Berry J A, Osmond C B, Lorimer G H
Department of Environmental Biology, Research School of Biological Sciences, Australian National University, Box 475, Canberra City 2601, Australia.
Plant Physiol. 1978 Dec;62(6):954-67. doi: 10.1104/pp.62.6.954.
Mass spectrometric techniques were used to trace the incorporation of [(18)O]oxygen into metabolites of the photorespiratory pathway. Glycolate, glycine, and serine extracted from leaves of the C(3) plants, Spinacia oleracea L., Atriplex hastata, and Helianthus annuus which had been exposed to [(18)O]oxygen at the CO(2) compensation point were heavily labeled with (18)O. In each case one, and only one of the carboxyl oxygens was labeled. The abundance of (18)O in this oxygen of glycolate reached 50 to 70% of that of the oxygen provided after only 5 to 10 seconds exposure to [(18)O]oxygen. 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 labeling 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 (18)O in glycolate synthesized in vitro by isolated intact spinach chloroplasts in a system which permitted direct sampling 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 (18)O in 20- and 40-minute feedings with [(18)O]oxygen to intact leaves at the CO(2) compensation 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-phosphoglycerate was 14 +/- 4% of that of glycine or serine, 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 CO(2) compensation point.Oxygen uptake sufficient to account for about half of the rate of (18)O fixation into glycine in the intact leaves was observed with intact spinach chloroplasts. Oxygen uptake and production by intact leaves at the CO(2) 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 stable isotope techniques to study the rate of photorespiration and the balance between photorespiration and photosynthesis in vivo.
采用质谱技术追踪了[(18)O]氧掺入光呼吸途径代谢产物的情况。从处于CO₂补偿点时暴露于[(18)O]氧的C₃植物菠菜（Spinacia oleracea L.）、滨藜（Atriplex hastata）和向日葵（Helianthus annuus）叶片中提取的乙醇酸、甘氨酸和丝氨酸被(18)O大量标记。在每种情况下，羧基氧中只有一个被标记。乙醇酸中该氧的(18)O丰度在仅暴露于[(18)O]氧5至10秒后就达到了所提供氧的50%至70%。甘氨酸和丝氨酸分别在40秒和180秒后达到相同的最终富集程度。这证实了甘氨酸和丝氨酸是由乙醇酸合成的。完整叶片中光呼吸中间产物的标记平均达到了饲喂中所提供氧标记的59%。这表明至少59%的光呼吸乙醇酸是在分子氧固定的情况下合成的。由于光合氧在乙醇酸合成位点对标记氧的稀释，这个估计肯定是保守的。我们在一个允许直接对合成位点的氧同位素组成进行采样的系统中，检测了分离的完整菠菜叶绿体体外合成的乙醇酸中(18)O的产量。此类实验中乙醇酸的同位素富集程度为孵育期间存在的氧的90%至95%。在CO₂补偿点对完整叶片用[(18)O]氧进行20分钟和40分钟的饲喂后，3 - 磷酸甘油酸的羧基氧也被(18)O标记。对照实验表明，这种标记可能是由于光呼吸过程中乙醇酸直接合成3 - 磷酸甘油酸所致。在10次单独的饲喂实验中，3 - 磷酸甘油酸的平均富集程度为其光呼吸途径前体甘氨酸或丝氨酸的14±4%。有人认为，这种标记的持续稀释表明在CO₂补偿点光呼吸和光合来源的3 - 磷酸甘油酸之间存在恒定的化学计量平衡。用完整的菠菜叶绿体观察到的氧吸收量足以解释完整叶片中约一半的(18)O固定到甘氨酸中的速率。在CO₂补偿点完整叶片的氧吸收和产生表明，每光呼吸一个乙醇酸大约有1.9个氧交换。分子氧固定到乙醇酸中，加上乙醇酸在过氧化物酶体中氧化为乙醛酸以及甘氨酸在线粒体中转化为丝氨酸，最多可解释每乙醇酸吸收1.75个氧。这些研究提供了新的证据，支持了当前光呼吸途径及其与光合代谢关系的表述。所描述的实验还提出了使用稳定同位素技术研究体内光呼吸速率以及光呼吸与光合作用平衡的新方法。
