The effect of glycidate, an inhibitor of glycolate synthesis, on photorespiration and net photosynthesis

ARCHIVES

OF BIOCHEMISTRY

The Effect

AND

BIOPHYSICS

163,

of Glycidate,

367-377 (1974)

an Inhibitor

Photorespiration

Synthesis,

on

and Net Photosynthesis

ISRAEL Department

of Glycolate

ZELITCH

of Biochemistry,

The Connecticut Agricultural New Haven, Connecticut 06504 Received January

Experiment

Station,

24, 1974

Glycolate synthesis was inhibited 40-50s in illuminated tobacco leaf disks, which have rapid rates of photorespiration, when floated on 20 mM potassium glycidate (2,3-epoxypropionate), an epoxide similar in structure to glycolate. The inhibitor also decreased the release of photorespiratory CO, about 40%, and the specificity of glycidate was demonstrated by the 40-50s increase in rate of photosynthetic CO, uptake observed in its presence. The importance of glycolate synthesis and metabolism in the production of photorespiratory CO, and the role of glycolate in diminishing net photosynthesis in species with rapid rates of photorespiration was thus further confirmed. L-(or 2S)-Glycidate was slightly more active than or.-glycidate, but glycidate was more effective as a specific inhibitor in leaf tissue than several other epoxide analogs of glycolate examined. The products of photosynthetic “CO, fixation after 3 or 4 min of uptake were proportionately altered in the presence of glycidate, and the specific radioactivity of the [‘C]glycolate produced was closer to that of the “CO, supplied. Glycidate inhibited glycolate synthesis in tobacco leaf disks irreversibly, since the degree of inhibition was the same for at least 2 hr after the inhibitor solution was removed. Glycidate also blocked glycolate synthesis in maize leaf disks, tissue with low rates of photorespiration, but large increases in net photosynthesis were not observed in maize with glycidate, because glycolate synthesis is normally only about 10% as rapid in maize as in tobacco. The demonstration of increases in net photosynthesis of 40-50s when glycolate synthesis (and photorespiration) is blocked with glycidate indicates in an independent manner that the biochemical or genetic control of photorespiration should permit large increases in plant productivity in plant species possessing rapid rates of photorespiration.

Research since about 1960 has provided evidence that large quantities of CO, are respired by photosynthetic tissues of many plant species during illumination (1). This light respiration, or photorespiration, results from the oxidation of photosynthetic products, and it often occurs at rates three to five times faster than the rate of “dark” respiration. Rapid photorespiration, when it occurs, diminishes net CO, assimilation. Hence, slowing photorespiration by biochemical or genetic means may be expected to greatly increase net photosynthesis and plant productivity (2). Considerable evidence indicates that the oxidation of glycolic acid provides the main

source of the CO, released in photorespiration (1, 3). Glycolic acid is synthesized as an early product of photosynthesis (4) by one or more of several reactions (2, 5) and the properties of the biosynthesis and oxidation of glycolic acid in leaves have many characteristics in common with those of photorespiration (1). I have previously shown that blocking glycolate oxidation in viuo with a suitable cu-hydroxysulfonate such as a-hydroxy-2pyridinemethanesulfonate will cause glycolate to accumulate at initial rates that are sufficient to account for photorespiration if the glycolate were normally oxidized (5). This sulfonate also inhibits photorespi367

368

ISRAEL

ration (6), and under conditions of rapid photorespiration in tobacco leaf tissue, the rate of net photosynthetic CO, assimilation was increased several-fold in the presence of the sulfonate (3). Specific biochemical inhibitors of the biosynthesis of glycolate are potentially more interesting than inhibitors of glycolate oxidation, because they may act more directly in slowing photorespiration. Furthermore, they would help to confirm the importance of glycolate metabolism in this process. No specific inhibitor of glycolate biosynthesis has thus far been described, a specific inhibitor being defined as one that does not also decrease photosynthesis. According to my hypothesis, such as inhibitor should diminish the rate of glycolate synthesis and increase the rate of CO, fixation when rapid photorespiration is blocked. I have previously described an assay for detection of inhibitors of glycolate biosynthesis (5), and this assay has been further refined in the present paper. Ideally I was seeking an irreversible active-site-directed inhibitor of the in. uivo system mainly responsible for glycolate synthesis. The recent findings that epoxides similar in structure to the substrate can react with carboxyl groups at the active site of enzymes to inactivate the enzymes through formation of ester linkages (8, 9) prompted an investigation of epoxide analogs of glycolate. This paper describes the inhibition of glycolate biosynthesis in leaf tissue by glycidic acid (2,3-epoxypropionate), an epoxide similar in structure to glycolic acid (Fig.1). The inhibition of photorespiration by glycidate and the increase in net photosynthesis caused by this epoxide are also demonstrated. MATERIALS

AND

METHODS

Assay for inhibitors of glycolate biosynthesis. The principle involved in the assay is outlined in Fig. 2. Each sample consisted of six leaf disks (usually of tobacco) 1.6 cm in diameter (total fresh weight about 240 mg) taken from leaves with a fresh weight of at least 5.0 g. The disks were cut with a sharp punch, and were assigned to each sample by use of a latin-square design (10) so that different locations on the leaf were equally represented in each sample. The disks were floated top side up on 2.5 ml of water in

