My Journey from Horticulture to Plant Biology Jan A.D. Zeevaart MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824; email: [email protected]Annu. Rev. Plant Biol. 2009. 60:1–19 The Annual Review of Plant Biology is online at plant.annualreviews.org This article’s doi: 10.1146/annurev.arplant.043008.092010 Copyright c 2009 by Annual Reviews. All rights reserved 1543-5008/09/0602-0001$20.00 Key Words photoperiodism, florigen, flowering, grafting, gibberellin, abscisic acid, water stress Abstract The author describes the circumstances and opportunities that led him to higher education and to pursue a research career in plant biology. He acknowledges the important roles a few individuals played in guiding him in his career. His early work on flowering was followed by studies on the physiological roles and the metabolism of gibberellins and ab- scisic acid. He describes how collaborations and technical developments advanced his research from measuring hormones by bioassay to their identification and quantification by mass spectrometry and cloning of hormone biosynthetic genes 1
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My Journey from Horticultureto Plant BiologyJan A.D. ZeevaartMSU-DOE Plant Research Laboratory and Department of Plant Biology,Michigan State University, East Lansing, Michigan 48824; email: [email protected]
Annu. Rev. Plant Biol. 2009. 60:1–19
The Annual Review of Plant Biology is online atplant.annualreviews.org
This article’s doi:10.1146/annurev.arplant.043008.092010
photoperiodism, florigen, flowering, grafting, gibberellin, abscisicacid, water stress
AbstractThe author describes the circumstances and opportunities that led himto higher education and to pursue a research career in plant biology. Heacknowledges the important roles a few individuals played in guidinghim in his career. His early work on flowering was followed by studieson the physiological roles and the metabolism of gibberellins and ab-scisic acid. He describes how collaborations and technical developmentsadvanced his research from measuring hormones by bioassay to theiridentification and quantification by mass spectrometry and cloning ofhormone biosynthetic genes
The invitation to write a prefatory chapter forthe Annual Review of Plant Biology gives me anopportunity to look back at my career and thecircumstances that shaped it. I have always en-joyed reading prefatory chapters in Annual Re-views; they shed light on the human aspectsof science that are not found in research pa-pers. My career was shaped by opportunities,not by obvious design. As will become clear inmy story, I owe much to a few individuals, whosteered me in the right direction at the righttime.
THE EARLY YEARS
I was born in Baarland in the province ofZeeland, the Netherlands, on January 5, 1930.The coat of arms of Zeeland shows a lion half-emerged from water with the text “Luctor etEmergo” (I struggle and emerge). This phraseis symbolic of the inhabitants in their strugglewith the sea over the centuries. Zeeland origi-nally consisted of several islands (now all con-nected by dams to the mainland), which gaverise to isolation and the development of localdialects and costumes. My parents, Willem andJohanna (who proudly wore the local costumeall her life), my sister Anna, and I lived on asmall farm, which had no electricity or running
water. Our farm was located outside the vil-lage and besides my older sister and an oldercousin, there were no children living nearby, somy preschool years were mainly spent with myparents and uncle and aunt. Starting at a youngage, I learned about farming and was assignedsmall tasks, which instilled a strong work ethicin me.
My mother was the oldest of eight childrenand had wanted to become a school teacher, butafter six years of elementary school, she had toleave school to help her family make a living. Myfather, due to illness, had only two years of for-mal education and had been mainly taught byhis brothers. Despite their limited education,my parents were reasonably well versed in cur-rent events through reading the newspaper andmagazines. My father was an elected memberof the village council and was active in politics.
At age five, I learned to ride a bicycle, whichwould be my main means of transportation forthe rest of my life in the Netherlands. At agesix, I started elementary school. In a villagewith only approximately 800 inhabitants, thepublic school had only two teachers. My firstgrade had seven students, two of whom wouldlater have careers in academics. Our house waslocated close to the Western Scheldt, the es-tuary of the river Scheldt, which forms theonly shipping route to the port of Antwerp inBelgium. Once I had learned to ride a bicy-cle and read, the nearby lighthouse became afavorite place for me to visit. The lighthousekeeper let me use his binoculars and I enjoyedrecording the names of the passing ships andidentifying their flags. I spent many hours inthe lighthouse watching ships from all over theworld that traveled to and from Antwerp. Thisexperience opened my eyes to the world beyondour small village.
When I had completed six years of elemen-tary education, a decision had to be made: Whatwas next for me? As the only son, I was expectedto succeed my father on the farm. Althoughmy parents had had limited formal education,they were well aware that six years of educa-tion were no longer sufficient, not even for afarmer. Thus, I enrolled in secondary school
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in Goes, the main city on the island of SouthBeveland. This meant bicycling approximately12 km each way, every school day, regardless ofweather conditions. The curriculum was rigor-ous, with three foreign languages, mathematics,chemistry, and physics, among others. Some ofthe teachers had a M.Sc. or Ph.D. in their spe-cialties and I recall them in general as compe-tent in their subject areas, although not alwaysvery didactic.
While I was in secondary school, my fatherdied suddenly. I was too young to take on the re-sponsibility and so my brother-in-law replacedmy father in operating the farm. This ended myprospect of becoming a farmer, but it also raisedthe question of what to do after I completedsecondary school. My mother had the wisdomand foresight to realize that in order to succeedI should continue my formal education. I hadgood grades in all subjects, so I received a schol-arship and could continue in higher education.We had an orchard as part of the farm and I wasparticularly interested in fruit growing (to myparents’ delight, I could recite at an early age thenames of the apple and pear varieties we grew).Therefore, I decided to study horticulture atthe Agricultural University of Wageningen.
WORLD WAR II
I was ten years old when the Nazis startedthe blitzkrieg in Western Europe and invadedthe Netherlands, Belgium, Luxembourg, andFrance on May 10, 1940. It was a beautifulmorning, the fruit trees were in full bloom, andthe sky was filled with German planes. The nextday, ships were fleeing Antwerp as I watchedGerman planes bomb them. French troopsarrived to help the Dutch defend the country,but after five days the Dutch army capitulatedand for the next four and a half years we wouldlive under German occupation. The first effectsof occupation were food rationing and identifi-cation papers for everybody. With suppressionof a free press, newspapers were required topublish German propaganda. Radios had tobe surrendered, although a sufficient numberwere kept underground so that people could
listen to Radio Orange, a Dutch broadcast fromLondon, where the Dutch government was inexile.
