Introduction

Andrew A. Benson has been called “The Atomic Man” for his bold biomolecular explorations utilizing radiolabeled chemicals during the nuclear era of the 1940s—his concepts set the course for the discoveries of many metabolic pathways based on his eponymous protocol (see G. Lorimer in Nonomura et al. (2016) for Benson’s Protocol). Andy, indeed, developed the first protocols for peaceful uses of radioisotopes in many biological applications. His science has been hailed as elegant (Fomina and Biel 2016; Buchanan et al. 2016) and superb (Lichtenthaler et al. 2015). Initially, Andy called his radioactive-label tracer technique radio-autography, but this is now generally called autoradiography. Be that as it may, Andy’s legacy of over a thousand autoradiograms leads us to use this radio-autobiography to commemorate his life’s work. Thus, we will let his autoradiograms tell the story of historical achievements that were the stuff of Andy’s rigorous science and through which he opened enduring vistas (Buchanan et al. 2007; Lichtenthaler et al. 2008; Buchanan and Douce 2015). Andy spent over half a century at Hubbs Hall, Scripps Institution of Oceanography (see Fig. 1 for a 2007 photograph taken outside his office on the 4th floor balcony). To complete Benson’s story, we show here (and in the Supplementary Material; also see Online Appendix) a sample of the 50 autoradiograms he left with us, many of which have never been published.

Fig. 1
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image by Arthur Nonomura

A 2007 photograph of Andrew Alm Benson. He was on the balcony outside his office in Hubbs Hall, Scripps Institution of Oceanography, La Jolla, California USA. From the Image galleries, https://scripps.ucsd.edu/news/obituary-notice-andrew-benson-world-renowned-scripps-plant-biochemist; with the permission of Mario Aquilera, Scripps Communications Office, Scripps Institution of Oceanography;

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Charting the path

To address the question “How does one see the carbon?” Andy tracked the path of carbon metabolism by his treatment of plants and algae with accumulated concentrates of 14CO2. The radiation emitted from metabolites that were tagged with 14C could be captured as black spots and trails on X-ray films and, thus, be readily viewed. This process of developing X-ray film that was exposed to two-dimensional (2D) paper chromatograms of radiolabeled metabolites is autoradiography. Andy’s materials included Whatman No. 1 filter paper, which was as large as Kodak Photoradiography Medical X-Ray Film SB-54—single coated blue sensitive tinted Estar Safety Base, with interleaved rounded corner, 14 × 17 inch sheets (see Benson et al. 1950). Starting in 1946, Andy built the laboratory he worked in, ordered instrumentation to his specifications, and grew large volumes of algal cultures. Figure 2 shows Andy with a row of 1.8 L Fernbach flasks, illuminated with fluorescent light panels. He initially used dense cultures of Chlorella ellipsoidea or Scenedesmus obliquus, pouring them into special glass vessels (called “lollipops”; see Fig. 12 in Nonomura and Benson 2013), then adding 14CO2 and providing illumination. At specific intervals, he quickly dropped the entire contents of the lollipop into hot methanol to instantly stop the reactions at different steps of the Path to Carbon Fixation. Andy also grew higher plants and designed modified lollipops for radioisotope uptake by whole leaves. Figure 3 shows such a lollipop with a geranium leaf installed inside.

Fig. 2
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Andrew A. Benson with his microalgal culture system. Pure cultures of Chlorella or Scenedesmus were grown in illuminated Fernbach flasks. After reaching a high cell density, cell suspensions were transferred to a lollipop for irradiation. From the private collection of Dee Benson

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Fig. 3
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Advanced generation lollipop designed for treatment of whole leaves with radioisotopes; inside, a geranium leaf. Both glass faces were illuminated during an experimental run. See Nonomura and Benson (2013) for an overview of a glass lollipop. From the private collection of Dee Benson

