The Plant Journal (2015) 82, 365–369
doi: 10.1111/tpj.12794
SI CHLAMYDOMONAS
Historical perspective on Chlamydomonas as a model for basic research: 1950–1970 Ursula Goodenough* Department of Biology, Washington University, St. Louis, MO 63130, USA Received 22 December 2014; revised 28 January 2015; accepted 2 February 2015; published online 17 February 2015. *For correspondence (e-mail [email protected]).
SUMMARY During the period 1950–1970, groundbreaking research on the genetic mapping of Chlamydomonas reinhardtii and the use of mutant strains to analyze photosynthesis was conducted in the laboratory of R. Paul Levine at Harvard University. An account of this era, based in part on interviews with Levine, is presented. Keywords: Chlamydomonas, history of science, R. Paul Levine, Ruth Sager, Ralph Lewin, photosynthesis, genetics.
INTRODUCTION The coming of age of Chlamydomonas reinhardtii has been recounted in detail in two book chapters, one by Togasaki and Surzycki (1998), with a focus on the chloroplast, and one by Joel Rosenbaum (2009), with a focus on the flagellum. Presented here is a version of the story based on conversations with Paul Levine, a pioneer in analyzing the genetics and photosynthesis of C. reinhardtii. The story has multiple plot lines, originating in the early 1950s, that come together on a beach at Woods Hole, Massachusetts, USA, in 1955. We start at Stanford University. After a lifetime of writing such comprehensive treatises as Phytoplankton of the Inland Waters of Wisconsin (Smith, 1920) and The Freshwater Algae of the United States (Smith, 1933), phycologist Gilbert Morgan Smith returned to an earlier passion, the isolation of pure algal clones from field specimens (Smith, 1916), and chose Chlamydomonas reinhardi (now spelled reinhardtii) as his target species. Bill Eversole, a student with fungal geneticist Edward Tatum, and Bill Ebersold, a student with population geneticist David Regnery, each launched PhD thesis projects that entailed mutagenizing these isolates and following the sexual inheritance of mutant traits (e.g. arginine-requiring, acetate-requiring, pigment-deficient; Eversole, 1956; Ebersold, 1956). Upon graduation in the mid-1950s Eversole left the field, but Ebersold took a postdoctoral position at Dartmouth and continued with his project. © 2015 The Author The Plant Journal © 2015 John Wiley & Sons Ltd
Next, we look to Scripps Oceanographic Institute, where phycologist Ralph Lewin, a man with seemingly countless interests (https://scripps.ucsd.edu/news/2423), spent several years focused on Chlamydomonas moewusii, a distant relative of C. reinhardtii, following the inheritance of paralyzed-flagella traits in sexual crosses (Lewin, 1954). His original interest had been to find mutants disabled in photosynthesis, but his lab discovered that C. moewusii is an obligate phototroph (Lewin, 1950), and hence mutations affecting photosynthesis would be lethal. Meanwhile, at Rockefeller University, Ruth Sager joined botanist Sam Granick’s lab as a postdoc in 1949. In 1951, as a staff member, she launched her own study of C. reinhardtii, independently obtaining a wild-type strain from Gilbert Smith that turned out to be different from the strain studied by Eversole and Ebersold, a salient difference being its ability to grow with nitrate as the sole nitrogen source (Harris, 2009: 11–16). She published definitive papers on its ultrastructure (Sager and Palade, 1957) and the nutritional control of its sexuality (Sager and Granick, 1954), and initiated several decades of research on the inheritance of its non-Mendelian traits (Sager, 1955, 1960), which, as she and others went on to document, are encoded in its chloroplast DNA. Finally, the story moves to Harvard University. Paul Levine, an assistant professor trained in Drosophila genetics at UC Berkeley, was interested in recombination 365
366 Ursula Goodenough mechanisms and decided to switch to a fungal system. He therefore went to the Marine Biological Laboratories in Woods Hole in the summer of 1955 to develop his skills in Neurospora tetrad analysis, a technique that he first learned as a summer undergraduate researcher at Cal Tech in George Beadle’s lab. This takes the story to Stony Beach. One afternoon, Paul ran into Ruth and Ralph out taking a stroll, and the three began to talk science. Paul described his plans to study recombination in fungi, and Ralph huffed. ‘Wrong organism’, he announced. He and Ruth proceeded to tout the advantages of C. reinhardtii, and Ralph dropped in the line that were Paul to follow their advice he should recruit a hot-shot guy named Bill Ebersold. Paul took the bait, and Ebersold moved to Cambridge in 1956 with the goal of developing a high-density genetic map of C. reinhardtii for recombination studies. The next few years can best be described as a genetics orgy, with technicians Elizabeth Levine and Peggy Olmstead to the fore. Some 250 crosses were performed and more than 20 000 tetrads were analyzed, with the results recorded on countless file cards. Replica plating was used to score
growth requirements, including strains that required acetate, and drops of water on colonies were used to score paralyzed mutants, many of which were provided by Ralph Lewin (a full list of strains is provided in Harris, 1989). The first map, of linkage group I, was published in 1959 (Ebersold and Levine, 1959). A map of 11 linkage groups, reproduced in Figure 1, was published in 1962 (Ebersold et al., 1962). A more detailed map was available by 1965 (Hastings et al., 1965) and now, of course, a full map has been generated with genome sequencing (http://www. phytozome.net/search.php?show=blast&method=Org_Creinhardtii). In the midst of all this, in 1957 Paul took a spring sabbatical at the Ephrussi lab in Paris where, in conversation with geneticist Georges Rizet, he outlined his plans to study recombination mechanisms in Chlamydomonas using the marked chromosomes that were being generated. ‘Wrong question’, Rizet huffed. ‘You should be studying photosynthesis’. Paul realized that C. reinhardtii, unlike C. moewusii, was able to grow in the dark in the presence of acetate, and that the acetate-requiring mutants that they had been mapping might be blocked in photosynthesis. Intrigued by this
Figure 1. Genetic maps of 11 linkage groups of Chlamydomonas reinhardtii. c, centromeres. From Ebersold et al. (1962).
