André Tridon Jagendorf(Courtesy of Division of Rare & Manuscript

Collections, Cornell University Library)

Photosynthesis Research57: 215–229, 1998.© 1998Kluwer Academic Publishers. Printed in the Netherlands.

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Personal perspective

Chance, luck and photosynthesis research: An inside story

Andre Tridon JagendorfPlant Biology Section, Plant Science Building, Cornell University, Ithaca, NY 14853, USA

Received 27 October 1997; accepted in revised form 31 May 998

Key words:chemiosmosis, photophosphorylation, proton gradients, Reca in chloroplasts, thylakoid ATPase

Abstract

A major theme in my career has been photophosphorylation; especially contributions to the early work on chemios-mosis, and later involvement in CF1 activation and function. A second theme has been interest in chloroplastbiogenesis, with work ranging from translation in chloroplasts to discovery of the enzyme which may contributeto strand exchange, homologous recombination and DNA repair in chloroplasts. Throughout, I try to point outthe major contributions of graduate students and postdocs, and help from friends and colleagues. Without them Iwould have had no career at all.

The perspective for these recollections comes from theunderlying theme in at least one book by StanislawLem (1978). The probability of any one event oc-curring is amazingly small; in retrospect, each stepseems like a minor miracle. Any person’s career hasto be shaped by interactions with other people; minewas certainly very much shaped by faculty colleagues,postdocs, graduate and undergraduate students. Tostart with the punch line – whatever happened wasvery much the result of luck, and chance, and thesewonderfully helpful influences.

Starting in early youth – my move into plant sci-ences was a compromise between two forces. Myfather, a New York City dentist with broad interests,loved the look of well-kept farms and wanted me tobe a farmer. But I was reading science fiction, andwas in one of the first classes at the Bronx HighSchool of Science. I compromised, and went into PlantPhysiology.

Education, and some deficiencies

In college at Cornell, I was strongly influenced byLoren Petri, teaching General Botany. Later I realizedhe’d done a fine job of showmanship; and that ap-proach was perfect for fresh-persons. He made Botanycome alive for us; a tradition followed in a highly

competent way by at least 2 succeeding paleobotanists(Harlan Banks; Karl Niklas) teaching the introductorycourse at Cornell. Other outstanding teachers helpedkeep my enthusiasm alive – especially Otis Curtis inPlant Physiology, Lester Sharp in Cytology, and BobSmock in Pomology.

Going to Yale for graduate work in Plant Science,I was very fortunate to be taken into the group run byDavid Bonner; essentially because nobody else in thatdepartment wanted to take a chance on having me.David Bonner was one of the early founders of Bio-chemical Genetics. Everybody else in his lab workedwith either Neurosporaor E. coli mutants and theirbiochemistry. However David’s PhD thesis, under theguidance of his older brother James Bonner, had beenon radish cotyledon growth. He turned me loose on acontinuation of that work, which ended up as a thesisdescribing effects of 2,4-D on seedling growth of cab-bage. My training in the lab was aided materially byAubrey Naylor, in whose tissue culture facilities I didmost of my work. While it was tremendously excitingfor me at that time, by taking that pathway I did missout on getting very early into the main stream of ge-netic engineering and biochemistry (other students inthe Bonner group at that time, for instance, includedthe future distinguished scientists Charlie Yanofsky,Gabriel Lester, Bill Jakoby and Otto Landman). Be-

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ing in Bonner’s group was a wonderful experiencebecause we had a strong feeling of fellowship, andall of us benefited from David and Miriam Bonner’sstrong support. And I learned the most important thinga graduate student can learn – doing science is fun.

However, I realized later that my formal educa-tion was, at best, spotty. For instance, I was nevertold I must take a course in Biochemistry, and neverdid (but I did audit the one given by Joseph Fruton,over in the medical school). It was an era (1948–1951)when exciting events included the discovery of asym-metry in the proportion of bases in DNA (overturningthe ‘tetranucleotide’ theory); the announcement of en-zyme ‘action at a distance’ through a layer of gold leaf(I’ll forget the name of the scientist who advocatedthat); the discovery of the language of the bees; and thebeginning of some idea of the diversity and functionsof organelles. Certainly, one area that I heard aboutand felt was really exciting, was the mechanism ofoxidative phosphorylation.

David Bonner, not having continued with plantphysiology after his thesis, didn’t feel entirely com-fortable trying to direct my work with cabbages. Atone point he sent me to see Kenneth Thimann atHarvard, to consult on some results; then one happysummer I hitchhiked out to Cal Tech to learn PlantPhysiology at its true center. I was supposed to workwith James Bonner there; but he was busy and I spentmy time with Sam G. Wildman, isolating leaf pro-teins. That 2 month period actually resulted in a paper(Axelrod and Jagendorf 1951); Bernard Axelrod mea-sured some hydrolytic enzymes, and I measured totalprotein levels, as excised tobacco leaves senesced. Theexperience was very exciting, and gave me a visionof doing work at a deeper level than just overall plantgrowth.

The happiest years of my life (postdoctoral)

I received my PhD in 1951 (after 3.5 years) andwas lucky enough to receive a Merck PostdoctoralFellowship. I spent 2 postdoctoral years with SamWildman, who had moved from Cal. Tech. to UCLA’sgreenhouse (we worked in the concrete-floored headhouse, converted to a genuine laboratory). Again, itwas a wonderful, friendly environment in which towork. Together with Al Siegal, Irv Rappaport, MiltZaitlin and Morris Cohen I discovered the joys of3-dimensional tic-tac-toe, and checkers, during thelunch breaks. During alternative lunches I attended a

‘cynical seminar’ – discussion of politics by selectedfaculty and postdocs, all from the liberal, losing pointof view. There I met Jacob Biale, a scholarly, intrepidback-packer and mountain climber, and a very goodscientist who introduced me to avocados and respi-ration. During that first year I also was excited by‘discovering’ that leaves had mitochondria (defined bydifferential centrifugation) as well as chloroplasts.