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Andy, a highly capable chemist, synthesized radiolabeled compounds, developed chromatographic innovations, and initiated autoradiography. Within 4 years, Andy had not only tracked 14CO2 to its early metabolites following carboxylation in photosynthesis, but he had also utilized 32P to track its metabolism to phosphate esters. Figure 4a shows an autoradiogram of a soybean leaf hydrolysate of phosphorylated compounds, where the chromatographic spots could not be identified, underscoring the difficulty and complexity involved in identifying these compounds. As an outcome of studying with Andy, Alex Wilson identified comparable spots in Scenedesmus soon thereafter (see Buchanan et al. 1952). Figure 4b shows an autoradiogram of a marine red alga Porphyra umbilicalis, where the chromatographic spots were identified (see Fig. 5 in Govindjee 2010, showing Andy holding a labeled autoradiogram of Scenedesmus that was fed with 14CO2). For this purpose, Andy generated a number of keys that showed the positions of numerous compounds that he had meticulously and consistently chromatographed in 2D (for one of the keys, see Fig. 5). For Andy’s outstanding contributions to the elucidation of the carbon reduction cycle in photosynthesis, made primarily through his development of double labeling techniques employing 14C, 3H, and 32P, The Ernest Orlando Lawrence Award was conferred on him. This award read: “Laureate Andrew A. Benson, 1962, Life Sciences” (see DOE 1962) and was accompanied by a check for $20,000. We suggest that without Andy’s early creative approach, mentioned above, ribulose bis phosphate (RuBP; Ru1,5 DP in Fig. 5) would not have been identified then as the key intermediate in the carbon reactions of photosynthesis.

Fig. 4
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a Autoradiogram by Alex T. Wilson (Buchanan et al. 1952; ATW was a coauthor). The soybean leaf hydrolysate of phosphorylated compounds was not labeled and underscores the difficulty and complexity involved in identifying the compounds. b Previously unpublished autoradiogram by A. A. Benson; it shows locations of identified compounds after 5 h of 14CO2 photosynthesis by Porphyra umbilicalis

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Fig. 5
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Benson’s keys to autoradiograms. Only after comparing each chromatographic spot with a known standard could the position of each be identified. This took years of highly reproducible precision chemistry, the methods of which were the basis of Benson’s Protocol. One of his keys mapped the Rf for each of the metabolites using two different mobile phases, Phenol–Water and Butanol–Propionic Acid–Water. Note the position of sedoheptulose and RuDP (RuBP) in the lower right corner. Andy focused on the group of compounds in this corner because, since 1949, he had discovered that they appeared in the first few seconds of exposure to 14CO2 (Benson et al. 1950)

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Bold explorations

We ascribe the breadth of Andy’s research to his adventurous character. See, for example, investigations on lobsters by Cooney and Benson (1980). One of us (Arthur Nonomura) remembers Andy actively monitoring field research among thousands of acres of thorny roses (see Fig. 6). As we now probe into Andy’s scientific adventures, we show, in Fig. 7, Andy riding astride a crop foliar spray tractor: he clearly enjoyed his scientific and personal endeavors immensely (see Benson 1981).

Fig. 6
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Benson (left) with Arthur Nonomura (right; one of the authors) monitoring field modulation of glycoregulation in roses; October 1992, Woolf Farms, Waddell, Arizona, USA. Benson enjoyed the intense fragrance of rose blossoms covering thousands of acres in the Valley of the Sun. From the private collection of Dee Benson

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Fig. 7
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Benson enthusiastically participated in a demonstration of a row crop spray rig; October 1992, Litchfield Park, Arizona. From the private collection of Dee Benson

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Andy had the gift of scientific inquiry that he utilized to dig out the truth, recognizing unexpected results; and, trailblazer that he was, he would effectively show his colleagues how to get to the correct destination. A partial listing of Andy’s exemplary scientific investigations is presented in Tables 1, 2, 3 and 4, featuring the global scope of his bold explorations. His radioisotopes, listed in Table 1, are grouped according to the elements used and his experiments undertaken across a wide variety of live samples. Early in his research, Andy combined different radioisotopes, such as 14CO2 and 32P-phosphate (Benson et al. 1950); later, in the 1960s, Andy combined 35SO4 and 14CO2.

