

0)

E

CL

E0E

0

(UE

c

00a-

910 G EMEL AN D RAN DALL




Darknight

6

1

[ Light Light Light15 min 30 min 45 min

BZucchini-growth chamber

100 500 1000

100 500 1000I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

C

Zucchini-greenhouse

100 500 1000Ught intensity, PE m' s-'

Figure 2. Light-dependent PDC inactivation in two Brassica species(A) and growth chamber-grown (B) or greenhouse-grown (C) zuc-

chini. Plants were dark adapted overnight and then illuminated at250 ME m-2 s-1 for Brassica and at the designated light intensitiesfor zucchini. Time of light exposure varied from 15 to 45 min.

exhibited high PDC activity but did not exhibit light-depend-ent inactivation. Further experiments established that Arabi-dopsis mitochondria and cell sap have extremely active PDH-P phosphatase activity that could not be inhibited quicklyenough to allow us to use Arabidopsis for further studies. Incontrast, B. napus and B. campestris yielded high PDC activity,and we were able to easily demonstrate light-dependentinactivation of PDC activity (Fig. 2A).

Requirement for Photosynthesis and Photorespiration

To confirm that photosynthesis was required for the light-dependent inactivation of mtPDC, detached pea leaf pairswere allowed to take up 10, 50, or 200 jM DCMU for 1 h in

the dark before illumination for 1 h (Fig. 3). DCMU treat-ments decreased the mtPDC inactivation 76 and 69% for 10and 50 or 200 Mm, respectively. Incubation of purified mito-chondria under various levels of illumination from 25 to 500,uE m-2 s-1 had no effect on PDC activity.

Previous in situ studies from this laboratory have shownthat, when intact pea leaf mitochondria were oxidizing gly-cine in state 3, PDC was phosphorylated and inactivated (6).Furthermore, in vitro studies by Schuller and Randall (29)have shown that micromolar NH4' stimulates PDH kinaseactivity severalfold. Thus, it was logical to investigatewhether or not photorespiratory carbon metabolism could bea possible signal route connecting photosynthesis to mito-chondrial metabolism. If the light-dependent inactivation ofmtPDC is influenced by photorespiratory metabolism, inhi-bition of photorespiration should decrease the light-depend-ent inactivation of mtPDC, and enhancement of photorespir-ation or provision of glycine in the dark should result inincreased inactivation of leaf mtPDC.

Photorespiratory carbon metabolism can be inhibited atthe peroxisomal localized glycolate oxidase step by HPMS(35) and the mitochondrial localized glycine decarboxylasestep by INH (1) and AAN (33). When detached pea leaf pairswere allowed to take up these inhibitors in the dark for 1 hand then illuminated for 1 h, the light-dependent inactivationof mtPDC was inhibited more than 90% as shown in Figure4. None of these compounds inhibited PDC activity whenpurified intact mitochondria were incubated for 1 h in thepresence of inhibitor concentrations equal to those used forthe experiments with detached leaves described above (datanot shown).When 1- to 2-mm strips of pea leaf tissue were incubated

for 1 h in the dark with 20 mM glycine to mimic photorespir-ation and then assayed by the in vivo assay, mtPDC activitywas partially, but significantly, inhibited (Table II). However,

0 6

0 _ Dark (night) m Light

E4

E 3

C2

o 1--Z---t~~----|---- ----- ---- ---- -----

Control 10 ,t 50 , 200 FM

DCMU

Figure 3. Effect of DCMU on the light-dependent inactivation ofPDC. Detached leaf pairs of pea seedlings were fed DCMU (waterfor control) via the transpiration stream in the dark for 1 h. PDCactivity was determined for dark-treated tissue or after 1 h ofillumination at 250 ME m-2 s-'. The dashed line shows the level ofestimated chloroplast PDC activity.

5

4

3

-20-W

0 1

0

E5-4c

- 4

E 3

c 2

> 1

0

005a.