Finding a job at that time was either easy or dif-ficult, depending on one’s connections. One day Ireceived a telephone call – ‘Would you like to be anAssistant Professor at the Johns Hopkins University?’I said ‘Yes, but could I please come East and give aseminar, first?’ (I was engaged, and that would havegiven me a chance to see my fiancée). The answer was‘No – just come next September.’ I was apparentlythe token botanist they were looking for. I workedwith plants, had taken (and enjoyed) Edmund Sin-nott’s courses on Plant Morphogenesis at Yale and socould be related to the growth and development biasin the Hopkins Biology Department (its chairman wasB. Willier, an embryologist). But I had also gottenmy degree with Bonner and so in theory should ap-preciate biochemistry and biochemical genetics. Thismade me acceptable to the McCollum–Pratt Institute(a biochemical group, devoted to trace element re-search, funded by Pratt in honor of Dr McCollum)which had just started recently with Bill McElroy incharge. I received a year’s reprieve; went East anywayto get married, and then spent a second happy conjugalyear at UCLA. This included a full 10 camping trips.During that second year Sam and I actually published apaper declaring among other things that ‘chloroplastshave no nucleic acid’ (Jagendorf and Wildman 1954).Of course, it was prior to development of really sen-sitive assays for nucleic acids, and also before weknew the difference between isolated chloroplasts andisolated thylakoids. Dim excuses!

The entry into photosynthesis; The Johns HopkinsUniversity

At The Johns Hopkins, my education in biochem-istry really began. From Bill Harrington I learned ofdensity gradient centrifugation; applied it to ‘chloro-plasts’ (really thylakoids), watched them float up on aglycerol gradient, and made them more pure than be-fore (Jagendorf 1955). McCollum–Pratt Institute hada lunch-time journal club in biochemistry, run by NateKaplan, Sid Colowick and Al Nason. Sitting in on it,

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I began to realize what an exciting field biochemistrycould be. Especially, the thrill of rigorous proof of aconcept, something often lacking in the plant physiol-ogy literature at that time. I also began to understanda little more biochemistry – for instance that enzymecatalyzed reactions are reversible.

Originally, I had thought to use the more pure‘chloroplasts’ to generate antibodies, which might beable to pull down proplastids selectively, and help inthe study of chloroplast biogenesis. But Bill McElroygave me a crucial push, by asking whether I might findout what the ‘pure chloroplasts’ could do. At aboutthat time Wolf Vishniac had presented evidence thatTPNH (today’s NADP) was an early intermediate inphotosynthetic electron flow. I went ahead and showedthat the isolated thylakoids could reduce TPN in thelight, but not DPN (Jagendorf 1956). And then, hav-ing learned that enzymes are reversible, and if TPNHwas an important early product of the light reactions –perhaps these chloroplasts might be able to use TPNHas substrate, to reduce something else? That led to thediscovery of an enzyme in isolated thylakoids, that didindeed reduce added dyes when fed TPNH.

This discovery was critical, because it permittedme to attract Mordhay Avron to my lab as a postdoc.He had gotten his PhD (in 2.5 years, if you can believeit – Sam Wildman said that if he’d realized, he neverwould have passed him) with Jacob Biale; so we had atie in common. At a meeting I offered him the position,to work on this enzyme. The enzymology appealed tohim more than working on CO2 uptake by roots, theheart of a competing postdoctoral offer to work withthe much more famous scientist, Kenneth Thimann.Mordhay joined my lab, as my second postdoc. I andJean were both immediately taken with the wonderfulpersonalities of these exotic people from Israel – Mordand Nira. We remained friends over many years, andMord did more for me than I can ever repay.

With no real trouble, Mordhay purified the enzymewhich turned out to be yellow, have FAD as a cofac-tor, and was properly described from its function as a‘diaphorase’ (Avron (Abramsky) and Jagendorf 1956).An aside – many of the enzymes in photosynthesiswere discovered 3 times in a row. This one was dis-covered just a little later by San Pietro, a colleague ofmine in the same department at the Johns Hopkins, asa transhydrogenase: it used NADPH to reduce analogsof NADP. Even later it was discovered for its trulyimportant functionin vivo, as the Ferredoxin-NADPReductase, or FNR, by Arnon, Whatley and Allen.

After purifying and characterizing the diaphorase,Mordhay said to me ‘André – we have to find its truefunction. It must be a part of photophosphorylation.’ Isaid ‘Mordhay – photophosphorylationdoes not exist.’This may need a little explanation.

Photophosphorylation had been discovered re-cently at the time, by Lipmann and Frenkel withbacterial vesicles, and by Arnon, Whatley and Allenwith thylakoids. The Arnon group was using AMPas phosphate acceptor (called ‘adenylic acid’ at thattime); perhaps to save money, and probably becauseit had not yet been proven that ADP was the real ac-ceptor. In trying to repeat their discovery, I went tothe shelf having left over chemicals from the previ-ous occupant of the lab, found a bottle labeled ‘YeastAdenylic Acid’, and used it. There was no Pi uptake.The point is, I was young and poorly trained at thattime, and did not know the difference between yeastadenylic acid (the name at that time for 3′-AMP, madefrom alkaline hydrolysis of yeast RNA) and muscleadenylic acid (5′-AMP).

Mordhay convinced me to let him try. He’d donehis thesis on oxidative phosphorylation with avocadomitochondria. Of course it worked for Mordhay, andwe were the first lab in the US to repeat Arnon’s dis-covery. Perhaps in part for a psychological reason. Anumber of established groups were distressed that inthe excitement of their discovery of cell-free ATP syn-thesis, Arnon et al. had failed to give proper credit tothose who had shown byin vivoexperiments that ATPsynthesismustbe a part of photosynthesis. At leastsome of these groups spent too much time trying toprove that Arnon was wrong; that he had discovered amixed reaction between 2 organelles. The chloroplastswould do their thing and produce O2, and mitochon-dria in the preparation would use the oxygen to makeATP. Which shows that sometimes it helps not to haveany stake in the field to begin with.