© 2015 The Author The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 365–369
A brief history of early Chlamydomonas research 367 Figure 2. Proposed pathway for TPN/NADP photoreduction in Chlamydomonas reinhardtii. The positions of the postulated blocks represented by the mutant strains are shown. The relative positions of the blocks given for ac-115 and ac-141 may be reversed. From Levine and Smillie (1962).
LIGHT
LIGHT
ac–115 ac–141 H2O +0.815 V
1/2 O2
possibility, he returned to Harvard and obtained access to an illuminated Warburg apparatus, a clunky device by today’s standards, with Norman Krinsky providing tutorials. None of the mutants gave any evidence of being defective in fixing CO2 or evolving O2. The experiments had been tedious and the results were discouraging. What he needed, he realized, was a rapid and definitive screen for identifying photosynthetic nulls. The idea for a screen came during a late-night lab chat with colleague Bill Sistrom, and Paul quickly tested it out: colonies of acetate-requiring mutants and wild-type controls were grown on agar; a solution of sodium 14C–bicarbonate was placed in a watch glass in the Petri dish; a drop of acid was added to release 14CO2; time was allowed for photosynthetic incorporation; and a filter-paper replica of the colonies was then exposed to X–ray film (Levine, 1960a). All of the colonies were proven to be radioactive except one, ac21, which, it turned out, had not been included in the mutants tested in the Warburg experiments. Further analysis documented that ac21 was indeed defective in CO2 fixation and, more precisely, photosynthetic electron transport (Levine, 1960b). Yet to be identified are the defects incurred in the many acetate-requiring but photosynthetically competent mutants in the collection (Levine and Goodenough, 1970: table 1).
ac–21 CYTOCHROME f +0.365 V
PLASTOQUINONE; CYTOCHROME b6 –0.030 V
TPN –0.320 V
DPIP +0.217 V
Repeating the screen with additional strains yielded two more photosynthetic mutants, ac115 and ac141, and in the summer of 1961 Paul and his graduate student George Russell spent 3 months at Brookhaven in the lab of Bob Smillie, who had a Carey spectrophotometer, and were able to identify the defects of all three mutant strains. Their scheme is reproduced in Figure 2 (Levine and Smillie, 1962; Smillie and Levine, 1963). Paul proceeded to acquire not only his own Carey apparatus but also an Aminco–Chance dual-wavelength spectrophometer, with tutorials provided by its lively inventor Britton Chance, and the stage was set for a comprehensive analysis of all of the non-photosynthetic acetate-requiring mutants in the Levine collection. This collection was augmented in 1966, when Pierre Bennoun moved to the lab and developed a more rapid screening technique based on the enhanced fluorescence of mutant colonies blocked in photosynthesis (Bennoun and Levine, 1967). The resultant strains were designated with the prefix F for high fluorescence. Donald Gorman joined the lab as a graduate student in 1964, and his physical-science background, combined with Paul’s biological instincts and Judy Armstrong-Surzycki’s technical prowess, came together to produce a series of papers that collectively provided the first genetically based
Figure 3. Electron transport and phosphorylation components affected in Chlamydomonas mutants. Upper scheme: depiction of the thylakoid membrane showing the four protein complexes involved in electron transport and phosphorylation. Lower scheme: components affected in Levine’s mutants. Abbreviations: cyt, cytochrome; Fd, ferredoxin; FP, FD/NADP reductase; FeSa, Rieske iron–sulfur protein; FeSb, iron–sulfur protein and the stable electron acceptor of PSI; P680, reaction center chlorophyll of PSII; P700, reaction center chlorophyll of PSI; PC, plastocyanin; PQ, plastoquinone; QA, stable electron acceptor of PSII and electron donor to PQ. From Togasaki and Surzycki (1998), adapted with permission from ‘Genetic dissection of photosynthesis in Chlamydomonas reinhardtii’, R.P. Levine Science 1968;162:768–771. ©1968 American Association for the Advancement of Science.
© 2015 The Author The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 365–369
A brief history of early Chlamydomonas research 367 Figure 2. Proposed pathway for TPN/NADP photoreduction in Chlamydomonas reinhardtii. The positions of the postulated blocks represented by the mutant strains are shown. The relative positions of the blocks given for ac-115 and ac-141 may be reversed. From Levine and Smillie (1962).
