Photosynthesis Research

, Volume 92, Issue 2, pp 181–185

From Chlorella to chloroplasts: a personal note


    • Robert Hill InstituteUniversity of Sheffield
Original Paper

DOI: 10.1007/s11120-007-9139-3

Cite this article as:
Walker, D.A. Photosynth Res (2007) 92: 181. doi:10.1007/s11120-007-9139-3


An historical and personal reflection on the function of the Benson–Calvin Cycle in isolated chloroplasts, the role of inorganic phosphate and the manner in which this might be best presented to students.


Benson–Calvin cycleChloroplastsPhosphate
It must have been about 1950. Sitting in a lecture theatre in King’s College, Newcastle upon Tyne, U.K., I heard Professor Meirion Thomas of Crassulacean Acid Metabolism fame (Thomas 1947; Osmond 1978) say that the latest news from far-off California (Benson et al. 1950) was that sugar phosphates were involved in photosynthetic carbon assimilation. Come to think of it, I suppose that I have been more or less wedded to them ever since. At the time, however, what Chlorella was doing in the light was of far less interest to Thomas than what Kalanchoe crenata was doing in the dark. So, in 1952, he told me that identifying the enzyme responsible for incorporating CO2 into malic acid in CAM leaves (Edwards and Walker 1983; Walker 1992) was just what I should work on for my Ph.D. Ochoa’s malic enzyme (Ochoa et al. 1950) seemed a good bet but its characteristics proved to be less than ideal for this purpose and it was only after Bandurski and Greiner (1953) described phosphoenolpyruvate carboxylase, an enzyme with an immense affinity for CO2, that everything started to fall into place (Walker 1956, 1957). By then, of course, the ‘Path of Carbon in Photosynthesis’ (Benson et al. 1950; Calvin and Benson 1948) was fully established. Following, the award of the Nobel Prize to Melvin Calvin in 1961, the ‘Calvin Cycle’ was, and is, as commonplace in the literature as the ‘Krebs Cycle’. Over the years, the regrettable imbalance in the acknowledgement of the part that Andy Benson and Al Bassham (see e.g. Bassham et al. 1954; Benson et al. 1950) played in its elucidation is slowly being redressed. For example, Clint Fuller (1999) has written:

“I would like at this point to express a personal note that represents my own feeling and the recollections of many of the scientists who with me experienced the research years at the ORL (Old Radiation laboratory) in Berkeley on photosynthesis. Calvin’s autobiography, ‘Following the Trail of Light’ (Calvin 1992), represents an extremely singular view of the research carried on in the laboratory particularly, in the area of the path of carbon for which he received the Nobel Prize. In all the 175 pages of his autobiography, there is not one sign of Andy Benson or a mention of him. There is not one picture of Andy in a book that contains 51 photographs ranging from graduate students to the King of Sweden. There is not the citation of a single paper with Benson as author or co-author in an extensive bibliography of over 150 references. Benson’s name appears nowhere in the text and consequently is absent in the 12-page index. This appears to be an undeserved slight to a great scientist both personally and professionally who had contributed in a major way to all of Calvin’s research and technology in the field of photosynthesis. Andy was a real leader in the laboratory both intellectually and experimentally. He should have been a partner in the Nobel Prize. Al Bassham’s contributions are also understated, although, he is pictured and cited through the text. I know that all of us who were colleagues at Berkeley agree that it was Andy and Al who contributed greatly to our own success in future endeavors. I have no idea what may have caused this unfortunate event, but, I think that history should record that the contribution of Andy Benson is not properly recognized in Calvin’s autobiography”.

But, regrettably, searching ‘Google’ still currently elicits 2,270,000 mentions of the ‘Calvin Cycle’ compared with a mere 644 for the ‘Benson–Calvin Cycle’.