Well, our first publication on photophosphoryla-tion was a mistake. It was a note in Nature (Avronand Jagendorf 1957), describing the function of an‘extractable factor’ – i.e. protein – in permitting morerapid rates of photophosphorylation. We were still us-ing AMP as substrate. Sid Colowick pointed out tome the protein might just be adenylic kinase (AMP +ATP⇐⇒ 2 ADP). I rushed to the shelf full of hand-me-down chemicals, found a bottle labeled ‘ADP’,used that as a substrate instead of AMP, and stillhad a big rate stimulation from the protein. So wesaid it wasn’t adenylic kinase; but we were wrong.Later work showed it was; and no doubt the ADP

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in the years old bottle sitting on the shelf at roomtemperature without desiccation, had largely decom-posed to AMP. But at least, the paper got us into thephotophosphorylation field.

Another fortuitous event helped move us forward.Symposia were rare in those days; the McCollum–Pratt Institute was pioneering in holding them. At thesymposium called ‘Light and Life’, I heard HowardGest mention that as the extra electron carrying dye,something called ‘phenazine methosulfate’ was justterrific, in bacterial photophosphorylation. Please re-call that in vitro photophosphorylation with isolatedthylakoids depends on some added dye to carry elec-trons around the cycle, for cyclic electron flow. Arnonet al. were using riboflavin and Vitamin K5, both nat-ural products, for this purpose. We threw in PMS andthe rates became faster. Then Mordhay noticed thatvessels exposed to the middle of the bank of lights(in an illuminated Warburg apparatus, left behind byConrad Yocum) showed faster rates than those at theends of the row. This led him to look at light intensi-ties more carefully.(historical note – measured in thosedays, as foot-candles). Sure enough, by raising thefluence greatly, the rates became much higher (Jagen-dorf and Avron 1958). This put us way ahead in the‘rate race’ with the Arnon crew (they got up to 2 to 3hundred; we were up to 7 hundredµmoles/mg chloro-phyll/hour), and helped show the enormous potentialsof the thylakoid system. We now know, of course, thatthis depends on the formation of a completely artifi-cial energy coupling site, as PMS itself takes electronsaway from the reducing end of PS I, is protonated,and moves back into the lumen; generating a protongradient in the process.

Other work in the early years at the Johns Hop-kins included participation in the definition of thy-lakoid electron transport pathways. Space limitationsimposed by a procrustean editor permit me to men-tion only that the associates doing this work includedGiorgio Forti, Maurice Margulies, John Brewer andTadashi Asahi. Their work included finding an enzymeto reverse inhibition by excess levels of CMU (Asahi),defining the roles for O2 and ascorbate (Forti), locat-ing sites for inhibition by DCMU and by incubation ofbean leaves in the cold and dark (Margulies), and byferricyanide with thylakoids in the dark (Brewer).

Al Nason, my mentor in plant biochemistry atHopkins, suggested the way to discover the electrontransfer pathway was to spend a week with BrittonChance. With his advanced electronic gadgetry (i.e.double beam spectrophotometers) the problem of the

nature and components in chloroplast electron flowwould be solved in a trice. I went to Philadelphia,spent hours in the dark lab watching Britt adding com-ponents and turn the light on and off. We discoveredan enormous peak in the 500 nm region when chloro-plasts were illuminated together with ADP and theputative inhibitor of electron flow,o-phenanthroline.Which turned out, of course, to be a matter of Fe3+contaminating the ADP. In the light the thylakoids re-duced it to Fe2+ which forms a deep red complex witho-phenanthroline. We also saw large spectrophotomet-ric changes at 518 nm, but didn’t understand them. Ittook Horst Witt to show this was the electrochromicshift, representing a membrane potential. Any life isfull of missed opportunities, of course.

The next aspect that we got into was that of ‘photo-synthetic control’ – i.e. the existence of coupled elec-tron flow in the Hill Reaction. Together with DavidKrogmann, then a graduate student, Mordhay showedthat FeCN reduction in the Hill reaction is faster, ifphotophosphorylationgoes on at the same time (Avronet al. 1958). David discovered (just a little before Nor-man Good did) that ammonium ions were uncouplersfor photophosphorylation (Krogmann et al. 1959). An-other graduate student, Richard McCarty, found thatfree fatty acids (generated in bean 1◦ leaves duringgrinding, by active lipases) were also uncouplers ofphotophosphorylation (McCarty and Jagendorf 1965).Later McCarty and his students, among others, pro-vided massive evidence for the existence of the1pHin illuminated thylakoids, for the stoichiometry of pro-tons/ATP formed, for the nature of coupling, and forstructure and function of CF1, in many publicationsover the years.

The existence of coupling in thylakoids sounds likeold hat today; and indeed it was that for oxidativephosphorylationat that time. But oxidative phosphory-lation had been discovered 18 years before (in 1937)and the Arnon group had just discovered photophos-phorylation by thylakoids only a year or two before. Iremember going to a Brookhaven Symposium, tellingabout the chloroplast reactions, and getting a truly star-tled reaction from Britton Chance. He could hardlybelieve that photosynthetic control had never beennoticed before.

Even more surprising, perhaps, was the reactionof Otto Warburg. For younger readers – he was theworld’s pre-eminent biochemist, without a doubt, forseveral decades. He discovered respiratory enzymes,studied many different biochemical reactions, and hadwon a Nobel prize. While a marvelous laboratory

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worker, his theories, especially later in his life, tendedto be way out of line. He had convinced himself, forinstance, that CO2 attached to chlorophyll, and was re-duced directly by energy from chlorophyll in the light.So to have electron transport generating ATP seemeda bit beside the point to him.

Warburg was dedicated to manometric methods tomeasure O2 exchanges and electron transport. Thesetake time, and Warburg found no difference betweenthe 30 minute output of O2 with and that withoutadded ADP, Pi & Mg2+. Probably the thylakoids werein bad shape due to their prolonged incubation withFeCN and at room temperature in the light. Warburgpublished that there was no such stimulation, and alsowrote me a letter to that effect. In a footnote to histable, he noted briefly and cryptically, that our ob-servation was probably due to a failure to control thepH.