When I enrolled in secondary school in1942, German troops were billeted in theschool building; my class first met in a roomat the Salvation Army. Soon a German offi-cer interrupted class, took measurements of theroom, and the next day the Germans comman-deered our classroom. That scene was repeatedonce more in a room near a theater. Finally, ourclass moved to a room in a church, which wasconsidered off limits by the occupying forces.During the occupation, many products becamescarce or were not available at all. Bicycle tireswere replaced by solid rubber cut from old cartires. This did not provide for a smooth ride,but it was good enough to get me to school.
As the war continued, young men wereforced to labor in the war factories in Germany,but many refused and went underground. Es-pecially in rural areas, almost every house orfarm accommodated an “onderduiker” (liter-ally “diving under”; hiding from the occupationforces). A reserve officer of the defeated DutchArmy was hiding at my uncle’s farm and I spentmany enjoyable hours playing chess with him.In the spring of 1944, he was joined for a coupleof weeks by an English pilot, whose plane hadbeen shot down and who had eluded capture.He was the first English-speaking person I evermet. Hiding downed Allied airmen was pun-ishable by execution, but I do not recall thatthe risks involved were ever discussed. It wasself-evident that this had to be done, regard-less of the possible consequences. Occasionally,there were raids by the Gestapo along with theirDutch collaborators, but there was an elaborateunderground warning system, so onderduikershad sufficient time to hide in more secret places.
Although it was illegal to possess a ra-dio, there were enough underground radios tospread news rapidly by word of mouth and viapamphlets. I remember very well learning of theNormandy invasion on June 6, 1944, before Ihad reached school that morning. By Septem-ber, the Germans were on the run in WesternEurope and the Allied Forces regained Antwerp
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with its harbor intact. But to make use of thisinland harbor, the lands surrounding the West-ern Scheldt, the gateway to Antwerp, had to befreed of German troops. That took another twomonths of heavy fighting. We were liberatedon October 26, 1944 following an amphibiouslanding across the Scheldt close to where welived. However, it was not until the followingMay that the Northern part of the Netherlandswas free and the war in Europe came to an end.My family was fortunate to survive the war, butit was a significant emotional event for our gen-eration that has stayed with us throughout ourlives. We lived through the Great Depression,and then the war, and for quite some time afterthe war we lived with its economic aftereffects.
WAGENINGEN AGRICULTURALUNIVERSITY
In September 1949, I enrolled at the Agri-cultural University of Wageningen [now Wa-geningen University and Research (WUR)].At that time, the study program consisted ofthree phases: 1) the “Propaedeuse,” which in-volved courses in the basic sciences and also wasmeant as a selection screen to weed out unmo-tivated students; 2) the “Candidaats,” more orless equivalent to a B.Sc. degree and involvingprograms in various aspects of agriculture, suchas crop science, tropical agriculture, forestry,and horticulture; and 3) the “Ingenieurs”(Ir. title) study, which was comparable to anM.Sc. program, involving independent study infour different areas.
The 1949 freshman class had approximately125 students. There were no student advisors,class attendance was optional, and no tests weregiven; we had only a written exam at the end ofthe first year. Not surprisingly, only approxi-mately 20% of the freshman class passed in allsubjects the first time. Obviously, it was “sinkor swim.” From this time, I remember favor-ably the lectures and laboratory course in Gen-eral Botany taught by Professor E. Reinders. Healso taught us didactic skills. After I completedhis course, he invited me to serve as a teachingassistant in the lab course, and I accepted.
Having completed the Propaedeuse, I couldproceed toward my goal of studying horti-culture. The Professor of Horticulture, S.J.Wellensiek, taught a two-year course on ba-sic principles underlying horticulture. One yeardealt with vegetative propagation, the next yeardealt with sexual propagation. The lectureswere meticulously organized and involved var-ious aspects of botany, physiology, genetics,and plant breeding. A recurring theme wasgenotype + environment = phenotype. Topicsranged from grafting and root stocks, to flo-ral biology and incompatibility, to vernaliza-tion and photoperiodism. The last two topicsin particular, with the associated florigen hy-pothesis, intrigued me and ultimately inspiredme to change my career goals.
For the final phase of my studies, I choseHorticulture, Genetics (Professor R. Prakken),Plant Physiology (Professor E.C. Wassink), andPhytopathology (Professor A.J.P. Oort). I car-ried out a research project in each area andsome of the results I obtained were featured inmy first publications. The professors were sup-portive and gave me freedom to pursue my in-terests. I came to realize that being a researcherwas an intellectually rewarding profession. I didnot hesitate, therefore, to accept when Profes-sor Wellensiek offered me an assistantship forwork toward my doctorate.
Ph.D. RESEARCH
My Ph.D. advisor, Professor Wellensiek(Figure 1), was a graduate of Wageningen.During the 1930s he had worked in the DutchEast Indies (now Indonesia) on the breedingof tea and cacao. After his appointment to theChair in Horticulture, he developed a basic re-search program on the physiology of flower-ing and the genetics of cyclamen and peas. Thegenetics of the pea plant was a topic he hadstarted as a student and continued long afterhis retirement. Besides his teaching duties, ad-ministrative responsibilities, and membershipson numerous committees, he continued to re-main personally active in research throughouthis lifetime. This was possible only through his
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methodical work habits and strong discipline.As was common at that time, he was addressedas Professor, but once one had obtained a Ph.D.,one was expected to call him by his first name,San (12).
The research topic for my dissertation wasthe result of a controversy that had arisenin 1954 at the eighth International BotanicalCongress in Paris at a session on the mechanismof photoperiodism. Anton Lang had presentedresults of grafting experiments with Hyoscyamusniger and tobacco in support of the florigen hy-pothesis. In contrast, Wellensiek had presentedthe idea, mainly based on work by his Ph.D.student Dick de Zeeuw, that flowering requiresremoval of an inhibitory effect and will takeplace when a certain balance is reached betweenthe quantity of available assimilates and growthsubstance level (11). Because of this contro-versy, Wellensiek suggested that I use graftingto see whether a transmissible flower-inducingstimulus could be demonstrated.
