An early Arabidopsis demonstration. Resolving a few issues concerning photorespiration.

One of the great discoveries of 20th century biology was the elucidation of the pathway of photosynthetic CO2 fixation by Calvin, Benson, and colleagues (18). Among the many loose ends that remained after the photosynthetic carbon reduction cycle had been defined was a series of observations showing that when CO2 was supplied to higher plants in the light, glycolate, Gly, Ser, and several other metabolites that could not be placed in the cycle were also rapidly labeled. By the late 1960s Ed Tolbert, Israel Zelitch, and others had identified the steps of a metabolic pathway in which two molecules of glycolate were converted in a series of enzymatic reactions through glyoxylate, Gly, Ser, and hydroxypyruvate to one molecule each of CO2 and phosphoglycerate (Fig. 1; 23, 24). It had also been established that the CO2 released from glycolate metabolism was the source of at least some of the CO2 released during a process that had become known as photorespiration (4, 25). However, there was no generally accepted explanation for the biosynthetic origin of glycolate or why it was rapidly labeled by CO2. Photorespiration was discovered shortly after the first infrared gas analyzers became available in the mid 20th century (6). The phenomenon was described as the light-dependent release of CO2, a difficult process to measure against a background of concurrent photosynthetic CO2 fixation and mitochondrial or “dark” respiration. To accurately measure the magnitude of photorespiration it was necessary to use elaborate pulse-chase isotope labeling methods that could distinguish recently fixed carbon from carbon fixed during an earlier time period (2). The best estimates suggested that under normal circumstances, a C3 plant could photorespire as much as 25% of the carbon fixed by photosynthesis. Thus, photorespiration was considered a potentially wasteful process that was limiting plant productivity. My interest in the problem was stimulated by a theory advanced by Bill Ogren and George Bowes that was the equivalent of the Grand Unified Theory of Photosynthesis and Photorespiration. In the late 1960s, Ogren had been intrigued by the observation that photosynthetic CO2 fixation is strongly inhibited by oxygen. This is simply demonstrated: Plants grown in 350 mL L CO2 and 2% (v/v) O2 have much higher rates of CO2 fixation than plants grown in 350 mL L CO2 and 21% (v/v) O2. Higher levels of CO2, however, suppress the negative effect of O2. These effects were exhaustively measured in a series of carefully executed experiments on photosynthetic gas exchange that became a scientific touchstone for Ogren (7). He resolved to try to find a mechanistic model of photosynthetic CO2 fixation that would explain the inhibitory effect of O2 and the salutatory effect of CO2. The recognition that O2 and CO2 had mutually competitive effects on photosynthesis led him invariably to the conclusion that O2 must compete with CO2 as a substrate for the enzyme responsible for photosynthetic CO2 fixation, RuBP carboxylase. I consider this to be one of the most brilliant examples of deductive reasoning in 20th century plant biology. On the basis of this theory, Ogren’s postdoc, George Bowes, carried out a protracted search for RuBP oxygenase activity. After more than a year of many failed attempts, RuBP oxygenase activity was at last detected and determined to be a property of RuBP carboxylase (1, 14). The enzyme was subsequently renamed RuBP carboxylase/oxygenase, or Rubisco. I think about this experiment frequently when something is not working in my lab—one of the great challenges of experimental science is to decide when to abandon a line of experimental work that is not progressing and when to keep trying. If there is a lesson from the RuBP oxygenase example I think it is that nothing substitutes for a good theory (and tenacity). It was not just George Bowes who initially had trouble demonstrating RuBP oxygenase activity. About a year after the oxygenase paper was published, George Lorimer, a student of Ed Tolbert’s at the time, reportedly burst into Ogren’s office with an armful of O2 electrode tracings from failed attempts to measure RuBP oxygenase activity and dumped them on Ogren’s desk with the words “It doesn’t work.” Lorimer was so inflamed with the idea that Bowes’ and Ogren’s paper was erroneous, and that he had wasted time testing their idea, that he had driven all the way from Lansing to Urbana to deliver the message in person. Of course it did work and Lorimer went on to show why with an elegant * E-mail [email protected]; fax 650 –325– 6857.