Birgit Vennesland, a highly competent biochemistin Chicago, and Mary Stiller worked hard to find outjust what ‘failure to control the pH’ might do. Theyfigured it out. Without proper buffering, if the pH wastoo low, the Hill reaction was rather slow. But ATPsynthesis leads to the consumption of protons; so thepH rises. (This rise in pH is often used as an indirectway to measure phosphorylation rates.) Venneslandthought that we had used a too low pH, and the rise dueto phosphorylation would then make the Hill reactionrun faster.

Their concept was published in Nature (Vennes-land and Stiller 1961). Luckily, Nature gave bothNorman Good (1961) and me (Jagendorf 1961) thechance to answer in accompanying letters. This I did ina chivalrous, and therefore gentle and mealy-mouthedway, and Norman Good in a highly straightforwardway. The point is, of course, wehad controlled thepH; and the Warburg/Vennesland criticism was totallyunjustified.

Mordhay Avron finally had to leave, and wenton to a most productive and distinguished career atthe Weizmann Institute of Science in Israel. Whilethere, among other things, he discovered the couplingfactor of chloroplast thylakoids, CF1. I had discov-ered how to uncouple thylakoids using EDTA at lowionic strength (Jagendorf and Smith 1962) but neverfound how to recouple them. Racker and Vambutashad found an extractable ATPase in thylakoids, andpresumed it would be the coupling factor; but Avronwas the first one to really show that it worked in ATPsynthesis (Avron 1963).

Looking into the mechanism of ATP synthesis:post-illumination, pH rise, and chemiosmosis

At that point in the history of bioenergetics, the shin-ing goal was to discover the link between electronflow, and the chemical reaction of ATP synthesis.There were many theories; but all of them involvedsome sort of high energy intermediate of one of theelectron carriers bonded to some other molecule (see,for instance, Chance and Williams 1956).

Every year, somebody else seemed to have anexperimental demonstration of the true high energyintermediate. A friend described it to me by sayingthat going to the Federation meetings was reminiscentof going to the Alchemists’ convention. Each year,somebody else would stand up and say thathe haddiscovered a fool-proof way of turning lead into gold.And of course, nobody did.

Well, I thought that using chloroplasts might makethe job of capturing a high energy intermediate a littleeasier, because you could turn off the light so quickly.So I thought I’d try to jump into the act.

First, I spent a summer at Brookhaven, trying andfailing to set up a stop-flow apparatus where the thy-lakoids would go from light to dark and meet thesubstrates (ADP, Pi, Mg2+) at the same time. How-ever, I did learn a great deal about photosynthesisthat summer; because Marty Gibbs was there also.Lunch after lunch we would eat outside on the grass,and Marty would ramble on and on about people,events, theories and ideas in photosynthesis. It was thefirst time I had actually had somebody talking aboutphotosynthesis, and it was very educational.

Then later, it occurred to me to use a simpler sys-tem - just a syringe in the light, with the thylakoidspushed down into an aluminum-foil covered dark testtube containing ADP,32Pi and Mg2+. That worked,and I discovered the existence of ‘XE’ – an unknown,high energy intermediate. Shortly after, I was fortu-nate to have Geoffrey Hind join my laboratory andhelp with this discovery. Now the only thing was, theamountof ATP made in the post-illumination dark (upto 50 nmoles/mg chlorophyll) was too high to fit withthe hypotheses current at that time. It represented upto 50 times the amount of any one electron transportenzyme. So turnover must have occurred, in the dark(Hind and Jagendorf 1963).

Geoffrey did scrupulous experiments defining thekinetics of rise and fall of XE. Of course, we neverused anything but spinach from the local A&P. Per-haps I shouldn’t have been surprised when the dark

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decay constant for this hypothetical high energy in-termediate in ATP formation decreased 3-fold, in ex-periments before and after April 1. I tried, and failed,to get an explanation (let alone an apology) from theA&P. They said it was a trade secret.

Keeping the record straight – XE was not a uniquediscovery. A little sooner, Y.-K. Shen in China hadalso discovered post-illumination ATP synthesis bythylakoids (Shen and Shen 1962). We did not knowof their work until we had sent our paper off for pub-lication. I corresponded with Shen a little bit; about2 letters in each direction. I remember, in the last let-ter, writing that I hoped we could meet, once politicalpassions died down. I didn’t hear from him again formany years – the Cultural Revolution had hit, and hewas unable to do research for at least 10 years. After,Shen rose high, headed the Shanghai Institute of PlantPhysiology, and led a distinguished group, exploringmany details of photophosphorylation. We did meet –Shen’s group of photosynthesis workers were the firstto make a tour of the U.S. after the Cultural Revolu-tion was over (see Shen, 1994 for a more completedescription of his fascinating career).

It was both fortunate and critical that GeoffreyHind was in my lab at that time. I had heard PeterMitchell talk about chemiosmosis at a bioenergeticsmeeting in Sweden. His words went into one of myears and out the other, leaving me feeling annoyed theyhad allowed such a ridiculous and incomprehensiblespeaker in. But – Geoffrey read Nature. Geoffrey wasfrom England, both better trained and more intelligentthan I was. He read Peter Mitchell’s paper, came tome, and said ‘André. could this possibly explain XE?’

During the discussion, it occurred to us that wemight be able to see the pH in the medium rise dur-ing light-driven electron flow. I stayed in the lab latethat same evening, put thylakoids in a beaker togetherwith PMS, inserted a glass electrode, and watched theneedle of the meter rise in the light and fall in thedark. It was the first time I remember an immedi-ately successful test of a working hypothesis – a mostexciting event! We published this first in the photo-synthesis symposium at Airlie House (Jagendorf andHind 1963).