Unlike PEP carboxylase, RUBISCO (ribulose 1–5 bisphosphate carboxylase-oxygenase), at least as first characterised, seemed to have a remarkably inadequate affinity for carbon dioxide (about 6% CO2 for maximum velocity). This soon ceased to be a problem in intact chloroplasts (Walker 1965; Jensen and Bassham 1968) and eventually the complex mechanism of light activation started to unfold (Buchanan 1980, 1991). In the meantime, my colleagues, Bill Cockburn, Chris Bucke, Carl Baldry and I (Walker 2003) were pre-occupied with the task of improving the functionality of isolated chloroplasts in other regards. Mostly, our chloroplasts did what might have been expected of them although, we were sorely disappointed (Walker 2003) when Dick Jensen and Al Bassham (1968) beat us in the race to achieve rates that might be fairly regarded as equal to those achieved by chloroplasts in situ. Nevertheless, our chloroplasts incorporated 14CO2 and 32P into sugar phosphates (Baldry et al. 1966) and evolved (CO2 dependent) oxygen (Walker and Hill 1967; Walker et al. 1968) as the Benson–Calvin Cycle predicted that they should. Moreover, they displayed an absolute requirement for external orthophosphate and in one, never to be forgotten series of experiments, yielded unequivocal evidence that when carbon dioxide dependent oxygen evolution had petered out through lack of external orthophosphate its restoration by the addition of external orthophosphate displayed, a stoichiometry of three molecules of oxygen evolved for every molecule of orthophosphate added (Cockburn et al. 1967a). By then it was already well established (Cockburn et al. 1967b, 1968; Walker et al. 1967) that higher concentrations of external orthophosphate were inhibitory and that this inhibition was readily reversed by triose phosphates. The conclusion that that there must be “an obligatory exchange between internal triose phosphate and external orthophosphate” (Walker and Crofts 1970) was inescapable. This was soon to be confirmed (Heldt and Rapley 1970), and subsequently amplified and characterised as the ‘phosphate translocator’ (Flügge and Heldt 1991).

Although, the Benson–Calvin Cycle is central to photosynthetic carbon assimilation in chloroplasts, phosphate rarely gets so much as mention in elementary texts and teaching. Instead, the emphasis is almost invariably put on glucose as its end-product. Ironically, this disregards the fact that that, like sucrose, free glucose is not a major product of carbon assimilation by photosynthesising chloroplasts if, indeed, it is formed in the light at all. In the 1960s, the absence of sucrose from radio-autographs (Walker 1965; Baldry et al. 1966) was taken, at first, as an indication that attempts to isolate fully functional chloroplasts had not yet been successful. The fact that such chloroplasts fail to make sucrose is now, long-since acknowledged to be because sucrose synthesis is a cytosolic event (Foyer et al. 1981, 1982; Robinson and Walker 1979). However, unless, we missed it back in the 1960s, buried in radio-autographs overburdened with sugar phosphates (Baldry et al. 1966), glucose is no more a product of carbon assimilation by illuminated chloroplasts than is sucrose. So, why do the textbooks insist that it is? Almost, certainly, (see e.g. Kelly and Latzko 2006) it derives directly from the historic observation by Sachs (1862) that leaves made starch inside (what he called) ‘chlorophyll corpuscles’. The starch disappeared in the dark and came back in the light. It was known that starches were mostly made up of glucose moietys, like beads on a string. Naturally, in Sachs’ day, everyone thought that was that. Bring together light, chlorophyll and CO2 and you get glucose. Join the glucose molecules end to and you get starch. So starch came to be regarded as the end product of photosynthesis despite, the fact that it is not made by all leaves or in all circumstances (Chapman 1924; Kelly and Latzko 2006). For half a century or more, however, it has been clear that the actual path of starch synthesis in chloroplasts starts with triose phosphates and progresses to polymer formation via glucose-1-P rather than free glucose. Nevertheless, in elementary teaching it has become customary to summarize photosynthetic carbon assimilation, in words, by something of this sort

$$ {\hbox{carbon di oxide}}\, + \,{\hbox{water}}\,\dynrightarrow{{Sunlight}}{{chlorophyll}}\,{\hbox{glucose}}\, + \,{\hbox{oxygen}} $$

This is an understandable simplification even though, it is more than a little economical with the known facts. The trouble starts when this sort of representation then invites an equation such as

$$ 6{\hbox{CO}}_2 \, + \,6{\hbox{H}}_2 {\hbox{O}}\, \to \,{\hbox{C}}_6 {\hbox{H}}_{12} {\hbox{O}}_6 \, + \,6{\hbox{O}}_2 $$

As so often pointed out, this may lead a reader to conclude, wrongly, that some of the O2 evolved is derived from CO2. Accordingly, additional molecules of water are often introduced on the left so that it now reads

$$ 6{\hbox{CO}}_2 \, + \,12{\hbox{H}}_2 {\hbox{O}}\, \to \,{\hbox{C}}_6 {\hbox{H}}_{12} {\hbox{O}}_6 \, + \,6{\hbox{O}}_2 \, + \,6{\hbox{H}}_2 {\hbox{O}} $$

(in which C6H12O6 is either identified as glucose or simplified to CH2O to emphasize its empirical nature). Sadly, in solving one problem, this last equation introduces yet another difficulty. Sooner or later, some thoughtful student (looking at the H2O on the right of the equation) inevitably asks “where does the water come out in photosynthesis?