ZELITCH

OH H-L--COOH

d

GLYCOLIC

ACI D

GLYCIDIC ACID FIG. 1. Structural formulas of glycolate and glycidate (2,3-epoxypropionic is a chiral center.

acid). Carbon-2 of glycidate

‘I Glycol~c

co

2

Ac,d Synthew lnh,b,tor

T-A -

I

d

Hydroxyrulfonate

Cti,OH-C00~ Glyrol~

CHO-COOHAc,d

+

Glycolore

Glyoxyl~c

Acid

Omdose

FIG. 2. Diagrammatic representation of the method used for assaying the activity of inhibitors of glycolate biosynthesis. During the first experimental period illuminated leaf disks were floated on solutions of the assumed inhibitor of glycolate synthesis. This solution was removed, and during the second period the leaf disks were floated on 10 mM a-hydroxy-2pyridinemethanesulfonate. The initial rate of glycolate accumulation was determined in comparison with leaf disks floated on water during the first experimental period. 50-ml beakers, three disks per beaker. The beakers were held in place in a cutout Plexiglas sheet in a 28°C water bath and illuminated from above with tungsten photoflood bulbs with a mirror beneath the beakers so that 2000 ft-candles were provided. The beakers were loosely covered with a Plexiglas sheet and illuminated for a preliminary period of 60 min to aid in the subsequent uptake of inhibitor. The water was then replaced with water (for the controls) or with solutions of an assumed inhibitor for an additional 60-min period with occasional shaking of the beakers. Then the solutions were replaced with 10 mM (Yhydroxy-2.pyridinemethanesulfonate, and the glycolate that accumulated in the leaf tissue during 2 min (for tobacco), or 6 min (for maize) was determined calorimetrically after separation of the glyolate on columns of Dowex 1 acetate anion-exchange resin (5). In this way the percentage of inhibition of the initial rate of glycolate accumulation (or synthesis) could be determined. Photorespiration assay. The details of this method

GLYCIDATE

EFFECT

which uses L’C-labeled tobacco leaf tissue have been described (6, 11). Illuminated leaf disks were labeled with a known quantity of “‘CO, in large Warburg flasks in a closed system. At zero time a rapid stream of CO,-free air was passed over the tissue and the “‘CO, released in the light and subsequently during a period in darkness was collected and measured. The results are usually expressed as the ratio of the ‘“CO, released in light to that in the dark during the same time interval. Photosynthetic “CO, uptake and photosynthetic products. Six leaf disks in each sample were strung together on a length of thread to facilitate rapid removal at the end of the experiment and placed in large (75ml) Warburg flasks. A thin layer of water (1.2 ml) was added carefully to avoid wetting the upper leaf surface, and the flasks were attached to manometers and maintained at 28°C and 2000 ft-candles for 60 min without shaking while open to the air. The water was then replaced with water (for controls) or inhibitor solution, and the flasks were shaken at 120 oscillations per min for 60 min. About 5 min before the end of this period, 50 11 of 50 mM NaH”C0, solution was placed in the sidearm of the vessel, and the sidearm was closed with a rubber serum stopper. At zero time, the system was closed and ‘“CO, was released by injection of acid through the serum stopper so that the initial total ‘“CO, concentration in the atmosphere was about 0.1%. At the end of the exposure to ‘VZO, (3-5 min), the leaf disks on their threads were taken from the vessels and rapidly plunged into boiling 20% ethyl alcohol and boiled for 3 min. The boiled disks were homogenized, the suspension made to 25 ml, and the radioactivity determined by scintillation counting of a 0.05-ml sample using either the sample channels ratio or external standard ratio method to obtain a constant cqunting efficiency. The “C-labeled products were separated into fractions using column chromatography on anion- and cation-exchange resins (12, 13). Paper chromatography was used to separate glycolate and glycerate from the glycolate fraction (12, 13), and high-voltage electrophoresis was used to isolate glycine and serine from the amino acid fraction (13). Glutamate and aspartate were separated from the aspartate fraction by elution from Dowex 1 acetate columns with 0.25 M acetic acid. Inhibitors. Potassium glycidate was prepared by treatment of 3-chlorolactate, which was synthesized by oxidation of glycerol a-monochlorohydrin with nitric acid (14), with KOH in absolute methanol as described by Blau et al. (15). Starting with 20 g of 3-chlorolactate, 14.7 g of K glycidate. l/2 H,O (16) were obtained (68% yield). The glycidate gave the correct analysis for oxirane oxygen (17) as expected from the above formula. Anal. Calcd for K: 28.9%. Found: 28.8%. Calcd neutral equivalent: 135. Found after passage through