I should point out that we were lucky on 3 ac-counts. First is that Mitchell, writing about mito-chondria, postulated electron flow causing H+ uptake.That’s true for chloroplasts, but not for right side outmitochondria. If we had seen the pH change in thewrong direction, it would have been too puzzling tofollow up. Secondly, we were lucky in that thylakoids,

unlike mitochondria, have active ion transporters intheir membranes. So that many protons can go in,their + charges neutralized by exiting cations but es-pecially by entering Cl−. Thylakoids accumulate HCl,rather like the stomach. Mitochondria, not having theappropriate translocator(s), rapidly form a membranepotential, and only a very few protons can move beforethe whole system stops.

The third point is, we were lucky in having pyocya-nine (the light-driven product of PMS non-enzymaticoxidation) to support a rapid cyclic electron flow, withno obscuring scalar production of H+ in the mediumas with ferricyanide or NADP+ reduction. The per-ceived change in pH of the (weakly or unbuffered)medium came to 0.5 pH unit, or more. This was trueif the medium pH was about 6.0 to begin with; muchless change is seen at pH 8. The extent of proton move-ment did seem large enough to suggest a real basis forthe high amount of ATP that could be formed in thepost-illumination darkness.

This discovery of H+ uptake in the light and re-lease in the dark was explored further by JosephNeumann, who was able to tie H+ uptake definitivelyto the coupling phenomenon. It only occurred in thelight, was prevented by uncouplers, and uncouplersadded once the protons were in allowed them to leakout very rapidly (Neumann and Jagendorf 1964).

At this point, I became rather convinced – al-though without completely rigorous evidence – thatthe chemiosmotic explanation was the correct one forthe connection between electron flow and ATP syn-thesis. This conviction helped me to avoid a fruitlesssearch for a chemical intermediate. I remember morethan one biochemical colleague (for instance, IsraelZelitch) saying ‘You have your fingers on the real in-termediate, André. What you should do is go in thereand fish it out!’ To which my response was – ‘Yes, butmaybe it’s just a pH gradient, and you can’t fish thatout.’ Of course he laughed.

Geoffrey Hind looked for spectral changes thatmight lead us to the nature of XE. The only thing hefound were changes in light scattering. Apparently inthe light, thylakoids shrink together and scatter lightmore strongly than when they’re swollen. This effectwas greater as the pH decreased, reminiscent of theyield of ATP in post-illumination experiments. Beinga careful scientist, Geoffrey ran a dark control. Hewas amazed to see that a little bit of ATP was indeedformed entirely in the dark, just because the thylakoidshad been moved from pH 4.6 to pH 8 (see Table 1,from Hind and Jagendorf 1965). The amount was only

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Table 1. Effect of pH on post-illumination phosphorylation by spinach thy-lakoids. Chloroplasts with 40µg chlorophyll per ml were illuminated for either5 sec or 1 min. Adjustment of pH was made by dilution of a concentratedparticle suspension in the appropriate buffer, just before use. Samples for deter-mining the dark XE level were kept in total darkness at pH 8.0 and 5◦ for 1 hbefore use, and were maipulated in total darkness. Results are shown in nmolesATP per Mg chlorophyll. Modified from Hind and Jagendorf (1965)

Row Measurement pH

4.5 5.3 6.0 6.7 7.2 7.7

A Yield from 5-sec flash 15 17 18 7.1 4.3 1.9

B Maximal yield 52 54 37 7.8 4.3 1.9

C Initial dark XE level 5.5 1.4 1.6 1.6 0.6 0

D Maximal XE level 58 55 39 8.0 4.9 1.9

5.5 nmoles of ATP/mg chlorophyll (see Row C, inTable 1). This was so little compared to the amountsof post-illumination ATP (‘XE’) that we hardly knewwhether to consider it significant. Now in retrospect,it was a very logical thing to look for. But if it hadnot been for the unlikely result in one control point,we probably would not have discovered acid-base ATPsynthesis.

Geoffrey moved on to Brookhaven, anxious to getinto spectrophotometric analyses of thylakoids doingphotosynthesis. I thought it would be important to looka little closer at this low level of dark XE. Geoffreyhad used hydrochloric acid to adjust the pH of the thy-lakoid suspensions. Being a mystical minded biologistinstead of a trained biochemist, I worried whether thestrong acid might be intrinsically harmful to the thy-lakoids. So I started using organic acids – first of all,the highly synthetic one, phthallic acid. The differencewas startling. Instead of 5 nmoles of ATP, I worked upto 50, 60 and eventually 100 or more nmoles of ATPper Mg chlorophyll. Explanation from hindsight – thedivalent acid at a pH where it was 50% protonated,moving into the thylakoid, brought along a store ofprotons far beyond the number of protonatable groupspresent endogenously in the thylakoid lumen.

At this point I began to communicate with PeterMitchell himself. He had recently moved into his newlaboratory and home in Cornwall (the Glynn ResearchFoundation) and invited me to spend a week there sohe could educate me about the chemiosmotic hypothe-sis in more detail. I was happy to go, and enjoyed verymuch meeting his family and the family donkey, andseeing his fascinating Regency house (half home, halflab) and tree-high rhododendrons in bloom in Corn-

Figure 1. Yield of ATP in acid-base phosphorylation as a functionof varying pH in the acid stage, at different base stage pHs (‘pHof ADP stage’). Reproduced with permission from Jagendorf andUribe (1966).

wall in the spring. I doubt that I learned enough aboutchemiosmosis, however.

Later that summer I did the experiment that con-vinced me (and I hoped, others) that we were re-ally seeing a chemiosmotic mechanism at work. Theamount of ATP that was made depended on the heightof the pH difference between acid and base stages,more than on their absolute values (Figure 1, fromJagendorf and Uribe 1966).

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These data helped very much in the scientific com-munity’s acceptance of the chemiosmotic principles. Ireported them first at a Gordon Conference in 1965.At the Federation meetings in the spring of the follow-ing year Paul Boyer was able to report that they wereindeed repeatable. With acceptance from importantquarters, the rest of the world had to pay attention.

Another bit of sociological fallout – it was good forthe morale of plant scientists at that time. It showedthat work with plant materials could contribute toproblems of importance for the general biologicalcommunity. We didn’t have to be restricted to eitherplant-specific problems, or trying to see if plants didthe same things as animals or yeast or bacteria, in anarea of general interest.