-

3

911

2

O




6

- Dark (nigjht) ZILightS-OL 5

E4

E53

c 2

(5-

Control I10mM HPMS8 10om M H 25mM AAN

Figure 4. Effect of inhibitors of photorespiration on light-dependentinactivation of PDC. Detached leaf pairs from pea seedlings were

fed inhibitors for 1 h in the dark via the transpiration stream. Waterwas supplied for control. PDC activity was determined for dark-treated tissue or after illumination at 250 ,E m-2 s-1 for 1 h. Thedashed line shows the level of estimated chloroplast PDC activity.

when inhibitors of glycine oxidation were also providedduring glycine-feeding experiments, mtPDC activity was notinactivated (Table II). These results support the hypothesisfor the involvement of photorespiratory metabolism in thelight-dependent inactivation of mtPDC.

Photorespiration can also be decreased by altering the 02

and CO2 levels in the atmosphere. When the in vivo PDCactivity was examined in illuminated tissue exposed to 1500,iL/L of CO2 and 1 to 2% 02, there was a 60 to 80% reductionin the light-dependent inactivation of mtPDC (data notshown). This further confirmed the results reported by Buddeand Randall (7).

DISCUSSION

The light-dependent and photosynthesis-dependent alter-ation in the steady-state level of pea leaf mtPDC activity (7,8) has been confirmed. This phenomenon has also beenshown to occur in other species, both monocots and dicots.Because light has no effect on PDC activity in purified intactmitochondria, and photosynthesis occurs in the chloroplast,we have concluded that the light signal is transduced throughsome product(s) of photosynthesis that is reaching themitochondrion.

Inhibition of mtPDC during photosynthesis should restrictKrebs cycle activity because both pyruvate and malate me-

tabolism by the cycle require an active PDC. Furthermore,this would limit the Krebs cycle as a source of reducingequivalents for ATP production by oxidative phosphoryla-tion. Because Kromer et al. (20, 21) showed that mitochon-drial ATP is essential for optimal photosynthesis (30-60%inhibition when mitochondrial ATP formation is inhibited byoligomycin), there must be an alternative source of reducingequivalents. Glycine oxidation by glycine decarboxylase thatoccurs in leaf mitochondria as part of the photorespiratorycarbon oxidation cycle (32) is well suited to be a componentof the signal transduction chain regulating mtPDC activity

and, at the same time, providing the reducing equivalents todrive mitochondrial ATP production by oxidative phos-phorylation. Glycine is an intermediate in the photorespira-tory metabolism of P-glycolate produced by Rubisco oxygen-ase action on RuBP, a process that only occurs in the lightbecause photosynthesis is required to generate RuBP and toactivate Rubisco (32). For a C3 plant, photorespiration, i.e.glycine oxidation, occurs at about 30% of the rate of photo-synthesis (32). Mitochondrial glycine oxidation is catalyzedby glycine decarboxylase and produces NADH, NH4', C02,and Cl-tetrahydrofolate (32). The NADH would be oxidizedby the mitochondrial electron transport chain to drive theformation of ATP for cytosolic needs (and to phosphorylatemtPDC). The NH4' from glycine oxidation could also stim-ulate the PDH kinase that phosphorylates the mtPDC as

shown in vitro (29). Consequently, the mtPDC would be verysensitive to glycine oxidation (as we have established in situ[6]). This working model for the light-dependent inactivationof mtPDC allows us to explain these key observations: (a)The need for mitochondrial ATP for photosynthesis. Reduc-ing equivalents for oxidative phosphorylation would be pro-

vided by glycine oxidation instead of from the Krebs cycle.(b) Failure to label Krebs cycle intermediates (except malate)in the light. mtPDC is inactivated in the light and preventsentry of carbon into Krebs cycle intermediates. (c) Light-dependent inactivation of mtPDC and lack of Krebs cycleactivity without concomitant loss of oxidative phosphoryla-tion. Glycine oxidation drives ATP formation.Gardestrom and Wigge (15) also concluded that glycine