369

ON PHOTOSYNTHESIS

Dowex 50 cation exchange resin, 131. Ethyl glycidate was synthesized by the classical procedure (18) by treating silver glycidate with ethyl iodide in ethyl ether and distilling off the ester. Reported bp (la), 161-163°C; found, 159~160°C. The glycidate as prepared above is racemic DL- or (2X)-glycidate (Fig. 1). In order to determine if one of the enantiomers was more active as an inhibitor, Lor (2S)glycidate was also prepared. m-Chlorolactate was first resolved by obtaining the crystalline brucine salt of L(+)-3.chlorolactate or (2R)-3-chlorolactate (19). L- or (2S)-Glycidate was then prepared by treatment with KOH in methanol as described above. A large number of precipitations was needed to purify the L-glycidate. The final preparation used had 83% of the theoretical oxirane oxygen content (17), and a neutral equivalent of 139 (calculated, 135). The other epoxides tested were obtained commercially. The a-hydroxy-2.pyridinemethanesulfonate was purchased from Columbia Organic Chemicals, Columbia, SC Plant material. Tobacco (various lines of Havana Seed) and Maize (hybrid Penn 602A) were grown in sand in a subirrigated greenhouse bench and were watered frequently with nutrient solution. The tobacco plants were generally assayed for photorespiration by the “C assay (6, ll), and only plants possessing high rates of photorespiration (light/dark ratio 3.0 or greater) were used. Excised leaves were maintained with their bases in water in darkness for 15-30 min before leaf disks were cut. RESULTS

Inhibition of glycolate accumulation by potassium glycidate. Illuminated tobacco leaf disks floated on water in the presence of sulfonate (Fig. 2) accumulated glycolate rapidly, rates of 52.8 to 91.7 pmoles/g fr wt.hr (Fig. 3), as found earlier (5). With increasing concentrations of glycidate provided during the preliminary period (Fig. 3), glycolate synthesis was increasingly inhibited. The average inhibition was about 47% with 20 mM and 38% with 10 mM potassium glycidate under the standard conditions of assay. Glycidate inhibited equally well at lower concentrations if it was not fully neutralized (the acid has a pK of about 3.0), presumably because it penetrated the leaf tissue more readily as the free acid. For example, 95% neutralized 10 mM potassium glycidate, at pH 4.0, gave about the same inhibition as fully neutralized 20 mM glycidate. However, the specificity of the inhibition is apparently less

370

ISRAEL

ZELITCH

reliable at pH 4.0 since the increases in net photorespiration. Tobacco leaf disks with CO, assimilation obtained were more vari- high rates of photorespiration (ratio of able than when neutralized potassium gly- “CO, release light/dark of 3.0 or greater) cidate was supplied. were floated on 20 mM glycidate under Effect of glycidate on photorespiration. conditions similar to those used to detect The “C assay of photorespiration provides inhibition of glycolate synthesis. Photoresa sensitive and reliable method of measur- piration was inhibited about 38% (Table I), ing photorespiration (6, ll), and it is a a value similar to the decrease in glycolate useful method for detecting inhibitors of synthesis, while the glycidate had little effect on the rate of “CO, released by respiration in darkness. This provides further evidence that photorespiration and dark respiration occur by different biochemical pathways. Narrowing the stomata1 pores of leaves would appear to bring about a slowing of photorespiration (6), hence it is important to establish that a presumed photorespiratory inhibitor does not just only close K GLYCIDATE. rn~ stomata. Experiments carried out under FIG. 3. The effect of potassium glycidate concenconditions similar to those used to measure tration on inhibition of glycolate accumulation in photorespiration showed that concentrailluminated tobacco leaf disks. The experiments were tions of potassium glycidate as great as 40 carried out as described under Materials and mM did not affect stomata1 apertures in Methods. Different symbols represent separate expertobacco leaf disks, hence the inhibition of iments with leaf tissue from different plants. The photorespiration by glycidate is not merely control values (without glycidate) for the initial rate an artifact of stomata1 closure. of glycolate accumulation were (in Kmoles/g fr wt. hr): open circles, circles, 52.8.

91.7;

closed

circles,

Increase assimilation.

65.9; half-open

TABLE EFFECT OF GLWDATE

Expt.

1

2

3

Treatment of leaf disks

ON PHOTORESPIRATION

in

photosynthetic

“CO,

My hypothesis requires that

I

OF TOBACCO LEAF DISKS

IN THE “C

“CO, Released in light (cpm)

“CO, Released in dark (cpm)

Ratio “CO 2 released light/dark

ASSA~”

Inhibition of Photorespiration by glycidate (‘%)

Water

497,000

117,000

4.2

-

Inhibitor

293,000

119,000

2.5

40

Water

747,000

118,000

6.3

-

Inhibitor

494,000

142,000

3.5

44

Water

408,000

114,000

3.6

-

Inhibitor

318,000

126,000

2.5

31

“The assay was similar to the one previously described (6, 11). Leaf disks were floated on water in large Warburg flasks for 1 hr at 28°C and 2000 ft-candles of illumination. The fluid was then replaced with water (controls) or with 20 mM K glycidate. After 15 min, 5 pmoles of *‘CO1 (about 3.5 x 10’ cpm) were released into the closed system, and after a further 30 min to insure complete uptake of the “CO,, the assay was begun. CO,-free air was swept through the flasks at a rate of five flask volumes per min, and the “CO, released between 5 and 20 min in the light and between 15 and 60 min in darkness was collected and measured by scintillation counting. The values given for the “COz released in the light have been multiplied by 3 to equalize the times of “CO, released in light and darkness. Under conditions similar to these experiments, independent tests showed that 20 rnM K glycidate did not close leaf stomata. Each experiment was performed with a leaf excised from a different plant.

