Cycle with Intact Leaves and Isolated Chloroplasts of C3 Plants1

Mass spectrometric techniques were used to trace the incorporation of 1180loxygen Into metabolites of the photorespiratory pathway. Glycolate, glycine, and serine extracted from leaves ofthe C3 plants, Spinacis okraces L., Atripex hastata, and Helianthus annuus which had been exposed to 1Oloxygen at the C02 compensation point were heavily labeled with 180. In each case one, and only one of the carboxyl oxygens was labeled. The abundance of 180 in this oxygen of glycolate reached 50 to 70% of that of the oxygen provided after only 5 to 10 seconds exposure to I"Oloxygen. Glycine and serine attained the same final enrichment after 40 and 180 seconds, respectively. This confirms that glycine and serine are synthesized from glycolate. The labelng of photorespiratory intermediates in intact leaves reached a mean of 59% of that of the oxygen provided in the feedings. This indicates that at least 59% of the glycolate photorespired is synthesized with the fixation of molecular oxygen. This estimate is certainly conservative owing to the dilution of labeled oxygen at the site of glycolate synthesis by photosynthetic oxygen. We examined the yield of 180 in glycolate synthesized in vitro by isolated intact spinach chloroplasts in a system which permitted direct samplng of the isotopic composition of the oxygen at the site of synthesis. The isotopic enrichment of glycolate from such experiments was 90 to 95% of that of the oxygen present during the incubation. The carboxyl oxygens of 3-phosphoglycerate also became labeled with 180 in 20and 40-minute feedings with 115Oloxygen to intact leaves at the CO2 compnsation point. Control experiments indicated that this label was probably due to direct synthesis of 3-phosphoglycerate from glycolate during photorespiration. The mean enrichment of 3-pbospboglycerate was 14 ± 4% of that of glycine or seine, its precursors of the photorespiratory pathway, in 10 separate feeding experiments. It is argued that this constant dilution of label indicates a constant stoichiometric balance between photorespiratory and photosynthetic sources of 3-phosphoglycerate at the C02 compensation point. Oxygen uptake sufficient to account for about half of the rate of 180 fixation into glycine in the intact leaves was observed with intact spinach chloroplasts. Oxygen uptake and production by intact leaves at the C02 compensation point indicate about 1.9 oxygen exchanged per glycolate photorespired. The fixation of molecular oxygen into glycolate plus the peroxisomal oxidation of glycolate to glyoxylate and the mitochondrial conversion of glycine to serine can account for up to 1.75 oxygen taken up per glycolate. These studies provide new evidence which supports the current formulation of the pathway of photorespiration and its relation to photosynthetic metabolism. The experiments described also suggest new approaches using ' C.I.W.-D.P.B. Publication No. 619. 2Permanent address: Department of Plant Biology, Carnegie Institute of Washington, 290 Panama Street, Stanford, California 94305. Partially supported by an A. N. U. visiting fellowship. 3Permanent address: Central Research Department, E. I. DuPont de Nemours & Co., Experimental Station, Wilmington, Delaware 19898. stable isotope techniques to study the rate of photorespiration and the balance between photorespiration and photosynthesis in vivo. Substantial uptake of 180 during photosynthesis of intact leaves of C3 and C4 plants in the presence of I'8OJoxoygen has been observed (15, 29, 43) and at least part of the 1 O absorbed is incorporated into the carboxyl oxygen atoms of glycine and serine (2, 14). The oxygenolytic cleavage of RuP24 by purified RuP2 carboxylase/oxygenase in the presence of [18OJoxygen in vitro forms P-glycolate labeled in one of the carboxyl oxygen atoms (24). The synthesis and excretion of [18O]glycolate by photosynthetic organisms can be explained by this reaction (13, 26) and because the oxygen ofcarboxyl groups exchanges only slowly with the oxygen of water at neutral pH (35), the 