Table 1 Benson’s isotopes
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Table 2 Benson’s microbes and algae
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Table 3 Benson’s plants (left column) with references (right column)
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Table 4 Benson’s heterotrophs
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Of mice and chemists

In the 1930s, Andy worked with Sam Ruben and Martin Kamen using 11C applied to rat lungs. He furthermore utilized this first, but short-lived, radioisotope of carbon in photosynthesis, after the publication by Ruben et al. (1939). With the advent of 14C, Andy synthesized the world’s supply of concentrated 14CO2 to be used for its fixation in living organisms. When Melvin Calvin invited Andy, in 1946, to set up the laboratory at University of California Berkeley, to find the path of carbon in photosynthesis, Andy began to apply “his” 14CO2 to photoautotrophic microbes and algae, listed in Table 2. Isotopes were selectively used based on the goal of the specific experiments. For this, different unicellular, colonial, and thalloid algae were treated with inorganic radioisotopes (see Benson and Bassham 1948; Calvin and Benson 1948; Benson and Calvin 1950; Calvin et al. 1951; also see Fig. 4b; Fig S5 in the Supplementary Material; Online Appendix; Table 2).

Membrane chemistry

Higher plants were fed primarily with 14CO2 (see Table 3). Using these plants, Andy discovered phosphatidylglycerol and sulfoquinovosyldiglyceride, and proved the importance of membrane phospholipids and sulfolipids in their function. Wada and Murata (2010) dedicated their book on “Lipids” to Benson for his discoveries in this area. Further, Andy studied membrane chemistry through the application of 32P and of 35S in many plants, listed in Table 3.

Benson’s biorefineries

Animals were tracked with radiolabeled organic products, such as alcohols, sugars, lipids, and organic acids, since these organisms were capable of metabolizing such compounds; for example, 14C-glucose. Andy obtained complex organic compounds by culturing algae in 14CO2 and isolating their specific radiolabeled metabolites; see, for example, 14C-Dunaliella (Cooney and Benson 1980). He looked into complex organisms such as zooxanthellae, symbionts in corals and clams (see, e.g., Benson 1990; Tables 2, 4). At that point Andy could have brought the investigation of zooxanthellae to completion, but he did not stop there, for he could now utilize these symbionts as bio-refineries to feed their animal hosts with radiolabeled products that he had isolated from the zooxanthellae. He investigated heterotrophs, such as bacteria and fungi listed in Table 4, and he studied animals including copepods, fishes, rats, hamsters, sheep, and cows (see, e.g., Strickland and Benson 1960; Patton and Benson 1966, 1975; Patton et al. 1977). Andy unveiled aging processes by applying 125I-calcitonin to porcine lung tissue (Fouchereau-Peron et al. 1981) and 32P + 45Ca to Oncorhynchus gorbuscha, pink salmon (Milhaud et al. 1977), as listed in Table 4.

Chemist and biologist

Since Andy was a chemist, partnership with biologists was critical for determining suitable species to explore (Buchanan et al. 2007; Lichtenthaler et al. 2008; Buchanan and Douce 2015; Nonomura et al. 2016). We note that our present historical article on Andy is indeed presented from a biological perspective. In order to define the path of sulfocarbohydrates, Andy investigated the coral tree because of its alkaloids and alfalfa for its accumulation of sulfolactates (Table 3; Lee and Benson 1972). For Andy’s step up to mixotrophy, he worked on Euglena (see Table 2; Figure S6). Foraminifera are amoeboid heterotrophs and some species may contain algal symbionts, while others are kleptoplastic (where chloroplasts are sequestered by the host organism), and Andy applied therefore autoradiography to Foraminifera vertebralis (see Table 4; Figure S19 in the Supplementary Material; also see Online Appendix).

By circumnavigating the seas to apply 74As to gossamer diatoms (Katayama et al. 1990; Busby and Benson 1973), clams (Benson 1990), lobster (Cooney and Benson 1980), and zooxanthellae, Andy found how arsenic is regulated in nature and the relationship of Si to S-metabolism (see Tables 1, 2, 4).

As is well known to most of us, Andy worked to define the carbon reactions of photosynthesis beginning in 1947 (Aronoff et al. 1947) to the end of 1955 (see, e.g., Lichtenthaler et al. 2015; Nonomura et al. 2016). Thereafter, Andy was also involved in studies on cell and organelle membranes (Benson 1966), and in particular chloroplast membranes (Douce et al. 1973). Plants used by Andy spanned a spectrum of diversity including barley (Benson and Calvin 1950; Benson 1951), soybean (Benson et al. 1952), Euphorbia (Nordal and Benson 1969), strawberry (Steim and Benson 1964), Lemna perpusilla (Shibuya et al. 1965), Botany Bay spinach, mangrove, tobacco, and wheat. Plants listed in Table 3 were the stars of his experiments.