At the start of my Ph.D. research in early1955, the short-day plant (SDP) Perilla (P. crispaor P. nankinensis) was extensively used in thelaboratory for various projects on floweringand rooting of cuttings. Perilla also turned outto be a very suitable plant for grafting anddemonstrating transmission of the floral stimu-lus. Whereas earlier workers had grafted shoots,I concentrated on grafting single leaves or partsthereof. In a three-year period, I made manythousands of grafts and found that a small partof an induced Perilla leaf was sufficient to in-duce flowering in a receptor shoot. A functionalphloem connection between donor and recep-tor was a prerequisite for transmission of thefloral stimulus. When detached, leaves of mostspecies senesce rapidly due to lack of cytokinins.However, I found that detached Perilla leavescould be maintained for a long time and ulti-mately regenerate roots at the base of the peti-ole. Thus, I could show that detached Perillaleaves could be induced in the absence of apicalmeristems. Furthermore, by successive graftingand regrafting of induced leaves onto a series ofreceptor stocks, I found that after three monthsin noninductive conditions, leaves could still
Figure 1My Ph.D. supervisor, San Wellensiek (1899–1990), at his desk with his ever-present pipe in May 1969. San got me started in my scientific career. He wasProfessor of Horticulture at the Agricultural University, Wageningen, theNetherlands, from 1946 until his retirement in 1969 (12). Courtesy of Mrs.Anneke Wellensiek and Ir. Henk Schouwink.
transmit the floral stimulus. However, flower-ing receptor shoots did not function as donors.This led to the concept that photoperiodic in-duction of the induced state is permanent inPerilla and gives rise to a transmissible stim-ulus, which dissipates (51). These grafting ex-periments were technically very simple and nospecial equipment was needed; the results wereclear cut and were some of the most gratifyingof my research career.
In addition to extensive work with Perilla,my thesis research also involved interspecificand intergeneric grafts with members of theCrassulaceae and Solanaceae. In each case, trans-mission of the floral stimulus could be read-ily established, thus demonstrating that the flo-ral stimuli in SDP and long-day plants (LDP)are similar, if not identical (51). Clearly, my re-sults provided further support for the florigenhypothesis and were readily accepted by Pro-fessor Wellensiek. The issue of removal of aninhibitory effect under an inductive daylengthinstead of production of a flower promoter wasnow moot.
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During my Ph.D. work, I had the privilege ofmeeting most of the prominent scientists study-ing flowering in Europe. On a trip to GermanyI met with Professor D. von Denffer, an earlyproponent of the flower inhibition hypothesis,in Gieszen, and in Gottingen I met with Profes-sor R. Harder, known for his elaborate studieson flowering in the SDP Kalanchoe blossfeldiana.In Tubingen, I visited Professor E. Bunning(endogenous rhythms) at the Botanical Insti-tute and Professor G. Melchers (vernalizationin biennial Hyoscyamus niger) at the Max PlanckInstitute. In the spring of 1957, I attended theSociety of Experimental Biology meeting inCambridge, United Kingdom, and presenteda paper on my results with Perilla. ProfessorF.G. Gregory, famous for his work on vernal-ization in winter rye, was in the audience andwas apparently favorably impressed, becausehe invited me to his club, the Athenaeum inLondon, the following Saturday. The nextweek, my wife and I visited Professor Gregory’scoworker, Dr. O.N. Purvis, in Chelsea. She in-vited us for lunch at her club, where I was aminority of one man in the presence of hun-dreds of women! I learned from these visits thatmy research was appreciated by established sci-entists and it gave me confidence that I couldperform at the international level.
In 1958, I defended my dissertation andwas awarded the Ph.D. degree cum laude.Throughout my studies I had been able todefer military service, but once my Ph.D. wascompleted, I was drafted and trained as anintelligence officer. During this interruptionof my academic career, Professor Wellensiekrecommended me for a postdoctoral fellowshipfrom the Netherlands Organization for PureResearch to work in the United States. Pro-fessor Wellensiek had worked with ProfessorE.C. Stakman at the University of Minnesotain 1926, supported by a Rockefeller fellowship.I wanted to continue working on flowering,so I decided to spend my fellowship at theCalifornia Institute of Technology (Caltech)with James Bonner. In 1959, Anton Langwas organizing a symposium at the ninthInternational Botanical Congress and invited
me to participate. The Army granted mefurlough and I made my first transatlanticflight in a propeller-driven plane. In Montreal,I learned that Anton Lang was moving fromUCLA to Caltech to become Director of theEarhart Plant Research Laboratory, usuallyreferred to as the Phytotron. Thus, I would beassociated with both Anton Lang and JamesBonner during my postdoctoral work. By earlyJanuary 1960, I had completed my militaryservice. The next week my wife and I (we hadmet while ice skating in 1953 and were marriedin 1956) embarked on the S.S. Rijndam boundfor New York.
CALTECH
As discussed in Montreal, at Caltech I wouldinvestigate the role of nucleic acids in flower-ing by the use of antimetabolites. These sub-stituted purines and pyrimidines were widelyused at that time to interfere with nucleic acidsynthesis. Frank Salisbury and James Bonner(Figure 2) had already shown that photoperi-odic induction of Xanthium was inhibited by 5-fluorouracil (5-FU) and we continued that work(5). I obtained the strain ‘Violet’ of Pharbitis nilfrom Professor S. Imamura, Kyoto University,Japan. Five-day-old seedlings of this strain canbe induced to flower by a single dark period.This attribute had obvious advantages to findout which step in the overall flowering processwas affected by the inhibitor. I found that ap-plied 5-FU had a strong flower-inhibitory ef-fect in Pharbitis and that its effect was in theshoot apex, not in the cotyledons. This ef-fect could be reversed by thymidine. Flower-ing was also inhibited by 5-fluorodeoxyuridylicacid (5-FDU). 5-FDU blocks thymidylate syn-thase, which results in a deficiency of thymidylicacid, one of the building blocks of DNA. Ourconclusion was that these inhibitors were notinvolved with the synthesis of the floral stimu-lus in the leaves/cotyledons, but instead actedin the shoot apex, where active DNA replica-tion was necessary for expression of the floralstimulus (52). We also worked with inhibitorsof steroid biosynthesis and found that these
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inhibitors suppressed flowering by acting in theleaf (4). Although this was initially an excitingfinding, it turned out later that the inhibitorblocked export of the stimulus from inducedleaves rather than its synthesis.
The earlier work by Anton Lang (Figure 3)on induction of flowering in LD rosette plantsby applied gibberellin (GA) led us to questionwhether GA induces production of the floralstimulus (20). This question could not be re-solved by grafting with LDP, because transmis-sion of flowering could be interpreted as dueto residual GA present in the donor induced toflower by GA. However, this problem could becircumvented by using the long-short-day plantBryophyllum daigremontianum, which can be in-duced to flower by GA in SD, but not in LD.Thus, by using plants induced to flower by GAin SD as donors in LD, transmission of flow-ering could not be caused by residual GA. Theresults showed unequivocally that GA-inducedflowering in Bryophyllum results in a trans-missible floral stimulus (67). We also showedthat inhibition of GA biosynthesis in Bryophyl-lum by the growth retardant (2-chloroethyl)-trimethyl-ammonium chloride (CCC) blockedflowering, thus providing further evidence thatGA is a factor required for flowering in thisspecies (68). Bryophyllum has a long juvenilephase and, by grafting, I showed that the ju-venile apex can respond to the floral stimuluswith flowering, but the leaves cannot produceit (53), as previously also found for Perilla (51).