18Q of glycolate is probably retained during the subsequent metabolism of glycolate in the glycolate pathway (for reviews see refs. 36 and 40) thus accounting for the incorporation of 18Q into glycine and serine. This fixation of 18Q from molecular oxygen into photorespiratory intermediates provides a specific and direct approach for tracer studies of the pathway of photorespiration. Here, we report studies which utilized MS to examine the isotopic composition of atmospheric or dissolved gases during incubation of intact leaves or chloroplasts, with labeled molecular oxygen and combined GCMS to examine the 18Q enrichment of photorespiratory intermediates extracted from intact leaves or chloroplasts. We have used these techniques: (a) to examine the rate and sequence of photorespiratory metabolism by following 18Q labeling of photorespiratory intermediates formed by intact leaves during exposure to ['80]oxygen; (b) to examine the yield of 180 incorporation into glycolate from [180]oxygen by intact leaves and isolated spinach chloroplasts in order to assess what proportion of glycolate synthesis in vivo occurs with fixation of molecular oxygen; (c) to examine the steady-state enrichment of components of the photorespiratory pathway in order to assess the stoichiometry of precursor-product relationships along the pathway of photorespiration; and (d) to examine the exchange of 02 and CO2 at the CO2 compensation point in relation to the pathway and kinetics of photorespiration studied in the same tissue. These studies provide additional support for the current formulation of the pathway ofphotorespiration (40) and its relation to photosynthetic metabolism at the CO2 compensation point (30). A preliminary report of this work has appeared (5). 'Abbreviations: RuP2: ribulose-1,5-bisphosphate; GC-MS: gas chromatography-mass spectrometry; 3-PGA: glyceric acid 3-phosphate; TCMS: trimethylchlorosilane. 954 www.plantphysiol.org on November 11, 2017 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. FIXATION OF 1802 IN PHOTORESPIRATION MATERIALS AND METHODS [1-'4C]Glycolic acid and [1-'4C]3-PGA were supplied by the Radiochemical Centre, Amersham, U.K. ['8O]Oxygen (99.9% 1802) was purchased from Norsk Hydro, Oslo, Norway. [180]Water was the kind gift of D. Staschewski, Kernforschungszentrum, Karlsruhe, West Germany. Catalase (EC 1.11.1.6) and 3-PGA were supplied by Boehringer Mannheim and triose-P-isomerase (EC 5.3.1.1), alkaline phosphatase (EC 3.1.3.1), glyceraldehyde 3P, and ribose-5-P by Sigma Chemical Co. Dowex I-X8 (100-200 mesh) formate form and Dowex 50-X8 (200 mesh) H+ form, both supplied by Bio-Rad, Richmond, Calif., were well washed before use. All formic acid solutions were prepared from redistilled formic acid. Bis(trimethylsilyl) trifluoroacetamide containing 1% trimethylchlorosilane was the product of the Regis Chemical Co., Chicago. Three per cent SP-2250 on 80/100 Supelcoport was obtained from Supelco Inc., Bellefonte, Pa. All other chemicals were reagent grade. Spinacia oleracea L. hybrid 102 and Atriplex hastata L. were grown hydroponically in a growth cabinet at 20/15 day/night temperature, with 30 nE cmsec' illumination from fluorescent lamps. Helianthus annuus L. Bronze hybrid was grown in soil in the same cabinet. A number of characteristics of leaves of these spinach plants were examined and are summarized here. Measurements with an open circuit gas exchange system (12) gave maximum rates of CO2 uptake in air of 4.8 nmol CO2 cm 2 sec' (290,umol CO2 mg Chl-' hr-') at 1,000 ,/l/ CO2. The initial slope of the CO2 response curve was 0.0159 nmol CO2 cm-2 sec-1 tl/l C02-1 (based on calculated intercellular CO2 concentrations) and the CO2 compensation point in air at 25 C was 45 yd/1 CO2. The leaves contained 60 ,ig Chl cm-2, estimated by the method of Amnon (3). This value was used to relate rates of chloroplast reactions measured on a Chl basis to a leaf area basis. The glycine and serine content of leaves which had been maintained at the CO2 compensation point was determined by analysis of leaf extracts using a Beckman model 