At the most elementary level, this representation:

$$ {\hbox{carbon di oxide}}\, + \,{\hbox{water}}\, + \,{\hbox{phosphate}}\,\dynrightarrow{{Sunlight}}{{Chlorophyll}}\,{\hbox{triose phosphate}}\, + \,{\hbox{oxygen}} $$

might suffice as a more acceptable alternative to one that implicates glucose. However, if there is a need to be a little more explicit, surely, a stylized summary (Walker 1992) such as this

might suffice. (For full details see, for example, Edwards and Walker 1983 or Walker et al. 1986). Historically, the carbon fixing reactions of photosynthesis have erroneously been designated ‘dark’ reactions or, in the period diagram above, ‘dark biochemistry’. Since, the “dark reactions” not only take place in the light, but also require light for regulation (e.g., for activation of certain enzymes), it is now generally accepted that the reactions be called “carbon reactions” (Buchanan et al. 2002).

In turn, this stylized summary simplifies to the following overall equation

$$ 3{\hbox{C}}_2 {\hbox{O}}\, + \,2{\hbox{H}}_2 {\hbox{O}}\, + \,{\hbox{H}}_3 {\hbox{PO}}_4 \, \to \,{\hbox{CH}}_2 {\hbox{OH}} - {\hbox{CO}} - {\hbox{CH}}_2 {\hbox{OPO(OH)}}_2 \, + \,3{\hbox{O}}_2 $$

which in itself is an accurate sum of the partial reactions of the Benson–Calvin Cycle and the photochemical events directly associated with it. Everything should then become clear. The three molecules of oxygen evolved are, indeed, derived from six molecules of water, as six molecules of NADP are reduced and as the known facts demand. The carboxylation of three molecules of RuBP consumes three molecules of water and a further two are used to hydrolyze fructose bisphosphate and sedoheptulose bisphosphate respectively, a total of 11 in all. This total is offset by the nine molecules of water released as nine molecules of ADP are esterified to ATP, so that only two appear in the final overall equation. No need to introduce purely hypothetical molecules of water to make an equation balance and there and therefore no need to agonize about where “they come out”.

In elementary teaching, to prefer triose phosphate to glucose as the immediate end product of photosynthetic carbon assimilation in chloroplasts could be regarded as mere pedantry. It has become increasingly clear, for example, that in dark catabolism the “neutral compounds released by hydrolysis of starch (glucose, maltose or larger malto-oligosaccharides) are exported from the plastid in vivo to provide substrates for sucrose synthesis” (Zeeman et al. 2004). Moreover, there may well be circumstances in which such mobilization of starch may occur in the light. Am I then straining at a gnat in order to swallow a camel or engaged in “a mad pursuit”? I think not. As one who has always favored simplicity of presentation, I would be the first to acknowledge the need to avoid unnecessary detail lest, it obscure the main message or frighten the students. However, the Benson–Calvin Cycle does, after all, consist of sugar phosphates rather than free sugars. Its continuous function depends on regeneration of the CO2 acceptor and autocatalytic feedback (Bassham et al. 1954, Edwards and Walker 1983, Leegood and Walker 1980)). Its regulation (and the manner in which it provides the feedstock for sucrose synthesis in the light (Robinson and Walker 1979; Foyer et al. 1981, 1982) is largely governed by the phosphate translocator (Stitt and Heldt 1985; Flügge and Heldt 1991; Kelly and Latzko 2006). These are fundamentals not to be carelessly overlooked or too lightly discarded in the quest for simplicity. In the present context, i.e., to join in a celebration of Andy Benson’s 90th birthday, without taking the opportunity to lament the absence of phosphate from so much of elementary teaching, would have been like describing the advent of the double helix without mentioning the fact that nucleotides contain phosphates as well as sugars.

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© Springer Science+Business Media B.V. 2007