In 1961, Lincoln and coworkers (26) re-ported that crude extracts from flowering Xan-thium could induce flowering when appliedto vegetative plants, a seeming breakthroughin the search for the elusive florigen. BothAnton Lang and James Bonner had testednumerous extracts with negative or irrepro-ducible results. They suggested that I repeatthe work by Lincoln, especially with Pharbitis,which might have advantages over Xanthium.However, testing extracts from Xanthium andPharbitis never gave reproducible results in myhands.
Caltech was an exciting place to work asa postdoc. The plant growth facilities in the
Figure 2James Bonner (1910–1996) was associated with Caltech during his entire careerand reached Professor Emeritus status in 1981. James was my example of ascientist working at the bench every day. He researched numerous topics,including root hormones, auxin, mitochondrial metabolism, flowering, andrubber synthesis. During my stay at Caltech, James started a “new” careerworking on chromatin and histones of pea and calf thymus (3). Courtesy of theArchives, California Institute of Technology.
Earhart and Campbell greenhouses were ex-cellent, there was a good stockroom, and mymentors left me ample room to pursue myown initiatives, while maintaining high scien-tific standards. The plant physiology group heldlectures at 8:00 am twice a week and everybody,regardless of rank, was expected to participate.This often provided for lively discussions. I re-call that in one such session, James Bonner andAnton Lang spent most of an hour discussingwhether thiamine in plants should be classifiedas a hormone or a vitamin. There were weeklyseminars and talks by visitors. The genetic codewas being deciphered in the early 1960s and allthe major contributors passed through Caltechand presented their recent discoveries. This alsowas the time when space exploration came ofage and science was held in high regard by thegeneral public. Scientists visited high schoolsand I remember one piece of solid advice they
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Figure 3Anton Lang (1913–1996) with his belovedHyoscyamus niger (black henbane) at the opening ofthe Campbell greenhouse at Caltech, May 1961.Hyoscyamus was referred to in the Phytotron asRussian spinach. Formally, I was not Anton’sstudent, but my research on flowering andgibberellins was closely related to his work. Antonwas Professor at Caltech and Director of theEarhart Plant Research Laboratory from 1959 to1965, then Director of the Plant ResearchLaboratory at Michigan State University from 1965until 1978 (21).
gave to prospective students: Study science,but also learn how to write and speak properEnglish, because you will always need it in writ-ten and verbal communications.
Apart from lab work, the Pasadena RotaryClub organized trips to help foreign visitingscientists and students get acquainted with theAmerican way of life. Also, we visited many na-tional and state parks in the Western UnitedStates, from Anza Borrego in the south toCrater Lake in the north and the Bryan and
Zion National Parks to the east. The presiden-tial election of 1960 introduced us to Ameri-can politics. A fellow postdoc took us to theLos Angeles Memorial Coliseum, where JohnF. Kennedy gave his New Frontier acceptancespeech as the presidential nominee for theDemocratic Party. Later that fall, a very par-tisan neighbor invited us to view the very firsttelevised presidential debate, between Kennedyand Nixon.
After more than three years at Caltech, myvisa was running out and it was time to moveon. Prospects for a job with adequate researchfacilities in the Netherlands were slim and bothAnton and James advised me to take a positionin Canada, which would give me a better op-portunity to return to the United States thanwould a position in Europe. From his formerstudent Dennis McCalla, James was aware of afaculty position available at McMaster Univer-sity in Hamilton, Ontario; I accepted an offerwithout a visit or interview. Southern Californiaand Caltech had been wonderful places to liveand work and it was with some regret that weleft. As Anton Lang wrote (21), “No one whohas ‘passed’ through Caltech has left it quite thesame person, and probably retains a trace of re-gret at having left.” To use a Bonnerism, I wasnow an ex-Caltechian.
McMASTER UNIVERSITY
My duties at McMaster University primarily in-volved teaching. My experience as a teachingassistant in Wageningen proved useful in devel-oping a General Botany course. I also taught theentire field of Plant Physiology; one semesterdealt with plant metabolism and nutrition, thesecond semester covered growth and develop-ment. I probably learned more plant physiol-ogy during that first year of teaching than inall my previous years combined. Initially, mylab was housed in an old building where facil-ities for my research were minimal, but withmy heavy teaching load there was little timefor research. After the first year, a new build-ing became available, which made it possibleto resume research. I continued work on theeffect of growth retardants on GA biosynthesis
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in plants. At Caltech, I had been involved with aproject in Lang’s group that showed that growthretardants block GA biosynthesis in the fun-gus Fusarium moniliforme, which produces co-pious amounts of GA. However, it remainedto be shown that this finding also applied tohigher plants. I used developing seeds of Phar-bitis nil, known to be a rich source of GAs.When plants were treated with CCC duringseed development, the GA content was greatlydecreased, although the seeds developed nor-mally. This result established that growth re-tardants cause a dwarfed growth habit, becausethey inhibit GA biosynthesis. Also, I found thatthe progeny of CCC-treated plants produceddwarfed seedlings as a result of CCC carriedover in the seeds (54).
In the summer of 1964, the tenth In-ternational Botanical Congress was held inEdinburgh. I was invited to participate in asymposium on flowering organized by Profes-sor Dennis Carr. Upon arrival in Edinburgh,I learned from Anton Lang that he was leav-ing Caltech to become Director of the newlyestablished Plant Research Laboratory (PRL)at Michigan State University (MSU) in EastLansing. Anton invited Hans Kende (whom Ihad befriended as a fellow postdoc at Caltech)and me to dinner, where he offered us bothfaculty positions at the PRL. We would haveample financial support and research would beour main duty. The prospect of greater researchsupport than I could ever expect in Canada wasobviously attractive. Additional job offers camemy way that fall, but in the end I chose to jointhe PRL. The new lab would be dedicated en-tirely to work on plants, with fellow plant sci-entists nearby to discuss mutual interests andproblems, a situation that appeared much lesslikely in other places.