Cornell again: Chemiosmosis, CF1 action, andchloroplast biogenesis

Just after this (1966) I was enticed to move back toCornell, especially by the persuasive Bob Morison,who was building the revitalized Biology Division. Ofcourse, part of the attraction was the memory frommy undergraduate days of what a beautiful spot Ithacais. Indeed, the continuity extended to the point of mybeing given, for my office, the room in which I wasadvised as an undergraduate.

A pH gradient is not simple to demonstrate di-rectly. Showing it was the true basis for ATP synthesisrequired ruling out alternatives as rigorously as pos-sible. Ernest Uribe, moving with me from Baltimoreto Ithaca, showed that entry of the organic acid wasthe only parameter that really mattered, for ATP for-mation (Uribe and Jagendorf 1967a, b). His resultsemphasized another lucky break in this project – Ge-offrey Hind had gotten into the habit of washing thethylakoids in 10 mm NaCl prior to use, and this meantwe were using swollen vesicles, with plenty of internalspace to store organic acids.

Another point – Donald Miles found that inhibitorsof electron flow (DCMU, HOQNO,o-phenanthroline,BDHB, simazine) either had no effect, or actuallyin-creasedthe ATP yield (Miles and Jagendorf 1970).We can ascribe that to blocking of reversed electronflow, which is driven by and would help use up, a pro-tonmotive force, as discovered by Mordahay Avron’slab (Rienits et al. 1973). Also relevant – Alice Gre-banier demonstrated that 2 different light-dependentinhibitions could be driven by either PS I only or PSII only. Then, the subsequent photophosphorylation

supported by the opposite photosystem was equallyinhibited (Grebanier and Jagendorf 1977). This clearlyimplied the absence of a specific association betweenthe electron flow and one particular ATP synthase pop-ulation. Many other laboratories contributed to theevidence for chemiosmosis. Maybe most prominentwas the demonstration that liposomes containing boththe H+-pumping purple membrane patch ofHalobac-terium halobium,a non-photosynthetic bacterium, to-gether with the Fo/F1 of mitochondria, accomplishedphotophosphorylation (Racker and Stoeckenius 1974)There was hardly any question left that this ruled outa direct, necessary interaction between mitochondrialelectron carrying enzymes (which were not there) andthe ATP synthase.

All in all, the bioenergetics community has donea good job of showing the reality of Peter Mitchell’sbrilliant concept of the nature of coupling betweenelectron flow and ATP synthesis. There are some con-troversial areas – it’s not totally clear that thylakoidsin vivo are swollen enough to drive ATP synthesis viaa bulk pH gradient. This, and some other data, leadssome to wonder whether there are "localized" protonsalong the membrane, communicating directly from theelectron carriers to the ATP synthase (see, for instance,Chiang and Dilley 1987). But this may be a matter ofdefinition, as to what a ‘local proton’ really is. Ba-sically, however, Mitchell’s concept of protonmotiveforce as the driving force for ATP synthesis stands,and I’ve been very pleased to have played some partin showing its validity.

On the photophosphorylation trail, I eventually de-veloped a stronger interest in CF1, and in its conforma-tional changes in the light when bound to thylakoids.A major contribution for that time was the tedious buteffective determination of the amino acid content ofall of the 5 CF1 subunits, by Andres Binder (Binderet al. 1978). I was inspired to think about confor-mational movements of these subunits by the earlierfinding of R. McCarty that one of the free -SH groupsof CF1 is attacked by N-ethylmaleimide only when thethylakoids are illuminated (McCarty et al. 1972)). Aprimary finding was that made by Ivan Ryrie, whoshowed that exchange between H atoms on CF1 andmedium tritiated water was more extensive in the lightthan in the dark, and returning thylakoids to the darkput up to 100 atoms per mole into hidden pocketswhere exchange out was very much slower (Ryrieand Jagendorf 1971, 1972). This was primary evi-dence for a major conformational change in thylakoidbound CF1. Studies of light-enhanced or -dependent

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inhibition by chemical reagents were also made byDipak Datta (permanganate inhibition), David Oliver(TNBS), and Ivan Ryrie and Alice Grebanier (sul-fate, which uncouples in the light if ADP and mg arepresent). Other contributions with chemical modifierswere made by Emanuel DeBenedetti and Joan Gar-barino, Roland Schmid and Tetsuko Takabe (Takabe etal.1982). Lawrence Posorske found significant effectsof adenylates in protecting CF1 against inactivation inthe cold. Some of the ionic interactions of thylakoidsas related to energy status were explored by Bill Co-hen, and by Yuichiro Nishizaki, who built a successfulsmall stop-flow apparatus to look at the kinetics ofproton efflux from thylakoids.

Studies of CF1 activation is another direction wefollowed. Joel Kaplan had found that the acid-basetransition activated thylakoid ATPase (Kaplan et al.1967). Gordon Anthon found a remarkable stimulationof the basal activity by methanol, with an incrediblysharp optimum at 35% (v/v) during the assay (An-thon and Jagendorf 1983). With solubilized CF1, amajor inhibition of the methanol-driven ATPase byfree Mg++ could be reversed by sulfite ions (Anthonand Jagendorf 1986). Eric Larson looked at the sulfitestimulation in much greater detail, and found that ithad remarkable effects on the reduced form of eitherthylakoid-bound or solubilized CF1 (Larson and Ja-gendorf 1989). Part of the effect is probably due torelease of the bound, inhibitory ADP (Larson et al.1989); but in any case it permitted observing ATPaserates 5 times faster than any seen before. Others work-ing in this area included Jia-mian Wei, Bruce Howlett,Ann Umbach and Rong-Long Pan.

Other work with CF1 has included experimentsby Geoffrey Chen, showing assembly of the full, ac-tive protein from over-expressed, denatured subunitsonly with the help of chloroplast chaperonins (Chenand Jagendorf 1994). Unisite hydrolysis of ATP bythylakoids was found by Shiying Zhang to differ insurprising ways from that shown by mitochondrialparticles (Zhang and Jagendorf 1995).