GLYCIDATE

EFFECT

if glycidate inhibits glycolate synthesis and photorespiration in a specific manner, then leaf tissue with normally high rates of photorespiration should show increases in net photosynthesis similar in magnitude to the inhibition observed. Such increases in net CO, uptake are shown in Fig. 4, and the stimulation of net photosynthesis increased over the same range of increasing potassium glycidate concentration as did inhibition of glycolate synthesis under similar experimental conditions (Fig. 3). The photosynthetic rate was increased by an average of 39% in the presence of 20 mM potassium glycidate, a value similar to the percentage of inhibition of photorespiration (Table I). The control rates (disks on water) for photosynthesis appear to be low in comparison with rates often obtained on intact leaves (about 150 /*moles COJg fr wt .hr), and this deserves comment. This undoubtedly results because the leaf disks are floating on water, and thus only the upper leaf surface (which contains fewer stomata) readily participates in rapid assimilation of CO,. Although the time of exposure to ‘“CO, is measured from the moment acid is added to the [“Clbicarbonate solution in the sidearm of the Warburg vessel, it requires a further % min before appreciable quantities of ‘*CO, diffuse to the leaf

K GLYCIDATE

mM

FIG. 4. The effect of potassium glycidate concentration on the increase in net ‘“CO, uptake by illuminated tobacco leaf disks. The experiments were carried out as described under Materials and Methods with 3.0-min exposure to “C0,. Different symbols represent separate experiments with leaf tissue from different plants. The control (without glycidate) values for the rate of “CO, uptake were (in @moles ‘“CO,/g fr wt.hr): (0) 42.4: (0) 43.5; (A) 36.1; (x) 35.5.

ON PHOTOSYNTHESIS

371

disks and can be assimilated. Finally, there is little atmospheric turbulence in the closed Warburg vessels, so that the CO, concentration next to the leaf disks was probably much lower than the calculated 0.1%. This also diminishes the measured assimilation rate. Nevertheless, all of these conditions are identical in control leaf disks and those supplied with glycidate, so that the percentage increase in net photosynthesis observed appears to be a reliable one. Comparison of DL- and L-glycidate on glycolate accumulation and photosynthesis. The glycidate used throughout this paper was Dr.-glycidate. In order to determine whether one of the enantiomers was more effective as an inhibitor of glycolate synthesis, L- or (XS)-glycidate was compared at equal molar concentrations with DL- or (2RS)-glycidate (Table II). The experimental conditions used were somewhat different from those described for the standard assay under Materials and Methods, and potassium glycidate neutralized to pH 4.0 (95g) neutralized) was supplied to the leaf disks. These experiments indicate that the L-enantiomer is more effective (Table II, Expt. 1 shows an exception) than DLglycidate, but probably only slightly so. Comparison of glycidate with other epoxides as specific inhibitors of glycolate synthesis. Other epoxides with structures somewhat similar to glycolic acid were also examined for their ability to inhibit glycolate synthesis and increase net photosynthesis in leaf disks. These compounds included ethyl glycidate, glycidol, glycidaldehyde, sodium and potassium phenylglycidate, and ethyl phenylglycidate. None of these was as satisfactory as potassium glycidate either because they did not inhibit glycolate accumulation as well, or because they failed to stimulate net photosynthesis as effectively. For example, 1 mM glycidaldehyde inhibited glycolate synthesis 24%‘, but it did not increase photosynthesis, while higher concentrations severely inhibited photosynthesis indicating the inhibitor lacked the specificity of potassium glycidate.

372

ISRAEL

ZELITCH

should approximate the metabolic pool sizes, since such metabolites as phosphoglyceric acid are essentially uniformly labeled in similar times (12). The distribution of l’C, given as the percentage of the total “CO, fixed, is shown in Table III Inhibition ofglycolate accumulation Expt. (synthesis) by glycidate when net CO, uptake was 48% and 42% faster in the presence of glycidate. Some Initial rate Inhibition Inhibition pool sizes were relatively unaffected by of glycolate of glycolate of glycolate synthesis, disks accumulation. accumulation, glycidate treatment, including those of in water 5rnM 5rnM phosphoglycerate, phosphoglycolate, and (~moles/g oL-glycidate L-glycidate fr wt hr) (%“r) (%) glycolate. The total radioactivity of some fractions was increased by glycidate treat1 108 26 9 ment, including the neutral compounds 2 87.8 22 36 (carbohydrates), aspartate, glutamate, 3 60.5 18 24 glycerate, and the fraction consisting of Increase in net photosynthetic “CO, uptake the strong acids eluted from the Dowex 1 by glycidate column (fructose diphosphate, phospho“CO, uptake. Increase in Increase in enolpyruvate, ribulose diphosphate). Dedisks in water “CO, uptake “CO1 uptake creases in the percentage of “C in leaf (fimolesig in5mM in5mM fr nt .hr) o&Iycidate L-glycidate tissue supplied glycidate were observed (P-x) (%I only in glycine and serine. Although glycidate clearly inhibits the 4 38.9 61 39 synthesis of glycolate, the small pool of 41.0 66 5 54 radioactive glycolate did not appear to a Initial rates of glycolate accumulation (synthesis) change in size. However, as shown below, and the rate of net photosynthetic “CO, uptake were the rate of turnover of some glycolate determined as described under Materials and precursors is decreased by the inhibitor. Methods with several modifications. The disks were Glycine and serine, products of glycolate maintained for 90 min at 30°C in either water or K metabolism and part of the glycolate pathglycidate solution (95% neutralized to pH 4.0 after way of carbohydrate synthesis (20), depassage through Dowex 50-H’) and illuminated at creased in total radioactivity in response to 1700 ft.c before adding the sulfonate for 2.0 min (Expts. l-3). The conditions were similar (1300 ftglycidate treatment, but glycerate, which candles) before supplying the “CO, for 3.0 min in the is also in this pathway, increased in pool photosynthesis experiments (Expts. 4 and 5). size. Thus, such experiments have limited usefulness in evaluating the important metabolic changes brought about by glyciEthyl glycidate was more effective than date in leaf tissue, probably because the potassium glycidate as an inhibitor of glybiochemical compartmentation of pathcolate synthesis, but it did not increase net photosynthesis as reproducibly in leaf ways and metabolite storage obscures some of the important changes that may occur. disks. Phenylglycidate did not greatly inMoreover, pool sizes at best provide limcrease CO, uptake, and ethyl phenylglyciited information since it is the rate of date strongly inhibited photosynthesis (in sunflower leaf disks) in experiments in turnover of a metabolite such as glycolate which large increases in “CO, uptake were that will determine rates of photorespiration and not the quantity present. An found in the presence of potassium glyciestimate of the relative turnover rate of date. endogenous precursors of glycolate has Change in distribution of “C after phobeen made (in addition to the direct meatosynthesis in “CO, in presence of with glycidate. After fixation of ‘“CO, for 3 or 4 surement of glycolate accumulation sulfonate described before) by determining min the distribution of “C in various the specific radioactivity of glycolate prometabolites of illuminated leaf disks TABLE