oxidation provides the electrons for mitochondrial ATP for-mation and that this ATP met energy requirements in thecytosol. Kromer and Heldt (20) concluded that the mitochon-dria would also oxidize excess reducing equivalents fromphotosynthetic electron transport. Consequently, there wouldbe no requirement to shuttle redox equivalents generatedfrom glycine oxidation out of the mitochondria to be used inthe peroxisomal reduction of photorespiratory hydroxypy-ruvate to glycerate. These redox equivalents would be sup-

plied by the excess from photosynthetic electron transport.We propose that the rapid and reversible nature of the

light-dependent inactivation of the mtPDC is the result ofreversible phosphorylation of the complex. It is also obviousfrom the results reported here that not all of the mtPDC isinactivated (mtPDC that is phosphorylated is inactive, not

Table II. Glycine Effect on mtPDC Activity in DarknessPea seedlings were kept in darkness overnight. Detached leaves

were cut into 1- to 2-mm strips and incubated in the dark in theindicated solution for 1 h. PDC was then assayed using the in vivoprocedure and the estimated chloroplast PDC activity of 1.4 nmolmin-1 mg of protein-' was subtracted to give the mtPDC activity.

mtPDC ActivityExperimental Conditions nmol min-' mg of Percent of

protein-' control

H20 4.0 10020 mm glycine 2.2 5520 mm glycine + 25 mm AAN 4.4 11020 mm glycine + 10 mm INH 3.8 95

GEMEL AND RANDALL912




simply less active), suggesting that some of the PDC is notphosphorylated. A possible explanation may be that not allleaf mitochondria are associated with peroxisomes and chlo-roplasts as part of the photorespiratory pathway, i.e. eventhough they are capable of oxidizing glycine, they are suffi-ciently and/or physically separated from these other organ-elles and do not receive glycine. An even more speculativehypothesis may be that some mitochondria may have func-tionally different roles, depending upon their location in thecell and in different cells, i.e. not all cells will be producingglycine, but all cells have mitochondria containing PDC, andall mtPDC is capable of undergoing reversible phosphoryla-tion-inactivation.

Others have recently concluded that the Krebs cycle wouldnot be inhibited significantly during photosynthesis (19, 22,34). McCashin et al. (22) fed slices of wheat leaves '4C-labeled succinate and acetate and concluded that the Krebscycle was operating at 80% of the dark rate. However, mostof the label accumulated in malate, suggesting that conden-sation of C4 acid with acetyl-CoA (from mtPDC) was greatlydecreased. If one subtracts the increased accumulation inmalate from the total, the result would suggest that the Krebscycle may be only 30 to 40% operational, which is more inline with what we report here. The 80% decrease in aspartatelabeling in the light suggests that malate is not being oxidizedin the mitochondria to yield oxaloacetate or possibly an excessof reducing equivalents in the mitochondria is keeping theoxaloacetate reduced. No label was found in isocitrate orfumarate. Even more curious was the labeling of succinate,glutamate, and malate from acetate (22), because the onlyway to activate acetate for metabolism is in the chloroplast(26), and the acetate then enters the fatty acid biosyntheticpathway.

Wiskich et al. (34) concluded that the Krebs cycle canoperate simultaneously with glycine oxidation by the exist-ence of metabolons, i.e. nonhomogeneous distribution ofenzymes or regions of the mitochondria where glycine oxi-dation enzymes occur and other regions where only the Krebscycle is operational. This would allow the mitochondrialmalate dehydrogenase to serve two roles, e.g. in the Krebscycle to produce oxaloacetate and also to use oxaloacetate asa substrate to shuttle excess NADH from glycine oxidationout of the mitochondria. This, too, is compatible with ourresults. We did not observe inactivation of all of the mtPDC,and previously we did observe inactivation of mtPDC in situwhen mitochondria were oxidizing glycine (6).Kirschbaum and Farquhar (19) reported that elevating the

internal partial pressure of CO2 did not relieve the light-dependent inhibition of nonphotorespiratory respiration. Wealso do not believe that this is in conflict with our results orconclusions but indicates that there are probably multiplesignals being received by the mitochondria during photosyn-thesis. In addition, Kirschbaum and Farquhar (19) did notdetermine the rates of photorespiratory glycine oxidation orglycine levels under their conditions.We recommend caution in interpreting studies of Krebs