Another strong interest for me has been under-standing something of chloroplast biogenesis. AlvaApp, in Baltimore, showed that the development ofchloroplasts inEuglenacould be repressed by metab-olizable substrates, such as ethanol. We had a quickfling at chloroplast RNA polymerase, with GideonPolya (Poly and Jagendorf 1971), and Holly Doremusdemonstrated synthesis of pyrimidine nucleotides inisolated chloroplasts (Doremus and Jagendorf, 1985).Quite a few students and postdocs worked on pro-

tein synthesis by isolated chloroplasts, with specialemphasis on activity of thylakoid-bound ribosomes.These included A. Gnanam, Mahtab Bamji, LindaGooding, Harry Roy, Kar-Ling Tao, Richard Patter-son, Len Fish, Josh Hurewitz, Helen Nivison, RuthAlscher, Taibo Yamamoto and Devaki Bhaya. Whilepursuing protein translation, Xiang-Qiu Liu discov-ered the ATP-dependent proteolysis of some of thenewly formed polypeptides in chloroplasts (Liu andJagendorf 1984). Unfortunately, the activity was notstable once the chloroplast envelopes were opened,and Liu studied other peptidases in chloroplast stroma,using synthetic substrates. An endopeptidase whichmay be able to attack Rubisco was purified further bythe brothers Bushnell (Bushnell et al. 1993), a pair ofvery talented undergraduates.

Probably no scientific career is without its near-misses. I had at least two, both related to my interest inchloroplast biogenesis. On sabbatic leave at the Weiz-mann Institute of Science in Rehovot, my ambitionwas to attach antibodies covalently to a solid matrix,in order to capture the specific antigen. Michael Sela’sdepartment was developing solid matrices which per-mitted covalent attachment of proteins. I eventuallytried brom-acetyl-bromide, which could attach to cel-lulose, and the second bromide could bind a protein.Six weeks before I had to leave Rehovot, I had a happyaccident. I left a magnetic stirrer run overnight in thecold room, and it ran amok. That way I discoveredthat if immunoglobulins are partially denatured, theycan bind to such a column (Jagendorf et al. 1963). Inretrospect – what was needed was a spacer arm (seeCuatrecasas 1970). We showed that the column withbound antibodies could in turn bind the antigen, eggalbumen (Jagendorf et al., 1963). While the work wassuccessful, it was a near-miss because I never did any-thing further about it. Fortunately for science, moreastute people such as Cuatrecasas developed affinitychromatography to the useful state we all know aboutnow. The moral is, follow-through, and publishingmore than one paper on a subject, is very often neededto allow something to become a real contribution toscience.

The other near-miss was coming close to, but fail-ing to, discover that proteins targeted for the chloro-plast are synthesized as a larger precursor. LindaGooding and Harry Roy, using antibodies, had shownthat isolated cytosol ribosomes apparently made a pro-tein of the expected size (12 kDa) for the small subunitof Rubisco (Gooding et al. 1973). But Roy also founda product that was quit a bit larger (about 20 kDa).

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Both proteins were isolated from the SDS gel, andlyophilized prior to analyzing peptides to see if theycorresponded to the expected primary sequence ofamino acids. During the lyophilizing, the flask con-taining the larger protein imploded, and the analysiswas never run. Roy had to leave my lab to go to anacademic job, and I never had anyone follow up onthat discovery.

Eventually, the era of DNA technology caught upwith me. I thought it would be interesting to try chloro-plast transformation; this interest was shared by a newgraduate student, Heriberto Cerutti. He first attemptedto see if DNA could actually enter intact chloroplasts.This turned out to be the case if they are subject toheat shock, probably forming hydrophilic pores in theenvelope via lipid HexII phase formation (Cerutti andJagendorf 1995). Trying to see if the imported DNAmight function in chloroplast metabolism, Heribertodiscovered DNA strand exchange activity in stromaextracts, with characteristics very much like those ofbacterial RecA protein (Cerutti and Jagendorf 1993).This led to a successful search for the gene for a RecAhomolog in anArabidopsiscDNA library. This wassequenced, and its close relationship to bacterial genesnoted (Cerutti et al. 1992). The genomic form of thegene was sequenced by Marie-Noëlle Binet (Binet etal. 1993), and further definition of its function withthe help of a magnificent series of DNA constructswas made by Jun Cao (Cao and Jagendorf 1997). Thiswas the pathway that brought my lab activities into thecurrent era, and incidentally gave a little more insightinto the capabilities of chloroplasts.

Concluding remarks

All of us in science know the deep satisfaction of doinga practical experiment to test a concept or model. Cer-tainly those with more than a year or two of experienceknow the feeling of wonder and pleasure at seeingmore and more of the workings of the plant worldexposed and understood; and the continual process ofchange and refinement of our understanding. Addingto these pleasures I have had warm, supportive andgenial companions in the arena of photosynthetic sci-ence. I have certainly heard and seen the dog-eat-dogatmosphere in some other fields, but rarely if ever inphotosynthesis. Maybe some colorful characters hereand there; but it’s been great to know, and interactwith, kind and thoughtful people like Jack Myers,Bessel Kok, William Arnold, Takashi Akazawa, Lou

Duysens, Marty Gibbs, Kazuo Shibata, George Hoch,Rod Clayton, Norman Good and so very many others,too numerous to mention.

Science is an activity done by human beings. Inany human activity, there are many turning points,chance events, lucky and unlucky accidents. Thereare some brilliant, hard-driving individuals who makeenormous contributions based on clear hypotheses andlogical reasoning. But others of us noodle along andby chance can add an important bit here and there.In my own career, chance and luck have had starringroles. I never would have accomplished much at all,except for the help of postdocs, graduate students andfriends. Most prominent among these were MordhayAvron and Geoffrey Hind. My thanks to them, and tothe United States public for its support of basic scienceover the years spanning my career. May these happyconditions, or at least a reasonable facsimile, continuefor all the patient readers of this wandering memoir.