MICHIGAN STATE UNIVERSITY
The PRL was established in 1964 at MSU bythe Atomic Energy Commission (AEC) as aninstitution devoted to fundamental research inplant biology and the training of graduate stu-dents and postdoctoral scientists (http://www.
prl.msu.edu/). Anton Lang was appointed asthe first Director. He set high standards for ex-cellence in science and most of the procedureshe initiated are still in use today. Except forJoe Varner, the initial PRL faculty was youngand Anton edited each of our manuscripts priorto submission to a journal. Anton’s editorialcomments were legendary and were sometimeslonger than the manuscript under review; thiswas one of the few rules established by Antonthat were not continued by subsequent Direc-tors. Following the example of Caltech, Antonalso assembled an excellent support staff. Com-bined with up-to-date facilities and equipment,this provided for an interactive research envi-ronment and collegial atmosphere, so the PRLquickly became more than the sum of its com-ponent parts. The regular evaluation of eachfaculty member (even full professors are re-viewed every four years) has contributed to theshared responsibility of the faculty for the entireunit.
Because the PRL is not a teaching depart-ment, each faculty member was and is also affil-iated with an MSU teaching Department. Myacademic department was the Department ofBotany and Plant Pathology (since 2001 theDepartment of Plant Biology), through which Idid my teaching. In line with our research inter-ests, Hans Kende (Figure 4) and I jointly taughtcourses on plant growth and development at theundergraduate and graduate levels. Starting inthe mid1980s, Hans taught the undergraduatecourse and the graduate course became my soleresponsibility, although we continued to give afew lectures in each other’s course. My prefer-ence was to teach at the advanced level, whereI could have regular interaction with studentsand advise them in the critical evaluation of cur-rent literature relevant to the course. My great-est reward in teaching has been hearing fromformer students who express their appreciationfor what they learned in our courses.
Flowering
Additional reports appeared that extracts fromflowering plants could induce vegetative plants
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Figure 4The author with Hans Kende (1937–2006) in the Plant Research Laboratory reading room, March 2004. Wespent many Saturday mornings together boning up on the latest journal articles and discussing what wefound interesting. We first met in 1961 at Caltech and through common interests became close friends untilHans’s premature death in 2006. Hans was an ardent promoter of plant biology; his altruism in helping andpromoting his colleagues and associates is still fondly remembered. Courtesy of C. Peter Wolk.
to flower (see References 57 and 59). Thisled me to think that there had been some-thing wrong or missing with our extractions atCaltech. But further negative results with ex-tracts convinced me that assaying plant extractswas not going to resolve the florigen enigma.Instead, in further work, we studied propertiesof florigen, including the most obvious aspect,its movement. David Kavon showed that lowtemperature applied to a localized region of thestem of Pharbitis nil plants inhibits both pho-tosynthate and floral stimulus transport (17).In grafting experiments with Perilla leaves incombination with translocation of 14C-labeledassimilates, Rod King demonstrated the strictcorrelation between floral stimulus and assim-ilate movement in the phloem (18). Thus, ifenough phloem exudate could be collected, onemight be able to identify florigen. Rod devel-oped a method whereby detached leaves wereplaced in an EDTA solution, so that instead ofsealing their sieve tubes, leaves continued to ex-ude from the cut surfaces (19). This methodhas been widely adopted by workers on phloemtranslocation. Still, the amount of exudate ob-
tained from Perilla leaves by the EDTA methodwas relatively small, and no positive resultswere obtained in assays. Much later, in a groupproject with my colleagues Hans Kende andLee McIntosh, Susanne Hoffmann-Benning(16) identified a series of peptides/proteins inPerilla exudate.
Starting in the 1970s, the florigen hypothe-sis was met with great skepticism, even border-ing on ridicule, as exemplified in the statement,“Flowering is a religion based on the totallyunfounded dogma of florigen” (8). The rea-soning was that if florigen cannot be isolatedand identified, it does not exist. The idea thatflowering is determined by a ratio between as-similates and certain hormones came to be ac-cepted. In my 1976 review on flowering (57),and also at a colloquium held in Gif-sur-Yvette,France, in 1978 (59), and in a chapter on hor-mones and flowering (58), I made the case forflorigen and against a ratio of certain chemicalsinducing flowering, but this seemed to have lit-tle impact. Ultimately, the solution to the flori-gen question came when the detailed analy-sis of genes/mutants involved in the flowering
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process of Arabidopsis converged with the classi-cal physiology of flowering (44). Interestingly,leading up to the discovery of FT protein asflorigen, citations of my 1976 review suddenlyshowed a marked increase. In hindsight, it isobvious that the florigen problem could neverhave been solved by extraction and applicationof extracts to vegetative plants. Instead of ex-tracts, the FT gene (or its orthologs) can beexpressed in different species and thus induceearly flowering. Although I was not involved inthe solution of the florigen enigma, it was grat-ifying both to see the florigen hypothesis cometo fruition after 70 years and to be invited toreview the recent work on florigen (61, 62).
Gibberellins
In 1965, the fields of GA chemistry and phys-iology were still in their infancy. Only nineGAs were known and the biosynthetic relation-ship among different GAs was not yet under-stood. Extracts from plant material were frac-tionated by thin-layer chromatography (TLC)and GA-like activity was measured by bioassay.Anton Lang (20) showed that GA applied toLDPs in the rosette stage in SD induces stemelongation (Figure 5) and ultimately inducesflowering. Therefore, it seemed that GA couldsubstitute for the LD requirement and, as acorollary, researchers postulated that LD treat-ment would promote the synthesis of endoge-nous GAs. This work was closely related tothe problems I had studied before, so I chosethe LDPs Silene armeria and spinach as exper-imental material to explore this hypothesis. InLD rosette plants, floral initiation and the be-ginning of stem elongation occur more or lesssimultaneously and the two phenomena wereonce considered part of the same process. How-ever, by application of GA or a growth retar-dant, we found that the two events representseparate processes. Stem elongation withoutflower formation can occur in rosette plants af-ter treatment with GA. Conversely, growth re-tardants can suppress stem growth, while flowerformation can take place normally (7, 55). Infurther work, extracts from spinach in SD and
Figure 5Anton Lang (–) and Jan Zeevaart (+). Joe Varner (1921–1995) wanted topresent a funny slide on the effect of gibberellin at a conference and made thispicture in March 1966.