II

COMPARISON OF EFFECT OF DL- AND L-GLYCIDATE ON (SYNTHESIS) INHIBITION OF GLYCOLATE ACCUMULATION AND INCREASE IN “CO, UPTAKE BY TOBACCO LEAF DISKS IN LIGH-P

GLYCIDATE

EFFECT

373

ON PHOTOSYNTHESIS

TABLE III EFFECT OF GLYCIDATE ON PERCENTAGEDISTRIBUTION OF “C IN ILLUMINATED TOBACCO LEAF DISKS SUPPLIED ‘“CO,” Fraction

or compound

Neutral compounds Aspartate fraction Aspartate Glutamate Phosphoglycerate Phosphoglycolate Fructose bisphosphate, phosphoenolpyruvate, ribulose diphosphate fraction Glycolate Glycerate Glycine Serine

Expt 1,3 min in “COz

Expt 2,4 min in “CO,

Leaf disks in water

Leaf disks in glycidat e

Leaf disks in water

Leaf disks in glycidate

4.4 3.8

5.1 6.2

6.0

11.5

3.1 0.18 17.5 2.3 2.0

5.4 0.68 17.6 2.9 3.0

0.28 3.1 15.3 22.8

0.34 5.2 7.6 6.8

29.0 3.2 4.4 0.31 4.1 6.3 16.9

26.9 2.5 6.4 0.37 6.1 4.7 5.7

“The results are given as the percentage of the total “CO, fixed by the leaf tissue in the times shown at 28°C and 2000 ft-c. In Expt 1 the leaf disks in water fixed “CO, at the rate of 40.8 and in glycidate they fixed 60.4 wmoles/g fr wt .hr (an increase of 48% in the presence of glycidate). In Expt 2 the leaf disks in water fixed “CO, at the rate of 57.1 and in glycidate they fixed 81.3 pmoles/g fr wt .hr (an increase of 428 in the presence of glycidate). About 80% of the total “C was accounted for in various fractions and compounds, and the “C content of only the more important ones are shown. The total “C fixed by the leaf disks (in cpm) was 359,000 and 532,000 in Expt 1, and 634,000 and 903,000 in Expt 2, respectively.

duced from “CO, when its synthesis is inhibited by glycidate. radioactivity Specific of C-l of [“Cjglycolate synthesized from “CO, in presence of glycidate. The specific radioactivity of the glycolate that accumulates in tobacco leaf tissue in the presence of sulfonate is about one-half of the initial specific radioactivity of the “CO, supplied to the closed system (5, 12), and is essentially constant after 5 min of exposure to “CO, (12). This dilution in specific radioactivity indicates that about one-half of the carbon found in glycolate under these conditions is derived from endogenous and largely nonradioactive carbon sources. Since some of this endogenous carbon might consist of nonradioactive photorespiratory CO, normally in the leaf, leaf disks treated with glycidate, an inhibitor of photorespiration, should contain less available endogenous CO, and perhaps lesser amounts of other carbon precursors of glycolate. If this is true, one would, therefore, expect the specific radioactivity of the glycolate carbon atoms to be still closer to that of the “CO,

supplied when glycidate is added to the leaf. The experiments summarized in Table IV show that when glycidate inhibited the initial rate of glycolate synthesis about 3596, the radioactive pool size was decreased less on a percentage basis. Therefore, the specific radioactivity of each of the carbon atoms of glycolate was increased (about 20%) by treatment with glycidate, showing that the turnover rates of endogenous sources of carbon that contribute to glycolate biosynthesis were diminished by the inhibitor. Comparison of the effect of glycidate on tobacco and maize. The relative importance of various possible pathways of glycolate biosynthesis apparently differs in tobacco leaf tissue in comparison with leaves of species possessing low rates of photorespiration such as maize. Part of the evidence in support of this view comes from the demonstration that isonicotinic acid hydrazide inhibited glycolate synthesis in tobacco but not in maize, while this compound induced a slow accumulation of