LIGHT
LIGHT
ac–115 ac–141 H2O +0.815 V
1/2 O2
possibility, he returned to Harvard and obtained access to an illuminated Warburg apparatus, a clunky device by today’s standards, with Norman Krinsky providing tutorials. None of the mutants gave any evidence of being defective in fixing CO2 or evolving O2. The experiments had been tedious and the results were discouraging. What he needed, he realized, was a rapid and definitive screen for identifying photosynthetic nulls. The idea for a screen came during a late-night lab chat with colleague Bill Sistrom, and Paul quickly tested it out: colonies of acetate-requiring mutants and wild-type controls were grown on agar; a solution of sodium 14C–bicarbonate was placed in a watch glass in the Petri dish; a drop of acid was added to release 14CO2; time was allowed for photosynthetic incorporation; and a filter-paper replica of the colonies was then exposed to X–ray film (Levine, 1960a). All of the colonies were proven to be radioactive except one, ac21, which, it turned out, had not been included in the mutants tested in the Warburg experiments. Further analysis documented that ac21 was indeed defective in CO2 fixation and, more precisely, photosynthetic electron transport (Levine, 1960b). Yet to be identified are the defects incurred in the many acetate-requiring but photosynthetically competent mutants in the collection (Levine and Goodenough, 1970: table 1).
ac–21 CYTOCHROME f +0.365 V
PLASTOQUINONE; CYTOCHROME b6 –0.030 V
TPN –0.320 V
DPIP +0.217 V
Repeating the screen with additional strains yielded two more photosynthetic mutants, ac115 and ac141, and in the summer of 1961 Paul and his graduate student George Russell spent 3 months at Brookhaven in the lab of Bob Smillie, who had a Carey spectrophotometer, and were able to identify the defects of all three mutant strains. Their scheme is reproduced in Figure 2 (Levine and Smillie, 1962; Smillie and Levine, 1963). Paul proceeded to acquire not only his own Carey apparatus but also an Aminco–Chance dual-wavelength spectrophometer, with tutorials provided by its lively inventor Britton Chance, and the stage was set for a comprehensive analysis of all of the non-photosynthetic acetate-requiring mutants in the Levine collection. This collection was augmented in 1966, when Pierre Bennoun moved to the lab and developed a more rapid screening technique based on the enhanced fluorescence of mutant colonies blocked in photosynthesis (Bennoun and Levine, 1967). The resultant strains were designated with the prefix F for high fluorescence. Donald Gorman joined the lab as a graduate student in 1964, and his physical-science background, combined with Paul’s biological instincts and Judy Armstrong-Surzycki’s technical prowess, came together to produce a series of papers that collectively provided the first genetically based
Figure 3. Electron transport and phosphorylation components affected in Chlamydomonas mutants. Upper scheme: depiction of the thylakoid membrane showing the four protein complexes involved in electron transport and phosphorylation. Lower scheme: components affected in Levine’s mutants. Abbreviations: cyt, cytochrome; Fd, ferredoxin; FP, FD/NADP reductase; FeSa, Rieske iron–sulfur protein; FeSb, iron–sulfur protein and the stable electron acceptor of PSI; P680, reaction center chlorophyll of PSII; P700, reaction center chlorophyll of PSI; PC, plastocyanin; PQ, plastoquinone; QA, stable electron acceptor of PSII and electron donor to PQ. From Togasaki and Surzycki (1998), adapted with permission from ‘Genetic dissection of photosynthesis in Chlamydomonas reinhardtii’, R.P. Levine Science 1968;162:768–771. ©1968 American Association for the Advancement of Science.
© 2015 The Author The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 365–369
368 Ursula Goodenough description of the photosynthetic electron transport pathway (Gorman and Levine, 1966a,b,c,d). Additional photosynthetic mutants and processes were characterized by graduate students Nam-Hai Chua (Chua and Levine, 1969), Alice Givan (Givan and Levine, 1969), Ursula Goodenough (Goodenough and Levine, 1969, 1970, 1971; Goodenough et al., 1969; Goodenough and Staehelin, 1971), Bernice Moll (Moll and Levine, 1970), Vicki Sato (Sato et al., 1971) and John Whitmarsh (Whitmarsh and Levine, 1974), and postdocs Norman Krinsky (Krinsky and Levine, 1964), Andy Paszewski (Levine and Paszewski, 1970) and Bob Togasaki (Togasaki and Levine, 1970). Figure 3 shows a full inventory of the mutants analyzed and their posited defects in photosynthetic pathways. Many of the genes marked by these mutations have been further characterized in subsequent studies: ac115 encodes the D2 protein of photosystem II (PSII; Rattanachaikunsopon et al., 1999), whereas the F–34 gene product is required to synthesize the chlorophyll a-binding protein of PSII (Zerges et al., 2003); F–1 lacks several components of PSI (Chua et al., 1975); and ac21 and ac206 encode components of the cytochrome b6f complex (Xie et al., 1998; de Vitry et al., 1999). With the Levine mapping group hunkering down in the transfer room and the photosynthesis group holed up in the spectrophotometer room, a third group took on various nucleic acid and chloroplast biogenesis projects. Noboru Sueoka performed the first Meselson and