Photosynthesis Research https://doi.org/10.1007/s11120-018-0600-2 HISTORY AND BIOGRAPHY Discovery of the canonical Calvin–Benson cycle Thomas D. Sharkey1 Received: 16 August 2018 / Accepted: 18 October 2018 © Springer Nature B.V. 2018 Abstract It has been 65 years since the Calvin–Benson cycle was first formulated. In this paper, the development of the concepts that are critical to the cycle is traced and the contributions of Calvin, Benson, and Bassham are discussed. Some simplified views often found in text books such as ascending paper chromatography and the use of the “lollipop” for short labeling are discussed and further details given. Key discoveries that underpinned elucidation of the cycle such as the importance of sedoheptulose phosphate and ribulose 1,5-bisphosphate are described. The interchange of ideas between other researchers working on what is now called the pentose phosphate pathway and the development of the ideas of Calvin and Benson are explored while the gluconeogenic aspects of the cycle are emphasized. Concerns raised about anomalies of label distribution in glucose are considered. Other carbon metabolism pathways associated with the Calvin–Benson cycle are also described. Finally, there is a section describing the rift between Calvin and Benson. 14 Keywords Bassham · Benson · Calvin · Calvin–Benson cycle · CO2 · Gluconeogenesis · Pentose phosphate pathway · Rubisco Introduction (Bassham et al. 1954). This series of reactions is the pri- mary mechanism by which the energy of sunlight is stored In 1947, Andrew Benson and Melvin Calvin published a as reduced carbon in plants, algae, and many bacteria. Here paper titled “The Dark Reductions of Photosynthesis”. Over I describe the development of core concepts of the pathway, the next seven years they published “The Path of Carbon the relationship to the discovery of the non-oxidative pen- in Photosynthesis” papers I–XXIII proposing many dif- tose phosphate pathway and the roles of Calvin, Benson, and ferent cycles. As late as 1952, in “The Path of Carbon in Bassham. Also included is some information on metabolic Photosynthesis XX” Calvin and Massini (1952) proposed pathways intimately associated with the cycle including pho- two carboxylations, a role for malate, and multiple, con- torespiration, non-photorespiratory respiration in the light, fusing suggestions for how carbon reduction occurred (no and a cytosolic bypass of the gluconeogenic reactions of less than five potential reduction steps). But then in 1954 the cycle. (submitted in 1953), with Al Bassham as first author, “The Path of Carbon in Photosynthesis XXI” was published lay- Setting the stage for the discovery of the Calvin– ing out the pathway as we understand it today, including Benson cycle the correct stoichiometry of reducing requirements and ATP During the early twentieth century, the basic mechanism of photosynthesis was under intense study. It became clear Electronic supplementary material The online version of this that conversion of carbon dioxide to sugars using the energy article (https ://doi.org/10.1007/s1112 0-018-0600-2) contains in light could be separated into two distinct phases, light supplementary material, which is available to authorized users. absorption and carbon metabolism. This led to the unfortu- * Thomas D. Sharkey nate practice of calling photosynthetic carbon metabolism [email protected] “the dark reactions” a practice some trace back conceptually to Blackman (1905). The terms “dark reactions” or “light- 1 MSU DOE Plant Research Laboratory, Plant Resilience Institute, Department of Biochemistry and Molecular independent” reactions are (too) slowly leaving common Biology, Michigan State University, East Lansing, MI 48824, usage (Buchanan 2016b). Two alternative names in use, USA Vol.:(0123456789)1 3 Photosynthesis Research “photosynthetic carbon metabolism” and “photosynthetic Public Service. Benson was a conscientious objector to war carbon reduction cycle,” are descriptive, but since there are and had met frequently with the photosynthesis expert Rob- a number of carbon metabolism pathways that allow for pho- ert Emerson while at Cal Tech for advice concerning this toautotrophic growth (Buchanan and Arnon 1990; Bar-Even status. Of the work with 11C, Benson concluded that “the et al. 2012) a more specific name is needed for the carbon many photosynthesis experiments performed by Ruben and metabolism found in plants, algae, and most bacteria. This is Kamen (Ruben et al. 1939) during 4 years of hectic effort sometimes called the reductive pentose phosphate pathway, yielded no real information on the path of carbon in photo- but this obscures the fact that most reactions are gluconeo- synthesis” despite also noting the importance of the work genic and the pentose phosphate pathway components of in moving the field away from the concept of direct interac- the Calvin–Benson cycle are not redox reactions. Melvin tion of CO2 with chlorophyll. Thus, Benson’s involvement Calvin was awarded the Nobel prize for elucidation of this with photosynthesis, interacting with Robert Emmerson at pathway (Calvin 1964), and so when textbooks avoid saying Cal Tech prior to 1942 and working with 11C and Ruben the dark reactions they usually call it the Calvin cycle. For and Kamen at Berkeley 1942–1943, significantly predated reasons given below I will call it the Calvin–Benson cycle. Calvin’s involvement. 