As early as 1949, Andy had investigated the contribution of the simplest organic compound, methanol, because he had observed that 14C-methanol (14C-MeOH) was metabolized as rapidly as 14CO2 in Chlorella and Scenedesmus (Calvin and Benson 1949). This interest in methanol had been ongoing: one of us (Robert Cooney, personal communication, 2017) had applied, in 1978, ~0.3% 14C-MeOH to cultures of Chlorella ellipsoidea, resulting in a single chromatographic spot (Fig. 8a); further, pretreatment of Chlorella ellipsoidea with low concentrations of MeOH was followed by 14CO2 treatment (Fig. 8b). Benson’s group resumed studies on plant responses to MeOH in 1992, when one of us (Nonomura) demonstrated visibly discernible reduction of midday wilt in fields of roses, cotton, and cabbage treated with 20–50% MeOH in modified Hoagland solution (for details, see: Nonomura and Benson 1992; Benson et al. 1992). This team of a chemist (Benson) and a botanist (Nonomura) followed through with experiments on Swiss Chard and barley leaves fed with 14CO2 and pretreated with 20% MeOH. When this experiment was followed by exposure of leaves to saturated O2, results showed unexpected inhibition of glycolic acid compared to that in controls without MeOH. In dozens of replicates, a dark glycolic acid (Schou et al. 1950) spot was clearly labeled in the control autoradiogram (Fig. 8c). In contrast, the MeOH autoradiogram (Fig. 8d) showed a faint gray trace in that location. Benson and Nonomura then joined with Roland Douce’s group in Grenoble, France, in tracking MeOH to methyl glucoside (MeG; Gout et al. 2000); and thereafter, observed plant and algal growth responses with Valrie Gerard (Benson et al. 2009). A decade later, sugar beets were treated with 14C-MeG (see Biel et al. 2010); after 22 h, autoradiography of whole sugar beet plants showed a single spot of ninhydrin-N-linked-14C-MeG (Fig. 9), while the untreated control showed nothing in that position. As captured in these autoradiograms, Andy’s pursuit of 14C-MeOH spanning 65 years led to new ideas on modulation of glycoregulation.

Fig. 8
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a Autoradiograms of Chlorella ellipsoidea fed with 14C-MeOH (a 1978 experiment); b treatment with ~0.3% MeOH was followed by 14CO2 autoradiograms from Robert Cooney; c photorespiration in Swiss chard leaf fed with 14CO2 revealed glycolic acid in controls in O2, without MeOH; d pretreated with 20% MeOH; leaves in O2 did not show glycolic acid (see Benson and Nonomura 1992 for the disclosure of these results, but in which these autoradiograms were not published)

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Fig. 9
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Autoradiography of Beta vulgaris (sugar beet) showed a single spot of ninhydrin-N-linked-14C-MeG (see Biel et al. 2010 in which the results were disclosed, but the 2D-paper chromatogram was not published)

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We believe that Andy’s greatest virtue was his perseverance, and we speculate that this may have been partly responsible for his longevity. Andy brought the finest details of life to the fore through development of isotopic applications (Benson 2011). He even investigated mushrooms (Vaskovsky et al. 1991), but why be limited to a crustacean, fish, or fungus when one can chart an entire ecosystem? After all, ecosystems are defined in part by the transfer of energy from one species to the next. Thus, the transfer of energy, for example, from corals to fishes (Benson and Muscatine 1974) or through wax from copepods, was charted by Andy and one of us (see, e.g., Holtz et al. 1973; Table 4) via radiolabels in the food chain.