Stahl-style demonstration of eukaryotic semiconservative DNA replication in C. reinhardtii in 1960 (Sueoka, 1960). Phil Hastings, Jean-David Rochaix and Steve Surzycki made major inroads in characterizing and manipulating chloroplast DNA (Surzycki and Hastings, 1968; Surzycki, 1969; Surzycki and Rochaix, 1971; Rochaix, 1972), and Nick Gillham made important contributions to our understanding of the chloroplast gene inheritance system pioneered by Ruth Sager (Gillham, 1963, 1965). By the early 1970s, Paul performed another of his selfreinventions and initiated a biochemical analysis of the complement proteins of the immune system, but many of his trainees (Bennoun, Chua, Ebersold, Gillham, Goodenough, Rochaix, Surzycki, Togasaki and Whitmarsh) continued with C. reinhardtii research in their own labs. The two other original C. reinhardtii training centers – the Rosenbaum lab at Yale and the Luck lab at the Rockefeller – bloomed in the 1970s, with a focus on flagellar motility and biogenesis, adopting strategies pioneered in Ralph Lewin’s original studies of C. moewusii. And Ursula Goodenough, Patrick Ferris, Jim Umen, JaeHyeok Lee, Bill Snell (a Rosenbaum graduate student) and their colleagues pursued an analysis of the sexual cycle (Goodenough et al., 1995, 2007; Snell and Goodenough, 2008), picking up where Gilbert Smith and Ruth Sager left off in their pioneering papers on C. reinhardtii
sexuality (Smith and Regnery, 1950; Sager and Granick, 1954). The articles in this volume document that it was most fortunate indeed that Paul, Ruth, and Ralph bumped into each other on Stony Beach that afternoon in 1955. REFERENCES Bennoun, P. and Levine, R.P. (1967) Detecting mutants that have impaired photosynthesis by their increased level of fluorescence. Plant Physiol. 42, 1284–1287. Chua, N.-H. and Levine, R.P. (1969) The photosynthetic electron transport chain of Chlamydomnonas reinhardi. VIII. The 520 nm light-induced absorbance change in the wild-type and mutant strains. Plant Physiol. 44, 1–6. Chua, N.-H., Matlin, K. and Bennoun, P. (1975) A chlorophyll-protein complex lacking in photosystem I mutants of Chlamydomonas reinhardtii. J. Cell Biol. 67, 361–377. Ebersold, W.T. (1956) Crossing over in Chlamydomonas reinhardi. Am. J. Bot. 43, 408–410. Ebersold, W.T. and Levine, R.P. (1959) A genetic analysis of linkage group I of Chlamydomonas reinhardi. Z. Vererbungsl. 89, 631–635. Ebersold, W.T., Levine, R.P., Levine, E.E. and Olmsted, M.A. (1962) Linkage maps in Chlamydomonas reinhardi. Genetics, 47, 531–543. Eversole, R.A. (1956) Biochemical mutants of Chlamydomonas reinhardti. Am. J. Bot. 43, 404–407. Gillham, N.W. (1963) The nature of exceptions to the pattern of uniparental inheritance for high level streptomycin resistance in Chlamydomonas reinhardi. Genetics, 48, 431–439. Gillham, N.W. (1965) Linkage and recombination between nonchromosomal mutations in Chlamydomonas reinhardi. Proc. Natl Acad. Sci. USA, 54, 1560–1567. Givan, A.L. and Levine, R.P. (1969) The photosynthetic electron transport chain of a mutant strain of Chlamydomonas reinhardi lacking P700. Biochim. Biophys. Acta, 189, 401–410. Goodenough, U.W. and Levine, R.P. (1969) Chloroplast ultrastructure in mutant strains of Chlamydomonas reinhardi lacking components of the photosynthetic apparatus. Plant Physiol. 44, 990–1000. Goodenough, U.W. and Levine, R.P. (1970) Chloroplast structure and function in ac-20, a mutant strain of Chlamydomonas reinhardi. III. Chloroplast ribosomes and membrane organization. J. Cell Biol. 44, 547–562. Goodenough, U.W. and Levine, R.P. (1971) The effects of inhibitors of RNA and protein synthesis on the recovery of chloroplast ribosomes, membrane organization and photosynthetic electron transport in the ac-20 strain of Chlamydomonas reinhardi. J. Cell Biol. 50, 50–62. Goodenough, U.W. and Staehelin, L.A. (1971) Structural differentiation of stacked and unstacked chloroplast membranes: freeze-etch electron microscopy of wild type and mutant strains of Chlamydomonas reinhardi. J. Cell Biol. 48, 594–619. Goodenough, U.W., Armstrong, J.J. and Levine, R.P. (1969) Photosynthetic properties of ac-31, a mutant strain of Chlamydomonas reinhardi devoid of chloroplast membrane stacking. Plant Physiol. 44, 1001–1012. Goodenough, U.W., Armbrust, E.V., Campbell, A.M. and Ferris, P.J. (1995) Molecular genetics of sexuality in Chlamydomonas. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 21–44. Goodenough, U., Lin, H. and Lee, J.-H. (2007) Sex determination in Chlamydomonas. Semin. Cell Dev. Biol. 18, 350–361. Gorman, D.S. and Levine, R.P. (1966a) The photosynthetic electron transport chain of Chlamydomonas reinhardi. IV. The purification and properties of plastocyanin. Plant Physiol. 41, 1637–1642. Gorman, D.S. and Levine, R.P. (1966b) Photosynthetic electron transport chain of Chlamydomonas reinhardi. V. Purification and properties of cytochrome 553 and ferridoxin. Plant Physiol. 41, 1643–1647. Gorman, D.S. and Levine, R.P. (1966c) The photosynthetic electron transport chain of Chlamydomonas reinhardi. VI. Electron transport in mutant strains lacking either cytochrome 553 or plastocyanin. Plant Physiol. 41, 1648–1656. Gorman, D.S. and Levine, R.P. (1966d) Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc. Natl Acad. Sci. USA, 54, 1665–1669.