374

ISRAEL

ZELITCH

glycolate (by blocking glycolate oxidase in viuo) in maize but not in tobacco (5). Glycidate (2 mM) did not inhibit partially purified glycolate oxidase from spinach leaf (21) when they were allowed to react for 30 min at pH 7.6 and then assayed with 5 mM glycolate. Potassium glycidate (20 mM) also did not inhibit glycolate oxidase in tobacco leaf tissue, since glycolate accumulation could not be detected in the presence of the inhibitor (Table V, Expts. 1 and 3). However, an inhibition of glycolate oxidase by glycidate was observed in maize leaves, where a slow accumulation of glycolate was found (Table V, Expts. 1 and 2). Glycidate also inhibited glycolate synthesis to a similar extent in maize as in tobacco on a percentage basis (compare Table V, Expt. 2 with Fig. 3). It should be emphasized that the rate of glycolate synthesis (initial rate of accumulation with sulfonate) of about 5 pmoles/g fr wt .hr in maize was, as before (5), only about one-tenth as rapid as the rates of synthesis in tobacco (Fig. 3). The effect of 20 mM glycidate on the rate of net photosynthesis in maize leaf disks

was measured many times, and increases in photosynthesis such as are shown with tobacco in Fig. 4 were never observed. Table VI shows “CO, uptake assays carried out with tobacco and maize leaf disks at the same time, and confirms the large increases in net photosynthesis in tobacco (as in Fig. 4) but not in maize. Although glycidate effectively inhibits glycolate synthesis in maize (Table V, Expt. 2), the rate of glycolate biosynthesis (and thus photorespiration) is probably already so low that blocking it further has little effect on net photosynthesis. Glycidate as an irreversible inhibitor of glycolate synthesis in tobacco leaf disks.

Studies with isolated enzymes (8, 9) have shown that epoxides similar in structure to the substrate inactivate the enzyme by specific reaction with exposed groups near the active site of the protein. If glycidate acts in a similar manner in leaf tissue by reacting with one or more of the enzymes responsible for glycolate synthesis, one would expect that once the glycidate had reacted the removal of the inhibitor solution from the leaf disks would not alter the

TABLE EFFECT OF GLYCIDATE

IV

ON SPECIFIC RADIOACTIVITY OF [%]GLYCOLATE ACCUMULATED LEAF DISKS SUPPLIED WHYDROXYSULFONATE~

FROM “CO,

Expt 1 Disks in water, then sulfonate Photosynthetic rate (pmoles “COJg fr wt .hr) Glycolate accumulation (pmoles/6 disks) Total “C found in glycolate (% of “CO, fixed) Specific radioactivity in C-l of glycolate (cpm/pmole C) Increase in specific radioactivity in presence of glycidate (%)

72.3 0.244 12.0 263,000 -

BY TOBACCO

Expt 2

Disks in glycidate, then sulfonate

Disks in water, then sulfonate

73.8 0.166 9.6 315,000 20

77.3

70.6

0.224 16.5

0.137 13.1

422,000 -

Disks in glycidate, then sulfonate

497,000 18

a Six leaf disks were threaded together and floated on water in large (75ml) Warburg flasks at 28°C and 2000 ft-c for 1 hr in air, then the water was replaced with either water or 20 mM K glycidate for 1 hr. At zero time, the fluid was replaced with 10 mM a-hydroxy-2-pyridinemethanesulfonate and 3.5 pmoles of “CO, (740,000 cpm/rmole C) were released into the atmosphere in a closed system. At the end of 5.0 min, the disks were quickly removed by the thread and killed. Glycolic acid was isolated and determined as described under Materials and Methods, It was assumed that the [“C]glycolate was equally labeled after 5-min exposure to “CO, (12). It should be noted that increases in photosynthetic rate usually observed with glycidate were not found in the above experiments, but that the sulfonate was also present during the exposure to “CO,.

GLYCIDATE

EFFECT

functions inhibitor uiuo.

degree of inhibition for some time afterward. Table VII shows that when the glycidate solution was removed from leaf disks previously exposed to the inhibitor, for at least 2 hr thereafter the percentage of inhibition of the initial rate of glycolate synthesis was not changed. These results are consistent with the view that glycidate

OF EFFECT OF GLYCIDATE

Experimental Expt

Large increases in net photosynbhesis were previously found in tobacco leaf when glycolate oxidation was blocked with an V

ON GLYCOLATE ACCUMULATION LIGHF

IN TOBACCO AND MAIZE

Glycolate

conditions

Preliminary period

as a specific and irreversible of the synthesis of glycolate in DISCUSSION

TABLE COMPARISON

375

ON PHOTOSYNTHESIS

accumulation

(pmoles) Maize

Tobacco

Period in 10 mM sulfonate Per Expt

LEAF DISKS IN

Per g fr Inhibition wt.hr by glycidate

Per Expt

P;;,\f’