Greatest benefit

When Andy needed a new technology, he had the vision to design and construct it from the ground up and then improve upon it. In the early years, Andy worked closely with James A. Bassham in the Old Radiation Laboratory (Benson 2002; Bassham 2003; Govindjee et al. 2016; Dent et al. 1947). As example of their pioneering work, immediately after finding organic acid products of 14CO2, they (Benson and Bassham 1948) developed sophisticated chemical synthesis and biological methods to biosynthesize, isolate, and apply 14C-succinic and 14C-malic acids in their investigations. Within months, The Path of Carbon in Photosynthesis (Calvin and Benson 1948) was published. This profound innovative approach was repeatedly demonstrated, as in the case of his neutron activation studies (Benson 1987). As soon as a new technology became available, Andy made fresh discoveries in other areas with a multitude of colleagues. For example, refraction of light through glass microbeads was found to enhance its intensity at the phylloplane (Nonomura and Benson 2012). Investigations of in vivo 13C nuclear magnetic resonance kept Andy on the cutting edge of science with his past doctoral associates and colleagues (Gout et al. 2000), and this too when he was 83-years old. With one of us (Nonomura), the first recognition of a specific glycoconjugate pathway was translated into the field of modulation of glycoregulation (Nonomura and Benson 2014). We are thrilled to note that, at age 97, Andy was recognized by Brandt iHammer for “Conferring the Greatest Benefit on Mankind” (Buchanan and Douce 2015).

When Andy applied each of the different radioisotopes to plants or animals, he discovered The Path of Sulfocarbohydrates, The Path of Sulfolipids, The Path of Phospholipids, The Path of Arsinolipids, The Path via Neutron Activation, The Path of Photosynthates in Animals, The Path to Waxes, The Path from Waxes into the Marine Food Chain, The Path to Membranes, and The Path to Glycoregulation. Each stepping stone laid down by Andy paved the path to discoveries of higher and higher order.

We note that Andy’s legacy was never confined to photosynthesis since he had applied his protocol to mixotrophs, heterotrophs, and ecosystems. In fact, his earliest investigations with 11C-phosgene were on rats, while some of his later studies were on marine ecosystems. During his lifetime, Andy demonstrated wide-ranging application of his philosophy of isotopic tracers, while others followed him.

Benson’s protocol, then and now

Following World War II (1939–1945), the stage was set for attempts to reveal the hitherto mysterious processes of metabolism. A radioactive form of carbon with a suitably long half-life had been discovered (see Kamen 1963) and the technique of 2D-paper chromatography had been developed for resolving complex mixtures of closely related compounds (Martin and Synge 1941). Andy was the first to apply these methods to a complex metabolic system, the path of carbon in photosynthesis. In its generic form, Benson’s Protocol (Fig. 10) is quite straightforward, although demanding in technical skill and considerable chemical know-how.

Fig. 10
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Flow chart of Benson’s Protocol

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A particularly novel application of Benson’s Protocol that took advantage of Andy’s extensive knowledge of the path of carbon in photosynthesis is exemplified by his novel discovery of the mechanism through which the herbicide methane arsonate acts in the eradication of Johnson grass (Sorghum halepense) near sugar cane fields (Knowles and Benson 1983). Methane arsonate is toxic to the C4 photosynthetic pathway employed by Johnson grass, yet it does not affect C4 Bermuda grass. Investigation of the pattern of photosynthesis by autoradiography revealed accumulation of malate and significant reduction of sucrose in methane arsonate-treated leaves. This was attributed to an inhibition of NADP+-malic enzyme. Their studies suggested that oxidation of key sulfhydryl groups by methane arsonate inhibited CO2 release from malate in bundle sheath cells, depriving the plant of its source of carbon for sucrose production. In the case of Bermuda grass, differential mechanisms of carbon transport to the bundle sheath cells and high transaminase activity may be responsible for the insensitivity of Bermuda grass to methane arsonate. In the hands of other skilled chemists, complex pathways leading to the synthesis of sterols were determined (see Cornforth 2002). In this manner, Benson’s Protocol was to form the basis for what might be considered the golden age of metabolic biology.

Science and technology march on; even today, however, the elements of Benson’s Protocol can still be recognized. Radioactive isotopes are being replaced with stable isotopes (e.g., 13C, 15N, and 18O) that are now available in highly enriched form. The limited resolution of paper chromatography has been largely replaced by liquid and gas chromatographic methods that have the additional advantage that they can be coupled to mass spectrometry. This allows the more-or-less unequivocal identification of the compounds in question and, from the fragmentation pattern, the quantitative distribution of the isotope. Beyond the ability to interpret a mass spectrum, next-to-no chemical knowhow is needed. Examples of the modern application of Benson’s Protocol, fittingly from the field of photosynthesis/photorespiration, with 13C (Gout et al. 2000; Flanagan et al. 2006) and another with 18O (Berry et al. 1978), illustrate the point.