© 2015 The Author The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 365–369
doi: 10.1111/tpj.12794
SI CHLAMYDOMONAS
Historical perspective on Chlamydomonas as a model for basic research: 1950–1970 Ursula Goodenough* Department of Biology, Washington University, St. Louis, MO 63130, USA Received 22 December 2014; revised 28 January 2015; accepted 2 February 2015; published online 17 February 2015. *For correspondence (e-mail [email protected]).
SUMMARY During the period 1950–1970, groundbreaking research on the genetic mapping of Chlamydomonas reinhardtii and the use of mutant strains to analyze photosynthesis was conducted in the laboratory of R. Paul Levine at Harvard University. An account of this era, based in part on interviews with Levine, is presented. Keywords: Chlamydomonas, history of science, R. Paul Levine, Ruth Sager, Ralph Lewin, photosynthesis, genetics.
INTRODUCTION The coming of age of Chlamydomonas reinhardtii has been recounted in detail in two book chapters, one by Togasaki and Surzycki (1998), with a focus on the chloroplast, and one by Joel Rosenbaum (2009), with a focus on the flagellum. Presented here is a version of the story based on conversations with Paul Levine, a pioneer in analyzing the genetics and photosynthesis of C. reinhardtii. The story has multiple plot lines, originating in the early 1950s, that come together on a beach at Woods Hole, Massachusetts, USA, in 1955. We start at Stanford University. After a lifetime of writing such comprehensive treatises as Phytoplankton of the Inland Waters of Wisconsin (Smith, 1920) and The Freshwater Algae of the United States (Smith, 1933), phycologist Gilbert Morgan Smith returned to an earlier passion, the isolation of pure algal clones from field specimens (Smith, 1916), and chose Chlamydomonas reinhardi (now spelled reinhardtii) as his target species. Bill Eversole, a student with fungal geneticist Edward Tatum, and Bill Ebersold, a student with population geneticist David Regnery, each launched PhD thesis projects that entailed mutagenizing these isolates and following the sexual inheritance of mutant traits (e.g. arginine-requiring, acetate-requiring, pigment-deficient; Eversole, 1956; Ebersold, 1956). Upon graduation in the mid-1950s Eversole left the field, but Ebersold took a postdoctoral position at Dartmouth and continued with his project. © 2015 The Author The Plant Journal © 2015 John Wiley & Sons Ltd
Next, we look to Scripps Oceanographic Institute, where phycologist Ralph Lewin, a man with seemingly countless interests (https://scripps.ucsd.edu/news/2423), spent several years focused on Chlamydomonas moewusii, a distant relative of C. reinhardtii, following the inheritance of paralyzed-flagella traits in sexual crosses (Lewin, 1954). His original interest had been to find mutants disabled in photosynthesis, but his lab discovered that C. moewusii is an obligate phototroph (Lewin, 1950), and hence mutations affecting photosynthesis would be lethal. Meanwhile, at Rockefeller University, Ruth Sager joined botanist Sam Granick’s lab as a postdoc in 1949. In 1951, as a staff member, she launched her own study of C. reinhardtii, independently obtaining a wild-type strain from Gilbert Smith that turned out to be different from the strain studied by Eversole and Ebersold, a salient difference being its ability to grow with nitrate as the sole nitrogen source (Harris, 2009: 11–16). She published definitive papers on its ultrastructure (Sager and Palade, 1957) and the nutritional control of its sexuality (Sager and Granick, 1954), and initiated several decades of research on the inheritance of its non-Mendelian traits (Sager, 1955, 1960), which, as she and others went on to document, are encoded in its chloroplast DNA. Finally, the story moves to Harvard University. Paul Levine, an assistant professor trained in Drosophila genetics at UC Berkeley, was interested in recombination 365
366 Ursula Goodenough mechanisms and decided to switch to a fungal system. He therefore went to the Marine Biological Laboratories in Woods Hole in the summer of 1955 to develop his skills in Neurospora tetrad analysis, a technique that he first learned as a summer undergraduate researcher at Cal Tech in George Beadle’s lab. This takes the story to Stony Beach. One afternoon, Paul ran into Ruth and Ralph out taking a stroll, and the three began to talk science. Paul described his plans to study recombination in fungi, and Ralph huffed. ‘Wrong organism’, he announced. He and Ruth proceeded to tout the advantages of C. reinhardtii, and Ralph dropped in the line that were Paul to follow their advice he should recruit a hot-shot guy named Bill Ebersold. Paul took the bait, and Ebersold moved to Cambridge in 1956 with the goal of developing a high-density genetic map of C. reinhardtii for recombination studies. The next few years can best be described as a genetics orgy, with technicians Elizabeth Levine and Peggy Olmstead to the fore. Some 250 crosses were performed and more than 20 000 tetrads were analyzed, with the results recorded on countless file cards. Replica plating was used to score
growth requirements, including strains that required acetate, and drops of water on colonies were used to score paralyzed mutants, many of which were provided by Ralph Lewin (a full list of strains is provided in Harris, 1989). The first map, of linkage group I, was published in 1959 (Ebersold and Levine, 1959). A map of 11 linkage groups, reproduced in Figure 1, was published in 1962 (Ebersold et al., 1962). A more detailed map was available by 1965 (Hastings et al., 1965) and now, of course, a full map has been generated with genome sequencing (http://www. phytozome.net/search.php?show=blast&method=Org_Creinhardtii). In the midst of all this, in 1957 Paul took a spring sabbatical at the Ephrussi lab in Paris where, in conversation with geneticist Georges Rizet, he outlined his plans to study recombination mechanisms in Chlamydomonas using the marked chromosomes that were being generated. ‘Wrong question’, Rizet huffed. ‘You should be studying photosynthesis’. Paul realized that C. reinhardtii, unlike C. moewusii, was able to grow in the dark in the presence of acetate, and that the acetate-requiring mutants that they had been mapping might be blocked in photosynthesis. Intrigued by this
Figure 1. Genetic maps of 11 linkage groups of Chlamydomonas reinhardtii. c, centromeres. From Ebersold et al. (1962).