(%) Water 120 min Water 60 min; 20 glycidate 60 min

1

2

mM

3

Water 90 min 10 mM K glycidate Water 90 min 10 mM K glycidate

0.078

None None

None

-

K

None

0.052

0.22

K

6 min 6 min

0.124b 0.052b

5.2b 2.2b

None None 2 min 2 min

90 min 90 min

0

-

K

Water 120 min Water 60 min; 20 mM glycidate 60 min Water 120 min Water 60 min; 20 mM glycidate 60 min

0

Inhibition by glycidate (S)

0.33

-

58

-

0 0.579 0.281

0 72.4 35.1

52

“Illuminated leaf disks were floated for a preliminary period as described under Materials and Methods under the conditions shown. The effect of glycidate on glycolate accumulation was determined without any further additions as part of each experiment, and accumulation was calculated by comparison with leaf tissue on water. In Expts 2 and 3, glycolate accumulation was also determined after the addition of 10 mM cu-hydroxy-2-pyridinemethanesulfonate for the times shown. b A correction has been made in these values for the glycolate accumulation during the preliminary period TABLE COMPARISON

OF GLYCIDATE

Expt

‘“CO,

VI UPTAKE

IN TOBACCO AND MAIZE

Tobacco Disks in water Cm;l~ TpJg

i

ON PHOTOSYNTHETIC

40.8 57.1

LEAF DISKSO

Maize

Disks in 20 mM K glycidate (pmoles “COJg fr wt .hr)

Effect of gly;i&te c

Disks in water trmoles “CO$g fr wt hr)

Disks in 20 mM K glycidate (pmoles “COdg fr wt hr)

60.4 81.3

t48 1-42

60.8 62.3

62.8 58.3

a The experiments were carried out as described under Materials for 3.0 min in Expt 1 and 4.0 min in Expt 2.

and Methods,

Effect of glycidate (SIo) t4.6 -6.4

The exposure to ‘“CO, was

376

ISRAEL

ZELITCH

TABLE EFFECT OF GLYCIDATE

ON INHIBITION

Expt

1 2

VII

OF GLYCOLATE ACCUMULATION IN ILLUMINATED AFTER REMOVAL OF GLYCIDATE“

Glycolate Leaf disks in water 2 hr

Leaf disks in water 1 hr; glycidate 1 hr

38.3 32.9

15.6 21.0

accumulation Inhibition (%)

59 36

TOBACCO LEAF DISKS 2 HR

(Fmolesig fr wt .hr) Leaf disks in water 4 hr

Leaf disks in water 1 hr; glycidate 1 hr; water 2 hr

41.1 50.3

23.1 20.4

Inhibition (%)

44 59

a Four samples of leaf disks were treated for a preliminary period as described in the assay for inhibitors of glycolate biosynthesis under Materials and Methods, with 20 mM K glycidate as the inhibitor. Two samples were treated in the usual way as shown above, and in the other two samples the fluid was removed and replaced with water for an additional 2 hr before a-hydroxysulfonate solution was used to measure the initial rate of glycolate accumulation.

a-hydroxysulfonate (3), and this constitutes part of the evidence that glycolate metabolism is largely responsible for the production of photorespiratory CO,. The sulfonate also inhibited photorespiration in the “C assay (6), and glycolate accumulated rapidly in the leaf, showing glycolate oxidation was inhibited in vivo. Glycidate did not inhibit extracted glycolate oxidase from spinach leaves nor did it inhibit glycolate oxidase in tobacco leaf disks, since no glycolate accumulation could be detected in the presence of glycidate alone (Table V). Clearly, glycidate inhibited the synthesis of glycolate (Fig. 2), and brought about increases in net photosynthesis without affecting the carboxylation efficiency or photochemistry. This suggests that photorespiration is largely responsible for the decreased net photosynthesis observed in inefficient species, such as tobacco, compared with efficient species, such as maize. The specificity of glycidate as an inhibitor of glycolate synthesis in illuminated leaf tissue of a species with normally high rates of photorespiration has been documented in this paper. Thus, a concentration of glycidate that inhibited glycolate synthesis 40-50% (Fig. 3, Table V, Table VII) also inhibited photorespiration in the “C assay about 40% (Table I), and it increased net photosynthetic CO, uptake by 40-50% (Fig. 4, Table II, Table VI). The increase in net photosynthesis, similar to the degree of inhibition of glycolate synthe-

sis and of photorespiratory COZ, again demonstrates the importance of these reactions in decreasing net photosynthesis. The role of glycolate metabolism in photorespiration is further indicated in an independent way since glycidate inhibited both processes to a similar extent. Glycidate did not affect the release of CO, during respiration in darkness, showing that dark respiration and photorespiration have biochemically distinct pathways. Glycolate synthesis in illuminated leaf tissue is probably inhibited by any substance that greatly interferes with net photosynthesis. Thus 3-(4-chlorophenyl)-l , ldimethylurea, an inhibitor of photosynthetic electron transport (22) strongly inhibited glycolate synthesis and photorespiration in tobacco leaf disks (6), but CMU decreased rather than increased photosynthetic “CO, uptake unlike the effect of glycidate. The presence of 3-(4-chlorophenyl)-1, 1-dimethylurea also decreased the specific radioactivity of [“Clglycolate synthesized from “CO, present in the atmosphere, indicating that endogenous sources of carbon were favored for synthesis over the “CO,. Glycidate, on the other hand, increased the specific radioactivity of the carbon atoms of glycolate (Table IV), suggesting that the inhibitor decreased the turnover rates of endogenous sources of carbon that contribute to glycolate synthesis. Multiple pathways for the biosynthesis of glycolate undoubtedly occur simultane-