Acknowledgments

These recollections would never have been writtenwithout the persistent urging of Govindjee over manyyears. Apologies to others who have worked in my lab,and whose contributions could not be described dueto lack of space. Above all, my gratitude to a warmand understanding family, without whose support andpatience my career could never have grown as it did.

References

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Avron M (1963) A coupling factor in photophosphorylation.Biochim Biophys Acta 77: 699–702

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Bushnell Timothy P, Bushnell David and Jagendorf André T (1993)A purified zinc protease of pea chloroplasts, EP1, degrades thelarge subunit of Rubisco. Plant Physiol 103: 585–591

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Chiang G and Dilley RA (1987) Evidence for calcium-gated protonfluxes in chloroplast thylakoid membranes: Calcium ion con-trols a localized to delocalized gradient switch. Biochemistry 26:4911–4916

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Gooding L, Roy H and Jagendorf AT (1973) Immunological iden-tification of nascent subunits of wheat ribulose diphosphatecarboxylase on ribosomes of both chloroplast and cytoplasmicorigin. Arch Biochem Biophys 159: 324–335

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Jagendorf AT and Uribe E (1966) ATP formation caused by acid-base transition of spinach chloroplasts. Proc Natl Acad Sci (US)55: 170–177

Jagendorf AT and Wildman SG (1954) The proteins of green leaves.VI. Centrifugal fractionation of tobacco leaf homogenates andsome properties of isolated chloroplasts. Plant Physiol 29: 270–279

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Postscript

Photophosphorylation and the greenhouse at Johns Hopkins University, 1959–1964

Richard E. McCartyDepartment of Biology, Johns Hopkins University, Baltimore, MD 21218, USA

The headhouse (storage and working space) for theJohns Hopkins greenhouse should have a plaque com-memorating a scientific breakthrough. Converted tolab space, it was the original home of the McCollum–Pratt Institute (starting faculty: W. McElroy, S. Colow-ick, N. Kaplan, A. Nason and R. Ballentine). AndréJagendorf, after a 2-year start in one room of the mainbuilding, was moved to the greenhouse headhouse.Then, one by one, the McCollum–Pratt faculty movedto fancier, renovated labs in the main building, Mer-genthaler Hall. By the late 1950s, André had inheritedall three floors of this small, separate building. It washere that André with his postdoctoral associates, Geof-frey Hind, Joseph Neumann and Ernest Uribe, carriedout the remarkable series of experiments that pro-vided strong evidence in support of Peter Mitchell’schemiosmotic hypothesis.

I started as an undergraduate in the Jagendorf lab-oratory as a result of a happy accident. I was the

only person to sign up for a laboratory course in plantphysiology that André was offering during the springsemester that year. I stayed on to do my thesis workand left the lab in the summer of 1964. I was thusin André’s lab when Geoff Hind was investigating thelight and dark stages of photophosphorylation, workthat led to the finding that ATP formation by thy-lakoids in the dark could be driven by acid to basetransitions. Joe Neumann was in the process of show-ing that electron transport caused a rise in pH that wasclearly a consequence of proton transport into what wenow call the thylakoid lumen.

I would like to say that I was in the thick of thisexciting work, but I was not. André’s space in thegreenhouse was distributed over all three floors of thegreenhouse. His main lab was adjacent to his office onthe first floor. There was a wonderfully air conditionedlab in the basement that housed equipment, includingthe Radiometer pH meter and pH-stat and a spectro-

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graph. The graduate students were housed upstairs inwhat is essentially a garret. I was working on a projectof my own making, one much less significant thanthose being worked on downstairs. André was a won-derful mentor for graduate students. He gave us freerein and expected us to have our PhD theses completedin four years. It was the postdoctoral associates andAndré who did the most significant work in André’slab at the time.

Despite the fact that I was not directly involvedin the work on protons and photophosphorylation, Ihave clear memories of several aspects of this work.I recall that the two-stage phosphorylation and acidbath experiments were carried out in near darkness ina coldroom. Many of these experiments were carriedout by the exceptionally able and good natured techni-cian, Marie Smith. Marie would spend hours gropingaround in the coldroom clad in a parka that lookedas though it was meant for use in the Arctic and thatseemed entirely out of place in Baltimore, especiallyduring the torrid summer months.

I also recall Joe Neumann working in the semi-darkness of the instrument lab in the basement, shininglight from a slide projector on thylakoid membranesand measuring pH changes. Only after he left the labdid I realize the significance of his experiments. Icould always tell when Geoff Hind’s work was goingwell. He would sing what I think were motets in aclear, tenor voice. He sang very frequently.

André has a long standing interest in cell, molec-ular and developmental biology. He and Alva App, apostdoctoral associate, were investigating protein syn-thesis by what we then called intact chloroplasts but

were really thylakoids. I had more interactions withAl App than with either Joe or Geoff perhaps becauseI had aspired to do my thesis work on the developmentof the two photosystems and of photophosphorylationin greening etiolated beans. I never got that far.

André was actively involved in all aspects of thelab. In addition to contributing his keen insights to thework of others in the lab, André spent as much time ashis often hectic schedule would allow working at thebench. He still does.

By the time I left André’s lab I had assimilated agreat deal of advanced knowledge about photophos-phorylation and how to study the process. Thanksto my wonderful experiences there, I was able makerapid progress in defining the role of a coupling factorfor photophosphorylation discovered by Vida Vambu-tas, a graduate student in Ef Racker’s lab. Thus, theexcitement of the results from the labs in the green-house rubbed off on a graduate student more interestedthen in developmental biology than in biochemistry.

Every chance I get I point out the greenhouse tostudents and visitors to the Hopkins campus and in-form them that the greenhouse is the place where thefamous acid bath and related experiments were carriedout. Many seem incredulous. The building is small andnot that impressive. Today, the greenhouse is occu-pied by Facilities Management, although one of thetwo glasshouses still is sometimes used. André’s mainlab on the first floor has been converted to a con-ference room. I like to attend meetings in this roomas it is a wonderful place to evoke the extraordinaryexperiences we all had in André’s lab.