© 2015 The Author The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 365–369
A brief history of early Chlamydomonas research 367 Figure 2. Proposed pathway for TPN/NADP photoreduction in Chlamydomonas reinhardtii. The positions of the postulated blocks represented by the mutant strains are shown. The relative positions of the blocks given for ac-115 and ac-141 may be reversed. From Levine and Smillie (1962).
LIGHT
LIGHT
ac–115 ac–141 H2O +0.815 V
1/2 O2
possibility, he returned to Harvard and obtained access to an illuminated Warburg apparatus, a clunky device by today’s standards, with Norman Krinsky providing tutorials. None of the mutants gave any evidence of being defective in fixing CO2 or evolving O2. The experiments had been tedious and the results were discouraging. What he needed, he realized, was a rapid and definitive screen for identifying photosynthetic nulls. The idea for a screen came during a late-night lab chat with colleague Bill Sistrom, and Paul quickly tested it out: colonies of acetate-requiring mutants and wild-type controls were grown on agar; a solution of sodium 14C–bicarbonate was placed in a watch glass in the Petri dish; a drop of acid was added to release 14CO2; time was allowed for photosynthetic incorporation; and a filter-paper replica of the colonies was then exposed to X–ray film (Levine, 1960a). All of the colonies were proven to be radioactive except one, ac21, which, it turned out, had not been included in the mutants tested in the Warburg experiments. Further analysis documented that ac21 was indeed defective in CO2 fixation and, more precisely, photosynthetic electron transport (Levine, 1960b). Yet to be identified are the defects incurred in the many acetate-requiring but photosynthetically competent mutants in the collection (Levine and Goodenough, 1970: table 1).
ac–21 CYTOCHROME f +0.365 V
PLASTOQUINONE; CYTOCHROME b6 –0.030 V
TPN –0.320 V
DPIP +0.217 V
Repeating the screen with additional strains yielded two more photosynthetic mutants, ac115 and ac141, and in the summer of 1961 Paul and his graduate student George Russell spent 3 months at Brookhaven in the lab of Bob Smillie, who had a Carey spectrophotometer, and were able to identify the defects of all three mutant strains. Their scheme is reproduced in Figure 2 (Levine and Smillie, 1962; Smillie and Levine, 1963). Paul proceeded to acquire not only his own Carey apparatus but also an Aminco–Chance dual-wavelength spectrophometer, with tutorials provided by its lively inventor Britton Chance, and the stage was set for a comprehensive analysis of all of the non-photosynthetic acetate-requiring mutants in the Levine collection. This collection was augmented in 1966, when Pierre Bennoun moved to the lab and developed a more rapid screening technique based on the enhanced fluorescence of mutant colonies blocked in photosynthesis (Bennoun and Levine, 1967). The resultant strains were designated with the prefix F for high fluorescence. Donald Gorman joined the lab as a graduate student in 1964, and his physical-science background, combined with Paul’s biological instincts and Judy Armstrong-Surzycki’s technical prowess, came together to produce a series of papers that collectively provided the first genetically based
Figure 3. Electron transport and phosphorylation components affected in Chlamydomonas mutants. Upper scheme: depiction of the thylakoid membrane showing the four protein complexes involved in electron transport and phosphorylation. Lower scheme: components affected in Levine’s mutants. Abbreviations: cyt, cytochrome; Fd, ferredoxin; FP, FD/NADP reductase; FeSa, Rieske iron–sulfur protein; FeSb, iron–sulfur protein and the stable electron acceptor of PSI; P680, reaction center chlorophyll of PSII; P700, reaction center chlorophyll of PSI; PC, plastocyanin; PQ, plastoquinone; QA, stable electron acceptor of PSII and electron donor to PQ. From Togasaki and Surzycki (1998), adapted with permission from ‘Genetic dissection of photosynthesis in Chlamydomonas reinhardtii’, R.P. Levine Science 1968;162:768–771. ©1968 American Association for the Advancement of Science.
© 2015 The Author The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 365–369
A brief history of early Chlamydomonas research 367 Figure 2. Proposed pathway for TPN/NADP photoreduction in Chlamydomonas reinhardtii. The positions of the postulated blocks represented by the mutant strains are shown. The relative positions of the blocks given for ac-115 and ac-141 may be reversed. From Levine and Smillie (1962).