cycle activity in the light when one is feeding exogenouspyruvate or Krebs cycle intermediates that can generate py-ruvate at significant levels (16). Pyruvate is the most effectiveinhibitor known for the PDH kinase and phosphorylation of

mtPDC (26). The results reported here and those of Kromerand colleagues (20, 21) support both sides of the controversyconcerning whether mitochondrial respiration occurs in leaftissue during photosynthesis. Mitochondrial respiration isoccurring and mitochondrial ATP formation is occurring;however, the Krebs cycle activity is quite likely to be signifi-cantly curtailed in C3 plants during photosynthesis underconditions when photorespiration is occurring.

ACKNOWLEDGMENTS

We gratefully acknowledge the superb technical assistance ofNancy R. David and we thank Jan A. Miemyk and A.L. Moore forinsightful discussions of the results.

LITERATURE CITED

1. Asada K, Saito K, Kitoh S, Kasai Z (1965) Photosynthesis ofglycine and serine in green plants. Plant Cell Physiol 6: 47-59

2. Bennett J (1991) Protein phosphorylation in green plant chlo-roplasts. Annu Rev Plant Physiol Plant Mol Biol 42: 281-311

3. Benson AA, Calvin M (1950) The path of carbon in photosyn-thesis. VII. Respiration and photosynthesis. J Exp Bot 1:63-69

4. Bradford MM (1976) A rapid and sensitive method for quanti-tation of microgram quantities of protein utilizing the princi-ples of protein-dye binding. Anal Biochem 72: 258-264

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

6. Budde RJA, Fang TK, Randall DD (1988) Regulation of thephosphorylation of mitochondrial pyruvate dehydrogenasecomplex in situ. Plant Physiol 88: 1031-1036

7. Budde RJA, Randall DD (1990) Pea leaf mitochondrial pyruvatedehydrogenase complex is inactivated in vivo in a light-de-pendent manner. Proc Natl Acad Sci USA 87: 673-676

8. Budde RJA, Randall DD (1990) Light as a signal influencingthe phosphorylation status of plant proteins. Plant Physiol94:1501-1504

9. Burnell JN, Hatch MD (1983) Dark-light regulation of pyruvate,Pi dikinase in C4 plants: evidence that the same protein cata-lyses activation and inactivation. Biochem Biophys Res Com-mun 111: 288-293

10. Camp PJ, Miernyk JA, Randall DD (1988) Some kinetic andregulatory properties of the pea chloroplast pyruvate dehydro-genase complex. Biochim Biophys Acta 933: 269-275

11. Camp PJ, Randall DD (1985) Purification and characterizationof the pea chloroplast pyruvate dehydrogenase complex. Asource of acetyl-CoA and NADH for fatty acid biosynthesis.Plant Physiol 77: 571-577

12. Campbell WJ, Ogren WL (1990) A novel role for light in theactivation of ribulose bisphosphate carboxylase/oxygenase.Plant Physiol 92: 110-115

13. Dry IB, Day DA, Wiskich JT (1983) Preferential oxidation ofglycine by the respiratory chain in pea leaf mitochondria.FEBS Lett 158: 154-158

14. Fang TK, David NR, Miernyk JA, Randall DD (1987) Isolationand purification of functional pea leaf mitochondria free ofchlorophyll contamination. Curr Top Plant Biochem Physiol6: 175

15. Gardestrom P, Wigge B (1988) Influence of photorespiration onATP/ADP ratios in the chloroplasts mitochondria and cytosol,studied by rapid fractionation of barley (Hordeum vulgare)protoplasts. Plant Physiol 88: 69-76

16. Graham D (1980) Effects of light on 'dark respiration.' In DDDavies, ed, The Biochemistry of Plants: A ComprehensiveTreatise, Vol 2. Academic Press, New York, pp 525-579