120 amino acid analyzer. The pool of glycine was found to be 13 ± 2 nmol cm-2 and that of serine was 87 ± 17 nmol cm2. PREPARATION OF CHLOROPLASTS Intact chloroplasts were isolated as previously described (20, 23). The integrity of the chloroplasts was determined by the K3Fe(CN)6 procedure (18). The maximum rate at which each preparation of chloroplasts evolved 02 in response to 2 mm NaHCO3 was determined by established procedures (23) and is reported in the text. All reactions were performed at 20 C in the basic medium previously described (23) with additions as indicated. Light intensities were measured with a quantum sensor from Lambda Instruments Inc., Lincoln, Nebr. 02 EXCHANGE MEASUREMENTS AND FEEDINGS The isotopic composition and concentration of gases such as 02, CO2 and argon were determined by MS. All 02 exchange measurements were conducted in cuvettes which incorporated a Teflon membrane (1.2 x 10-2 mm thickness) to separate the reaction medium from the high vacuum inlet to the ion source of the analyzer. This inlet system has the advantage, as pointed out by Hoch and Kok (19), that it is impermeable to water and can be used with aqueous as well as gaseous media. The response was linear with partial pressure and the rate of gas sampling was quite small, permitting continuous monitoring of the gas composition without significant depletion. The GD 150/4 mass spectrometer used in these studies has four separate ion collectors which were appropriately positioned to monitor 1602 (m/e = 32), 1802 (m/e = 36), Ar (m/e = 40), and CO2 (m/e = 44). Steady-state labeling experiments with whole leaves were done in the closed circuit gas exchange system outlined in Figure 1. This comprised a thermostatted leaf chamber, a metal bellows pump (Metal Bellows Corp. Sharon, Mass.), a two-way ball valve which served to open or close the circuit and an inlet system for sampling the composition of the air within the chamber for MS. The total gas volume of the chamber and associated plumbing was 74 ± 2 ml. The petiole of a detached leaf was recut under water and then immersed in water in a potometer compartment within the chamber. Water was continuously replenished during the experiment by means of a syringe. The leaf was preequilibrated with 40 nE cm-2 sec' illumination with air flowing through the chamber. At intervals the system was closed and the rate of decline in the CO2 concentration from near 300 td/1 to compensation was observed. When a leaf was fully active (no further response to preillumination) the compensation point was reached within I min from closure (Fig. 2). The system was then quickly flushed with Ar or He and closed. The pump was stopped and the two-way valve opened. A quantity of ['"O]oxygen was injected from a syringe through a septum on one port of the two-way valve, and an equal volume of Ar was displaced from the system and escaped through the other port of the two-way valve. The valve was closed and the pump started. In this way labeled 02 was introduced within a min, with little dilution from 1602. The isotopic composition and total concentration of 02 were either monitored continuously using separate collectors or by repetitively scanning mass 32 to 40 using a single collector (Fig. 2). Changes in these signals with time were used to calculate the net exchanges of 02 by the leaf, and the isotopic composition of the 02 was calculated for comparison with the abundance of 180 found in photorespiratory intermediates at the end of the feeding experiment. Short term non-steady-state feedings were done by an alternate technique which permitted more rapid experimental manipulation and avoided exposure of the leaf to air before killing. These short term feedings with ['8O]oxygen were done in small (10 x 20 cm) plastic bags which could be sealed at the top. A short length of tubing was attached with a leak-tight seal to the bag. The leaf to be fed was placed in the bag with its petiole immersed in a small amount of water, and preconditioned for 5 min at 40 nE cm 2 sec-1 light with a continuous flow ofcompressed air. In the feeding