LD were assayed in the dwarf-5 maize bioas-say (which did not differentiate between pre-cursor and bioactive GAs); only a small differ-ence existed in total GA content (55). One ex-planation for these results was that it was notso much an increase in GAs that caused stemgrowth and flowering in LD, but rather a de-crease in inhibitor, possibly the newly discov-ered plant hormone abscisic acid (ABA). Thisline of research is discussed in the next section.Two other aspects of GA metabolism becameapparent from this early work: First, that GA ismuch more rapidly metabolized in LD than inSD (55), as was later confirmed with radioactiveGA20 (29), and second, that the sensitivity to GAis increased in plants grown in LD as comparedwith those grown in SD (7, 55). The molecularbasis for this phenomenon remains unknown,but it is probably related to the status of the GAreceptor.
In 1976, my lab acquired the first high-performance liquid chromatograph (HPLC) oncampus. When operated with reverse-phaseC18 columns, this instrument became indis-pensable for purifying and fractionating plant
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hormone extracts. In the meantime, moreGAs were discovered, so Jim Metzger couldidentify six GAs in spinach by combined gaschromatography–mass spectrometry (GC-MS)(27). Five of these GAs formed a biosyntheticsequence: GA53 → GA44 → GA19 → GA20 →GA29. These results also allowed us to rein-terpret the earlier results obtained by bioassay.Upon transfer of spinach from SD to LD, GA19
decreased and GA20 strongly increased, thusshowing that LDs play a crucial role in the con-version of GA19 to GA20 (28). In this early work,no GA1 was identified, but later we showed itto be the bioactive GA in spinach (65). At thispoint, radioactive GAs were required to furtherelucidate GA biosynthesis and its regulation bythe photoperiod in spinach. Jan Graebe (15)at the University of Gottingen, Germany, hadmade great progress in cell-free biosynthesisof GAs, using liquid endosperm from pumpkinfor the incorporation of 14C-labeled mevalonicacid into GA12, and immature pea seeds for theconversion of GA12 to GAs native to spinach.I spent part of two summers in Jan’s lab learn-ing the in vitro techniques and preparing 14C-labeled GAs. Sarah Gilmour then prepared cell-free extracts from spinach leaves for conversionof GAs. She found that enzyme activities foroxidation of GA53 and GA19 increase in plantsgrown in LD and decrease in SD and darkness,but the enzyme activity that oxidizes GA44 re-mains high irrespective of light or dark treat-ment (14). Sarah further partially purified fourdifferent GA oxidases from spinach, but becauseof low abundance and lability it appeared un-likely that homogeneous enzymes could be ob-tained by classical biochemical methods (13).Further progress with this problem had to awaitthe application of molecular techniques.
During a visit to Wageningen in 1982, Imet Maarten Koornneef, who had generateda host of mutants in Arabidopsis, including mu-tants that appeared to be impaired in GA orABA biosynthesis or response. A fruitful col-laboration followed from this visit. Maarten hadthe expertise to generate and map mutations inArabidopsis and my lab had the expertise to bio-chemically characterize some of these mutants.
By that time, Lew Mander of the Australian Na-tional University at Canberra had generouslymade available a collection of 2H2-labeled GAscommonly occurring in green plants. TheseGAs were extremely useful as internal stan-dards for quantifying endogenous GAs in plantmaterial by combined gas chromatography–selected ion monitoring. Manuel Talon usedthis methodology to quantify GAs present inSilene armeria (40), and in wild-type Arabidopsisand the semidwarf ga4 and ga5 mutants (38).Twenty GAs were identified in Arabidopsis andthese could be arranged in three parallel path-ways. Increases or decreases in certain GAs inthe mutants made it possible to propose whichsteps were blocked in the pathways. Results withthe ga4 mutant suggested that the GA4 geneencoded 3β-hydroxylase. The ga5 mutant hadincreased levels of C20-GAs and reduced lev-els of C19-GAs, which led us to conclude thatthe GA5 product catalyzes oxidation and elim-ination of C-20 (the product was later identi-fied as GA 20-oxidase). At the time this workwas conducted, the dogma was that the biolog-ically active GA in higher plants was GA1. Butwe found that the content of GA4 was muchhigher than that of GA1 and that GA4 was atleast ten times more active than GA1, thus sug-gesting that GA4 is the biologically active GA inArabidopsis (38). We further found that anotherdwarf mutant, gai (insensitive to GA), containedlow levels of C20-GAs and high levels of C19-GAs (39). Our conclusion that GAI, encodinga DELLA protein, probably acts in the signaltransduction pathway between the GA receptorand stem elongation, was later confirmed (45).
By the early 1990s, no GA oxidases hadbeen cloned. Therefore, to clone the GA5gene of Arabidopsis, we followed an indirectapproach. We first cloned a 20-oxidase fromliquid endosperm of pumpkin. This gene wasalso cloned from developing pumpkin cotyle-dons by Graebe’s group (22). In our approach,Li Li prepared antibodies against purified 20-oxidase and used them for immunoscreening acDNA library from liquid endosperm, whichyielded a clone encoding GA 20-oxidase. Thisclone was subsequently used by Yun-ling Xu to
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screen a genomic library of Arabidopsis, whichled to the isolation of the GA5 clone. Heterol-ogous expression in Escherichia coli showed thatthe recombinant protein was multifunctionaland catalyzed the conversion of GA53 → GA44
→ GA19 → GA20. Consistent with a functionin GA biosynthesis, the ga5 mutant containeda G → A point mutation that resulted in apremature stop codon (49). At the same time,Peter Hedden’s lab (31) reported the cloning ofthree cDNAs encoding 20-oxidases from Ara-bidopsis, one of which was identical to our GA5clone. The cloning of GA5 of Arabidopsis soonwas followed by the cloning of a gene encodinga 20-oxidase from spinach. Its expression wasstrongly upregulated in LD (48). In later work,other GA oxidases were isolated from spinach,but the day length had little effect on their ex-pression (23). Surprisingly, GA53, the substrateof 20-oxidase, does not accumulate in spinachin SD, because GA53 is 2β-hydroxylated to giveGA97. The gene encoding this 2β-hydroxylasewas later cloned by Dong Ju Lee and it wasfound that this enzyme is specific for C20-GAs.Thus, GA53 is at a branch point: In SD it is pri-marily deactivated to GA97, whereas in LD in-creased 20-oxidase activity converts it predom-inantly to GA20, and by 3-oxidase to bioactiveGA1 (24).
Our research with GAs demonstrates howthe application of new techniques is essentialfor making continued progress. For measur-ing endogenous GAs we advanced from TLCcombined with bioassay to HPLC, followedby GC-MS with internal GA standards. Oncethe physiological-biochemical background wasin place, mutants in Arabidopsis and molecular-genetic techniques became available and thesethen provided definitive answers about the reg-ulation of GA biosynthesis in spinach.