17. Huber JLA, Huber SC, Wielsen TH (1989) Protein phosphoryl-ation as a mechanism for regulation of spinach leaf sucrosephosphate synthase activity. Arch Biochem Biophys 270:681-692

913




18. Jiao J-A, Chollet R (1991) Post-translational regulation of phos-phoenolpyruvate carboxylase in C4 and Crassulacean acidmetabolism plants. Plant Physiol 95: 981-985

19. Kirschbaum MUF, Farquhar GD (1987) Investigation of theCO2 dependence of quantum yield and respiration in Eucalyp-tus pauciflora. Plant Physiol 83: 1032-1036

20. Kromer S, Heldt W (1991) On the role of mitochondrial oxida-tive phosphorylation in photosynthesis metabolism as studiedby the effect of oligomycin on photosynthesis in protoplastsand leaves of barley (Hordeum vulgare). Plant Physiol 95:1270-1276

21. Kromer S, Stitt M, Heldt W (1988) Mitochondrial oxidativephosphorylation participating in photosynthetic metabolismof a leaf cell. FEBS Lett 226: 352-356

22. McCashin BG, Cossins EA, Canvin DT (1988) Dark respirationduring photosynthesis in wheat leaf slices. Plant Physiol 87:155-161

23. Miernyk JA, Randall DD (1987) Some kinetic and regulatoryproperties of pea mitochondrial pyruvate dehydrogenase com-

plex. Plant Physiol 83: 306-31024. Miernyk JA, Randall DD (1987) Some properties of pea mito-

chondrial phospho-pyruvate dehydrogenase phosphatase.Plant Physiol 83: 311-315

25. Nimmo GA, Nimmo HG, Fewson CA, Wilkins MB (1984)Diurnal changes in the properties of phosphoenolpyruvatecarboxylase in Bryophyllum leaves: a possible covalent modi-fication. FEBS Lett 178: 199-203

26. Randall DD, Miernyk JA, Fang TK, Budde RJA, Schuller KA

(1989) Regulation of the pyruvate dehydrogenase complexesin plants. Ann NY Acad Sci 573: 192-205

27. Randall DD, Williams M, Rapp BJ (1981) Phosphorylation-dephosphorylation of pyruvate dehydrogenase complex fromleaf mitochondria. Arch Biochem Biophys 207: 437-444

28. Schopfer P, Bayracharya D, Falk H (1975) Photocontrol ofmicrobody and mitochondrial development: the involvementof phytochrome. In H Smith, ed, Light and Plant Develop-ment. Butterworths, Boston, pp 193-212

29. Schuller KA, Randall DD (1989) Regulation of pea mitochon-drial pyruvate dehydrogenase complex. Does photorespiratoryammonium influence mitochondrial carbon metabolism? PlantPhysiol 89: 1207-1212

30. Schuller KA, Randall DD (1990) Mechanism of pyruvate inhi-bition of plant pyruvate dehydrogenase kinase and synergismwith ADP. Arch Biochem Biophys 278: 211-216

31. Servaites JC, Ogren WL (1977) Chemical inhibition of theglycolate pathway in soybean leaf cells. Plant Physiol 60:461-466

32. Tolbert NE (1983) The oxidative photosynthetic carbon cycle.Curr Top Plant Biochem Physiol 1: 63-77

33. Usuda H, Arron GP, Edwards GE (1980) Inhibition of glycinedecarboxylation by aminoacetonitrile and its effect on photo-synthesis in wheat. J Exp Bot 31: 1477-1483

34. Wiskich JT, Bryce JH, Day DA, Dry IB (1990) Evidence formetabolic domains within the matrix compartment of pea leafmitochondria. Plant Physiol 93: 611-616

35. Zelitch 1 (1959) The relationship of glycolic acid to respirationand photosynthesis in tobacco leaves. J Biol Chem 234:3077-3081

914 GEMEL AND RANDALL



Documents

Light Regulation of Leaf Mitochondrial Pyruvate ... · (pyruvate andsodiumthiamine PPi; refs. 26, 30) and PDH phosphatase (Triton X-100; ref. 6), thereby effectively trap-pingthein