LIGHT
LIGHT
ac–115 ac–141 H2O +0.815 V
1/2 O2
possibility, he returned to Harvard and obtained access to an illuminated Warburg apparatus, a clunky device by today’s standards, with Norman Krinsky providing tutorials. None of the mutants gave any evidence of being defective in fixing CO2 or evolving O2. The experiments had been tedious and the results were discouraging. What he needed, he realized, was a rapid and definitive screen for identifying photosynthetic nulls. The idea for a screen came during a late-night lab chat with colleague Bill Sistrom, and Paul quickly tested it out: colonies of acetate-requiring mutants and wild-type controls were grown on agar; a solution of sodium 14C–bicarbonate was placed in a watch glass in the Petri dish; a drop of acid was added to release 14CO2; time was allowed for photosynthetic incorporation; and a filter-paper replica of the colonies was then exposed to X–ray film (Levine, 1960a). All of the colonies were proven to be radioactive except one, ac21, which, it turned out, had not been included in the mutants tested in the Warburg experiments. Further analysis documented that ac21 was indeed defective in CO2 fixation and, more precisely, photosynthetic electron transport (Levine, 1960b). Yet to be identified are the defects incurred in the many acetate-requiring but photosynthetically competent mutants in the collection (Levine and Goodenough, 1970: table 1).
ac–21 CYTOCHROME f +0.365 V
PLASTOQUINONE; CYTOCHROME b6 –0.030 V
TPN –0.320 V
DPIP +0.217 V
Repeating the screen with additional strains yielded two more photosynthetic mutants, ac115 and ac141, and in the summer of 1961 Paul and his graduate student George Russell spent 3 months at Brookhaven in the lab of Bob Smillie, who had a Carey spectrophotometer, and were able to identify the defects of all three mutant strains. Their scheme is reproduced in Figure 2 (Levine and Smillie, 1962; Smillie and Levine, 1963). Paul proceeded to acquire not only his own Carey apparatus but also an Aminco–Chance dual-wavelength spectrophometer, with tutorials provided by its lively inventor Britton Chance, and the stage was set for a comprehensive analysis of all of the non-photosynthetic acetate-requiring mutants in the Levine collection. This collection was augmented in 1966, when Pierre Bennoun moved to the lab and developed a more rapid screening technique based on the enhanced fluorescence of mutant colonies blocked in photosynthesis (Bennoun and Levine, 1967). The resultant strains were designated with the prefix F for high fluorescence. Donald Gorman joined the lab as a graduate student in 1964, and his physical-science background, combined with Paul’s biological instincts and Judy Armstrong-Surzycki’s technical prowess, came together to produce a series of papers that collectively provided the first genetically based
Figure 3. Electron transport and phosphorylation components affected in Chlamydomonas mutants. Upper scheme: depiction of the thylakoid membrane showing the four protein complexes involved in electron transport and phosphorylation. Lower scheme: components affected in Levine’s mutants. Abbreviations: cyt, cytochrome; Fd, ferredoxin; FP, FD/NADP reductase; FeSa, Rieske iron–sulfur protein; FeSb, iron–sulfur protein and the stable electron acceptor of PSI; P680, reaction center chlorophyll of PSII; P700, reaction center chlorophyll of PSI; PC, plastocyanin; PQ, plastoquinone; QA, stable electron acceptor of PSII and electron donor to PQ. From Togasaki and Surzycki (1998), adapted with permission from ‘Genetic dissection of photosynthesis in Chlamydomonas reinhardtii’, R.P. Levine Science 1968;162:768–771. ©1968 American Association for the Advancement of Science.
© 2015 The Author The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 365–369
368 Ursula Goodenough description of the photosynthetic electron transport pathway (Gorman and Levine, 1966a,b,c,d). Additional photosynthetic mutants and processes were characterized by graduate students Nam-Hai Chua (Chua and Levine, 1969), Alice Givan (Givan and Levine, 1969), Ursula Goodenough (Goodenough and Levine, 1969, 1970, 1971; Goodenough et al., 1969; Goodenough and Staehelin, 1971), Bernice Moll (Moll and Levine, 1970), Vicki Sato (Sato et al., 1971) and John Whitmarsh (Whitmarsh and Levine, 1974), and postdocs Norman Krinsky (Krinsky and Levine, 1964), Andy Paszewski (Levine and Paszewski, 1970) and Bob Togasaki (Togasaki and Levine, 1970). Figure 3 shows a full inventory of the mutants analyzed and their posited defects in photosynthetic pathways. Many of the genes marked by these mutations have been further characterized in subsequent studies: ac115 encodes the D2 protein of photosystem II (PSII; Rattanachaikunsopon et al., 1999), whereas the F–34 gene product is required to synthesize the chlorophyll a-binding protein of PSII (Zerges et al., 2003); F–1 lacks several components of PSI (Chua et al., 1975); and ac21 and ac206 encode components of the cytochrome b6f complex (Xie et al., 1998; de Vitry et al., 1999). With the Levine mapping group hunkering down in the transfer room and the photosynthesis group holed up in the spectrophotometer room, a third group took on various nucleic acid and chloroplast biogenesis projects. Noboru Sueoka performed the first Meselson and Stahl-style demonstration of eukaryotic