Conceptually, these and other metabolic pathways arose from applying a variant of Benson’s Protocol. Beyond science, Andy was first and foremost our great friend, whether at home, in the laboratory, or abroad (see tributes from Govindjee 2010; Biel and Fomina 2015; Nonomura et al. 2016; Buchanan et al. 2016 for examples). Fondly remembered camaraderie from one of us (Barry Holtz) is shown in Fig. 11, and another (Govindjee) remembers the fun timeFootnote 1 he had with Andy when he was driven with his wife Rajni by Andy in his old BMW (Bavarian Motor Works automobile; see Fig. 12a). Figure 12b shows another memorable occasion in 2001 when Andy and Govindjee each gave their own historical lecture on “carbon fixation” and “water oxidation and NADP reduction” aspects, respectively, of oxygenic photosynthesis (see Govindjee 2010).

Fig. 11
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A 1976 photograph of Benson (left) with Barry Holtz (one of the authors). Benson is wearing one of his signature bowties and a broad smile; they were at a conference. From the private collection of Barry Holtz

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Fig. 12
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a A 2001 photograph of Andy Benson and Rajni Govindjee with Benson’s vintage BMW before Andy gave Govindjee and Rajni a ride (see footnote 1 for a story). From the private collection of Govindjee; b A 2001 photograph of Benson, wearing a bowtie, and one of the authors (Govindjee) at a dinner in Brisbane, Australia. (Fig. 3 from Govindjee 2010, reproduced with Springer’s permission)

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Concluding remarks

No phase of life’s unfolding story is more brilliant with the fire of heroic purpose than the crossing of these radioisotope tracers with the spectrum of life (Buchanan et al. 2016). Even the 1961 Nobel Prize to Melvin Calvin, in chemistry, was trifling beside these volumes of enlightenment of the interactions of air, minerals, water, light, and the food chains of life. And it was Benson (1981) who explored unknown metabolic worlds and adventurously persevered until he found answers to the most significant questions.

We end this Tribute by showing photographs of Andy with a few other scientists. Figure 13a shows him with Ralph Lewin, the two having had a hand in designing and “building” Hubbs Hall where they did their own research; Fig. 13b shows Andy with others including Barry Holtz (coauthor of this Tribute), Roland Douce, and Bob Buchanan who have written papers honoring Andy (see, e.g., Buchanan and Douce 2015 and a 2012 conversation with Buchanan at: https://www.youtube.com/watch?v=GfQQJ2vR_xE); Fig. 14 shows Andy actively involved in field research with ArthurFootnote 2; and Fig. 15 shows a fitting photograph to end Andy’s centenary celebration—it shows Andy in front of a black board where he had just written the chemical structure of a compound (a frequent happenstance, when he talked with others about his or their research). We all miss you, Andy.

Fig. 13
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a A 2007 photograph of Ralph A. Lewin (left) and Benson (right) discussing matters of common interest (algae in hairs of captive polar bears, plant physiology, and discoveries); b A 2014 photograph at Benson’s residence in La Jolla, California, USA. From left to right Roland Douce, Andrew A. Benson, Barry Holtz, Julian Schroeder, and Bob B. Buchanan (see Buchanan and Douce 2015). From the private collection of Arthur Nonomura

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Fig. 14
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photograph by George Lorimer

Never one to be lab-bound, Benson met one of us, George Lorimer, in San Diego, and trekked inland to agricultural fields in Arizona during a sweltering 45 °C day in 1990. Benson (left) and Nonomura (right) standing in an irrigated farm field (see footnote 2 for a story). Nonomura handed his camera over to Lorimer prior to crossing the irrigation ditch onto the field with Benson;

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Fig. 15
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A 2007 photograph of Andrew Alm Benson standing before the blackboard in his office, holding a copy of the Proceedings of the National Academy of Sciences of USA, which he had read cover to cover. From the private collection of Arthur Nonomura

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