Abscisic Acid
My interest in ABA arose from the results ob-tained with endogenous GAs in rosette plants,which showed only small quantitative differ-ences between material from SD and LD asmeasured by bioassay. We next investigated
whether an inhibitor (ABA) might be removedunder LD as suggested by Wareing and col-leagues (46). But contrary to expectations, theresults showed an increase in ABA in spinach inLD rather than a decrease. Furthermore, whenplants were moved from LD to SD, the ABAlevel had decreased after 8 h. Therefore, it wasexpected that an extended period of darknesswould drop the ABA level even further. Butafter 48 h of darkness, the ABA level had in-creased sixfold (56). I noticed, however, that atthat point the plants were wilting. Wright &Hiron (47) had just reported that wilting in-duced a rise in the level of ABA in wheat leaves.This also turned out to be the case in spinach(56). So, serendipity led me to an intriguing as-pect of ABA: that its level dramatically increasesduring dehydration of plants. Elucidating thebiochemical basis of this phenomenon then be-came a major objective of my research group.
During the 1973–74 academic year, I spenta sabbatical leave supported by a GuggenheimFellowship at the Shell-operated Milstead Lab-oratory for Chemical Enzymology in Sitting-bourne, United Kingdom. It was there thatBarry Milborrow and associates had originallyidentified dormin, which turned out to be iden-tical to abscisin II (both were later called ABA),isolated by Addicott and associates at Universityof California at Davis. I worked on catabolismof ABA, showing that bean leaves metabo-lize ABA to the newly discovered phaseic acid(PA) and dihydrophaseic acid (DPA) (69). Thechemists made me aware that their criteria foridentification of a compound were more rigor-ous than those used by many physiologists. Inmy later work, I tried to live by these standards.
Upon my return to MSU, my lab investi-gated various aspects of ABA chemistry andphysiology, such as the time course of ABAaccumulation after dehydration and its rapidconversion to PA upon rehydration of water-stressed leaves (60), and the transport of ABAand its catabolites (63). Katrina Cornish did anextensive study of the effects of leaf age and wa-ter stress on ABA distribution in Xanthium (9).Yet, the question of ABA biosynthesis remainedunresolved. ABA is a sesquiterpenoid and it was
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generally assumed that mevalonic acid (MVA)would be the precursor of ABA. (The plastid-localized methylerythritol phosphate pathwayhad not been discovered at that time.) But when14C-labeled MVA was fed to leaves, followedby dehydration to stimulate ABA biosynthe-sis, the ABA that we subsequently isolated con-tained no radioactivity. This is where the prob-lem remained until a new graduate student,Bob Creelman, came along and suggested theuse of 18O2 to determine which of the fourO-atoms of newly synthesized ABA would be-come labeled. The results of this experimentprovided a breakthrough in understanding ABAbiosynthesis. The electron impact mass spec-trum showed that one 18O atom had been incor-porated into the carboxyl group of ABA. Thisresult meant that ABA is the breakdown productof a larger precursor, presumably a xanthophyll,in which the ring oxygens are already presentand oxidative cleavage introduces a single Oatom in the side chain (10). This idea had beensuggested earlier by Taylor & Burden at WyeCollege, United Kingdom, when chemical ox-idation of xanthophylls yielded xanthoxin (42),which plants could convert to ABA (43). Oursubsequent review in the Annual Review of PlantPhysiology and Plant Molecular Biology (64) fur-ther expanded on the idea that ABA is a cleav-age product (apocarotenoid) of carotenoids andwas highly cited even when it had long been sur-passed by more recent reviews. Our review wastimely and stimulated research on carotenoidsand ABA by other workers, especially in thelaboratories of Roger Horgan (30) and DanWalton (25). (Both later left the field for dif-ferent reasons.) In addition, the ABA field hademerged from the narrow view that ABA wasinvolved only in abscission, and in dormancyin woody species (both ideas were later shownto be incorrect). We also discussed other rolesof ABA, such as stomatal control, adaptationto stress, embryo development, and seed dor-mancy (64).
In further work, in collaboration with DougGage, we used chemical ionization to determine18O incorporation and the position of the 18O inthe ABA molecule. With this ionization mode,
the molecular ion is the base peak of methyl-ated ABA, making it much easier than with elec-tron impact ionization to estimate 18O enrich-ment. We found invariably that in short-termlabeling experiments only a single 18O was in-corporated into the carboxyl group of ABA; inlong-term experiments, the two O atoms on thering also became slowly labeled, indicating thatthe xanthophyll pool was gradually being re-plenished by nonoxygenated precursors (66). Inthe meantime, we had obtained a wilty ABA-deficient mutant of Arabidopsis from MaartenKoornneef, who had become a regular visitorto East Lansing on his way to the Arabidopsismeetings in Madison, WI. Chris Rock showedthat this mutant, aba1, is impaired in the con-version of zeaxanthin to violaxanthin (34), thusproviding further evidence that carotenoids areprecursors of ABA. Later, Steve Schwartz char-acterized the aba2 and aba3 mutants as beinginvolved in the two steps involved in the con-version of xanthoxin to ABA (35).
The main step missing in the ABA biosyn-thetic pathway at that stage was the carotenoidcleavage reaction. Considering the lability ofcarotenoids and probable low abundance ofthe enzyme, it seemed unlikely that this prob-lem could be solved by classical biochemi-cal methods. But a mutant in Arabidopsis forthe presumptive cleavage enzyme also hadnot been isolated. The solution to the prob-lem came from an unexpected source. DonMcCarty (University of Florida) reported atthe fifteenth International Conference on PlantGrowth Substances in Minneapolis in 1995that he had isolated a viviparous mutant inmaize, vp14. The corresponding gene wascloned and Don found that the sequenceshowed similarity to lignostilbene dioxyge-nases from Pseudomonas paucimobilis, which cat-alyze a cleavage reaction similar to the pro-posed cleavage reaction in ABA biosynthesis(41). Don’s lab prepared recombinant VP14protein and Steve Schwartz in my lab devel-oped an assay for the enzyme (37). Indeed,the enzyme cleaved the 11,12-double bondof 9-cis-epoxycarotenoids to give 2-cis,4-trans-xanthoxin (C15) and a C25-apo-aldehyde. The
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reaction, catalyzed by nine-cis-epoxycarotenoiddioxygenase (NCED), required ferrous ionsand molecular oxygen (37). The hypothesis thatthe cleavage reaction is the key regulatory stepin water stress–induced ABA biosynthesis couldfinally be tested. Working with primary beanleaves, Xiaoqiong Qin showed that water stressrapidly upregulated expression of NCED atboth the mRNA and protein levels in paral-lel with accumulation of ABA (32). These re-sults showed that the dramatic accumulationof ABA in response to water stress is primar-ily due to upregulation of the carotenoid cleav-age step. Large increases in NCED mRNA alsowere found in ripening avocado fruits prior toand during the period of rapid ABA accumula-tion (6). As expected, overexpression of NCEDin tobacco produced an increase in ABA andenhanced drought tolerance, but overexpres-sion was also accompanied by more rapid ABAturnover (33). The next question concerned therapid decline in ABA following rehydration ofwilted leaves and the concomitant accumulationof PA. In work with bean leaves and immatureembryos, Seung-Hwan Yang showed that therapid breakdown of ABA is due to increased ex-pression of ABA 8′-hydroxylase, a cytochromeP450 (CYP707A) (50). Although the biosyn-thetic pathway of ABA is now well understood,it remains unknown how the stress signal is per-ceived and transmitted to the chloroplasts, thesite of the cleavage reaction.