semiconservative DNA replication in C. reinhardtii in 1960 (Sueoka, 1960). Phil Hastings, Jean-David Rochaix and Steve Surzycki made major inroads in characterizing and manipulating chloroplast DNA (Surzycki and Hastings, 1968; Surzycki, 1969; Surzycki and Rochaix, 1971; Rochaix, 1972), and Nick Gillham made important contributions to our understanding of the chloroplast gene inheritance system pioneered by Ruth Sager (Gillham, 1963, 1965). By the early 1970s, Paul performed another of his selfreinventions and initiated a biochemical analysis of the complement proteins of the immune system, but many of his trainees (Bennoun, Chua, Ebersold, Gillham, Goodenough, Rochaix, Surzycki, Togasaki and Whitmarsh) continued with C. reinhardtii research in their own labs. The two other original C. reinhardtii training centers – the Rosenbaum lab at Yale and the Luck lab at the Rockefeller – bloomed in the 1970s, with a focus on flagellar motility and biogenesis, adopting strategies pioneered in Ralph Lewin’s original studies of C. moewusii. And Ursula Goodenough, Patrick Ferris, Jim Umen, JaeHyeok Lee, Bill Snell (a Rosenbaum graduate student) and their colleagues pursued an analysis of the sexual cycle (Goodenough et al., 1995, 2007; Snell and Goodenough, 2008), picking up where Gilbert Smith and Ruth Sager left off in their pioneering papers on C. reinhardtii
sexuality (Smith and Regnery, 1950; Sager and Granick, 1954). The articles in this volume document that it was most fortunate indeed that Paul, Ruth, and Ralph bumped into each other on Stony Beach that afternoon in 1955. REFERENCES Bennoun, P. and Levine, R.P. (1967) Detecting mutants that have impaired photosynthesis by their increased level of fluorescence. Plant Physiol. 42, 1284–1287. Chua, N.-H. and Levine, R.P. (1969) The photosynthetic electron transport chain of Chlamydomnonas reinhardi. VIII. The 520 nm light-induced absorbance change in the wild-type and mutant strains. Plant Physiol. 44, 1–6. Chua, N.-H., Matlin, K. and Bennoun, P. (1975) A chlorophyll-protein complex lacking in photosystem I mutants of Chlamydomonas reinhardtii. J. Cell Biol. 67, 361–377. Ebersold, W.T. (1956) Crossing over in Chlamydomonas reinhardi. Am. J. Bot. 43, 408–410. Ebersold, W.T. and Levine, R.P. (1959) A genetic analysis of linkage group I of Chlamydomonas reinhardi. Z. Vererbungsl. 89, 631–635. Ebersold, W.T., Levine, R.P., Levine, E.E. and Olmsted, M.A. (1962) Linkage maps in Chlamydomonas reinhardi. Genetics, 47, 531–543. Eversole, R.A. (1956) Biochemical mutants of Chlamydomonas reinhardti. Am. J. Bot. 43, 404–407. Gillham, N.W. (1963) The nature of exceptions to the pattern of uniparental inheritance for high level streptomycin resistance in Chlamydomonas reinhardi. Genetics, 48, 431–439. Gillham, N.W. (1965) Linkage and recombination between nonchromosomal mutations in Chlamydomonas reinhardi. Proc. Natl Acad. Sci. USA, 54, 1560–1567. Givan, A.L. and Levine, R.P. (1969) The photosynthetic electron transport chain of a mutant strain of Chlamydomonas reinhardi lacking P700. Biochim. Biophys. Acta, 189, 401–410. Goodenough, U.W. and Levine, R.P. (1969) Chloroplast ultrastructure in mutant strains of Chlamydomonas reinhardi lacking components of the photosynthetic apparatus. Plant Physiol. 44, 990–1000. Goodenough, U.W. and Levine, R.P. (1970) Chloroplast structure and function in ac-20, a mutant strain of Chlamydomonas reinhardi. III. Chloroplast ribosomes and membrane organization. J. Cell Biol. 44, 547–562. Goodenough, U.W. and Levine, R.P. (1971) The effects of inhibitors of RNA and protein synthesis on the recovery of chloroplast ribosomes, membrane organization and photosynthetic electron transport in the ac-20 strain of Chlamydomonas reinhardi. J. Cell Biol. 50, 50–62. Goodenough, U.W. and Staehelin, L.A. (1971) Structural differentiation of stacked and unstacked chloroplast membranes: freeze-etch electron microscopy of wild type and mutant strains of Chlamydomonas reinhardi. J. Cell Biol. 48, 594–619. Goodenough, U.W., Armstrong, J.J. and Levine, R.P. (1969) Photosynthetic properties of ac-31, a mutant strain of Chlamydomonas reinhardi devoid of chloroplast membrane stacking. Plant Physiol. 44, 1001–1012. Goodenough, U.W., Armbrust, E.V., Campbell, A.M. and Ferris, P.J. (1995) Molecular genetics of sexuality in Chlamydomonas. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 21–44. Goodenough, U., Lin, H. and Lee, J.-H. (2007) Sex determination in Chlamydomonas. Semin. Cell Dev. Biol. 18, 350–361. Gorman, D.S. and Levine, R.P. (1966a) The photosynthetic electron transport chain of Chlamydomonas reinhardi. IV. The purification and properties of plastocyanin. Plant Physiol. 41, 1637–1642. Gorman, D.S. and Levine, R.P. (1966b) Photosynthetic electron transport chain of Chlamydomonas reinhardi. V. Purification and properties of cytochrome 553 and ferridoxin. Plant Physiol. 41, 1643–1647. Gorman, D.S. and Levine, R.P. (1966c) The photosynthetic electron transport chain of Chlamydomonas reinhardi. VI. Electron transport in mutant strains lacking either cytochrome 553 or plastocyanin. Plant Physiol. 41, 1648–1656. Gorman, D.S. and Levine, R.P. (1966d) Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc. Natl Acad. Sci. USA, 54, 1665–1669.
© 2015 The Author The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 365–369