GLYCIDATE

EFFECT

377

ON PHOTOSYNTHESIS

ously in a given tissue (1, 2, 23), and some of these pathways are derived from recently fixed (or even directly fixed) carbon dioxide while others do not directly involve CO, assimilation (5). There is evidence that glycolate might be synthesized by an oxygenation reaction catalyzed by ribulose bisphosphate carboxylase that produces phosphoglycolate (24). However, I have found that 5 mM glycidate has no effect on the formation of phosphoglycerate from “CO, by the isolated spinach enzyme at pH 9.3, when the reaction is carried out in air or in 100% 0,. It is still not known which of the biosynthetic reactions are inhibited by glycidate, and perhaps experiments with shorter times of ‘CO, fixation and in simpler systems than leaf tissue, such as isolated chloroplasts, will help determine this. Glycidate does, however, appear to react irreversibly with enzymes in living tissue, since inhibition of glycolate synthesis remained high for at least 2 hr after glycidate was removed from leaf disks (Table VII). Glycolate is synthesized only about 10% as rapidly in maize as in tobacco leaf (see (5) and Table V, Expt. 2). Hence blocking glycolate synthesis in maize with glycidate had little effect on net CO, fixation (Table VI). Earlier I found that isonicotinic acid hydrazide inhibited glycolate synthesis in tobacco, and inhibited glycolate oxidase slowly in maize but not in tobacco (5). Glycidate also did not inhibit glycolate oxidation in tobacco, but glycolate accumulated slowly with the inhibitor in maize (Table V, Expts. 1 and 2). This suggests that the glycolate oxidase of maize is more sensitive to control, perhaps because of the changed metabolite concentration (Table III) than is the enzyme in tobacco leaf. The failure to observe increases in the net photosynthesis in maize by inhibiting glycoIate synthesis with glycidate, while obtaining such increases in tobacco, provides further evidence to support the view that photorespiration does not account for an important part of the CO, budget in maize. The demonstration that glycidate blocks glycolate synthesis and increases net photosynthesis 40-50% in leaf tissue with a

shows high rate of photorespiration, further (1, 2, 11) that biochemical or genetic control of photorespiration, perhaps achieved by slowing glycolate synthesis, should make possible large increases in the productivity of many plant species. ACKNOWLEDGMENTS I thank Dr. K. R. Hanson for helpful discussion, Pamela Beaudette for technical assistance, and George R. Smith for growing the plants. REFERENCES 1. ZELITCH, I. (1971) Photosynthesis, Photorespiration, and Plant Productivity, Academic Press, New York. 2. ZELITCH, I. (1973) Proc. Nat. Acad. Sci. USA 70,

579. 3. ZELITCH, I. (1966) Plant Physiol. 41, 1623. 4. BENSON, A. A., AND CALVIN, M. (1950) J. Exp. Bot. 1, 63. 5. ZELITCH, I. (1973) Plant Physiol. 51, 299. 6. ZELITCH, I. (1968) Plant Physiol. 43, 1929. 7. BAKER, B. R. (1967) Design of Active-SiteDirected Irreversible Enzyme Inhibitors. The Organic Chemistry of the Enzymic Active-Site, Wiley-Interscience, New York. 8. TANG, J. (1971) J. Biol. Chem. 46, 4510. 9. SCHRAY, K. J., O’CONNELL, E. L., AND ROSE, I. A. (1973) J. Biol. Chem. 248, 2214. 10. VICKERY, H. B., LEAVENWORTH, C. S., AND BLISS, C. I. (1949) Plant Physiol. 24, 335. 11. ZELITCH, I., AND DAY, P. R. (1973) Plant Physiol. 52, 33. 12. ZELITCH, I. (1965) J. Biol. Chem. 240, 1869. 13. ZELITCH, I. (1972) Plant Physiol. 50, 109. 14. BAER, E. (1952) Biochem. Prep. 2, 25. 15. BLAU, N. F., JOHNSON, J. W., AND STUCKWISCH,C. G. (1954) J. Amer. Chem. Sot. 76, 5106. Chem. Ges. 13, 16. MELIKOFF, P. (1880) BeF De&. 271. 17. ROSS, W. C. J. (1950) J. Chem. Sot. 2257. 18. MELIKOFF, P., AND ZELINSKY, N. (1888) Ber. Deut. Chem. Ges. 21, 2052. 19. TSUNOO, S. (1935) Ber. Deut. Chem. Ges. 68B, 1341. 20. TOLBERT, N. E. (1971) Annu. Reu. Plant Physiol. 22, 45. 21. ZELITCH, I., AND OCHOA, S. (1953) J. Biol. Chem. 201, 707. 22. WESSELS, J. S. C., AND VAN DER VEEN, R. (1956) Biochim. Biophys. Acta 19, 548. 23. ZELITCH, I. (1973) Curr. Aduan. Plant Sci. Nov., p. 44. 24. ANDREWS, T. J., LORIMER, G. H., AND TOLBERT, N. E. (1973) Biochemistry, 12, 11.