The discovery of the role of NCEDs inABA biosynthesis had an impact far beyond theABA field. NCEDs are a subfamily of a muchlarger group of carotenoid cleavage enzymes(CCDs), which are present in organisms fromcyanobacteria to humans, and which produceapocarotenoids such as retinal, hormones, andflavor and fragrance molecules (1). The firstcharacterized CCD, CCD1 in Arabidopsis,cleaves carotenoids (C40) at the 9,10 (9′,10′)double bonds to give two products: two C13
fragments (e.g., β-ionone) and a central C14-dialdehyde (36). VP14 was the first identifiedmember of this new group of enzymes, whichamong others includes β-carotene 15,15′-dioxygenase for retinal and other members that
cleave carotenoids at the 7,8 (7′,8′) or 9,10(9′,10′) double bonds (1).
IN RETROSPECT
Looking back at my career, I have been priv-ileged to observe enormous advances in plantphysiology/biology. In my own areas of spe-cialization, I recall the first structural elucida-tion of a GA, GA3, in the mid-1950s when Iwas working on my Ph.D. Now there are atleast 136 characterized GAs. I listened to FredAddicott’s presentation on abscisin II at the An-nual Meeting of the American Society of PlantPhysiologists in Urbana, IL, in 1965, not anti-cipating that ABA would become a major themeof my research program. The biosyntheticpathways of the plant hormones have been elu-cidated and most of the hormone receptors havebeen identified. The mystery of crown gall is fi-nally understood. In fact, Agrobacterium has be-come an indispensable tool of our profession.All these discoveries were made possible by ad-vances in technology. My laboratory had accu-mulated an extensive body of information onGA metabolism in spinach as regulated by pho-toperiod. Likewise, the phenomenology of ABAaccumulation and degradation in relation to wa-ter stress had been worked out. In the 1990s,molecular-genetic techniques could be appliedto yield definitive answers to these problemsand I was fortunate to have competent asso-ciates to carry out this work. Much of the re-search in plant physiology has been about ana-lyzing individual processes or pathways withoutregard for whole-plant functioning, the idealgoal. But with the availability of whole-genomeexpression data and regulatory networks insteadof separate linear pathways (2), this ideal maycome closer to reality.
As the science has changed, so have theconditions and atmosphere in which science ispracticed. Over the years, added rules and reg-ulations have significantly increased the admin-istrative burden of the principal investigator.Some of these rules are, of course, necessary.Nobody wants to return to the old days whenall waste was poured down the drain. Research
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in plant biology has expanded so much that nosingle worker can claim to have all the expertise.As a result, multidisciplinary research involv-ing many workers from many institutions hasbecome commonplace. Some of these develop-ments may be to the detriment of curiosity-driven research. Proposed research in fundedproposals has become more of a mandate thanit used to be. I recall a science administrator be-ing asked whether one should strictly follow theproposed research schedule. His answer: “Fol-low your scientific intuition.” I doubt that wewould get the same answer today.
To be successful in academic research, a cer-tain level of intelligence and formal educationis required, but at least as important is an in-quiring mind, asking the right questions, moti-vation, and persistence in pursuing answers. Inmy case, good health and longevity were alsoimportant. The PRL was an ideal place for meto combine a career with a passion for research.I had continued funding [not only from theDepartment of Energy (DOE), but also fromthe National Science Foundation (NSF) andthe United States Department of Agriculture(USDA)], so work on projects was never inter-rupted. I never had a large group, which allowed
me to continue working at the bench. Havingtwo research topics also worked to my benefit;if there was stagnation in one area, we mightmake progress in the other. Science requires alot of hard work and passion. One morning mycar radio played the song, “Some Days Are Di-amonds, Some Days Are Stones.” John Denverwas not singing about scientists, but the wordsring true for us as well. We do not make greatdiscoveries every day. It is the day when a stu-dent shows an exciting result or when a paperappears in print that makes for a diamond.
The final stage of research is preparing theresults for publication in a professional jour-nal. Because English is not my native language,I found writing to be the hardest part of re-search. Most of my writing was done late atnight when I could best concentrate. I tried tofollow Anton’s interpretative style, not just pre-senting the dry facts, but integrating the newinformation with earlier work and hypotheses.The Instructions for Authors in Plant Physiology,which recommend short declarative sentences,were a useful guideline. Still, I definitely sympa-thize with the saying, “I hate to write, but I loveto have written.” After all, we are rememberedthrough our writings.
DISCLOSURE STATEMENT
The author is not aware of any biases that might be perceived as affecting the objectivity of thisreview.
ACKNOWLEDGMENTS
I wish to thank my teachers and mentors for their help and valuable advice, and all my colleaguesat the PRL (former and current) for their support and friendship. Without the contributionsof students and postdocs, much of the research discussed in this chapter would not have beenpossible. Because of space limitations, not every one could be mentioned, but I thank them all.Continued support for my research was provided by the Atomic Energy Commission (AEC) →Energy Research and Development Administration (ERDA) → DOE. My work on ABA and GAwas supported by NSF and USDA, respectively. Several readers provided helpful comments onearlier versions of this chapter. Finally, I also wish to acknowledge my wife Riet, who has been mycompanion and support during all these years. Our son, Scott, was born in Lansing after we hadmoved to Michigan. He has chosen a career in civil engineering. Scott and his wife, Brenda, havea son Luke, who continues the family name as the sole descendant.
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