14 This cycle is sometimes seen as synonymous with “C3” pho- In the first report of results with C, Benson and Calvin tosynthesis and indeed is at the heart of C3 photosynthesis. (1947) reported that they had held algae in the light while However, it is also at the heart of C4 photosynthesis and bubbling with N2 to keep out CO2. They then turned off the 14 even C2 (also called C 3–C4 intermediate photosynthesis). light and quickly added CO2 and incubated for 5 min. They These refer to mechanisms of CO2 supply but all rely on the found a small amount of label in sugars providing strong Calvin–Benson cycle. evidence that it was possible for sugars to be made after the Early thinking about carbon fixation in photosynthesis light was turned off. This paper was important because it was described by Willstätter and Stoll (1918) [cited in Ben- demonstrated “that the reduction of CO 2 to sugars and the son (2002)]. They hypothesized that photo-excited chloro- intermediates in that reduction does not involve the primary phyll would interact with carbon dioxide, likely making for- photochemical step itself” and that photosynthetic carbon maldehyde that would undergo further reactions to become metabolism “cannot be a simple reversal of the respiratory sugar. In 1938, Sam Ruben and Martin Kamen, working at system of reactions.” The CO2 fixation that occurs after turn- Berkeley, used 11C to study carbon fixation in photosyn- ing the light off was ascribed to the presence of reducing thesis. They found no support for formaldehyde as a first power but is now understood to result from an accumulation product and put forward that the first product should be a of RuBP when photosynthesizing material is deprived of carboxylic acid (Ruben et al. 1939). That work was very light (Eichelmann and Laisk 1999). In fact the accumula- important in moving the thinking away from formaldehyde tion of RuBP in low CO 2 was an important clue about its formation to carboxylation as the first step in carbon fixation. role in carboxylation (Buchanan and Wong 2013; Bassham The short half-life of 11C made it unsuitable since meth- et al. 1954). ods for separating the products of photosynthesis required Further experiments exploring the relationship between more time than the short half-life would allow. Nuclear phys- photosynthesis and respiration were described in Benson icists, including Ernest Lawrence, inventor of the cyclotron and Calvin (1950). In that paper, they reported that no and Director of the Radiation Laboratory (which became the isocitrate or glutarate became labeled in the light although Lawrence Berkeley National Laboratory) predicted that the label quickly appeared in these compounds in the dark. The isotope 14C should exist. With some effort, the method for occurrence of respiration in the light is still debated (Heskel making it was discovered (Benson 2002; Ruben and Kamen and Tang 2018; Tcherkez et al. 2017), but the lack of label 1940). Following the premature death of Ruben and the end in some of the citric acid cycle metabolites has been con- of World War 2, Lawrence recruited Melvin Calvin to make firmed frequently (Gauthier et al. 2010; Szecowka et al. use of the discovery of 14C. Calvin was more interested 2013). Benson and Calvin (1950) also reported the oxygen- in oxidation–reduction reactions and so recruited Benson dependent labeling of phosphoglycolate and glycine, which, to return to Berkeley to be Director of the Photosynthesis 20 years later, would be found to result from the oxygena- Laboratory within Calvin’s Bio-Organic Laboratory which tion of RuBP (Bowes et al. 1971) and to be the first step in was part of the Rutherford Laboratory at the University of photorespiration. California. Andrew Benson, who had gotten his Bachelor’s The 1947 paper (Benson and Calvin 1947) proposed three degree at Berkeley, returned from Cal Tech in 1942, where components of photosynthetic carbon metabolism; (1) that he had gotten his PhD in carbohydrate chemistry and con- the central cycle had four reduction steps, (2) that there were firmed some of the findings of Ruben and Kamen using11 C. two carboxylations, and (3) that it included several reac- Benson had a teaching appointment at Berkeley that ended tions from the tricarboxylic acid cycle. Many cycles were in 1943, whereupon he left Berkeley and entered the Civilian proposed before the correct one and most involved reactions 1 3 Photosynthesis Research Light is supplied by oxidation of water and I have added shading O2 to show the significance of gluconeogenic reactions. The H2O importance of ribulose 1,5-bisphosphate (RuBP) (called RDP in the original scheme) was also a critical step in CO2 [H] elucidation of the cycle, and notably this was published RuBP PGA Triose phosphate by Benson (1951), independent of Calvin.