Plant Physiology

Raikhel, Natasha V.

doi: 10.1104/pp.125.1.1pmid: N/A

The 75th anniversary of Plant Physiology comes at a very exciting time in the history of plant biology. We are currently experiencing an unprecedented acceleration in the pace of scientific progress (as our Society's recent publication of the 1,400-page textbook Plant Biochemistry and Molecular Biology [3] bears testament!). The completion of the genomic sequences of some of the most intensively studied multicellular organisms such as Drosophila melanogaster (1),Caenorhabditis elegans (13), Arabidopsis (12), and soon human and rice, has created new research directions, experimental approaches, and opportunities. For the first time, our laboratory toolbox is so powerful that it is now possible to envisage a whole-systems approach to gene and protein function and to study the function of all genes of a particular species within cellular, organismal, and evolutionary contexts. Equally dramatic have been the changes in the publishing landscape: online publication of journals has forever altered the way scientists relate to the literature. When the American Society of Plant Physiologists was 50 years old in 1974, the Journal (in its 48th year) published a series of eight retrospective articles that summarized 50 years of progress in plant biology (2, 4, 6–9, 14, 15). Many groundbreaking insights and original discoveries were made during those first 50 years (11), but plant physiologists still had insufficient mechanistic understanding of the biological processes they were studying. The advent of new molecular tools has changed all that: Virtually every day, plants become less and less of a “black box.” In this issue, we present 42 short commentaries that attempt to summarize conceptual breakthroughs in plant biology during the past 25 years. In a perfect world, we would have asked even more members of the Society to offer their perspectives, but alas, neither this world nor this project is perfect. Given limitations of space and resources, not every field could be covered. By selecting only 42 fields, many other areas in which there has also been substantial progress had to be omitted. Undoubtedly, many important individual contributions were not cited, particularly as the objective of the authors was not to write comprehensive reviews, but to illustrate how our thinking about plants and our experimental approaches have changed in their respective fields over the course of the past 25 years. Given the rapid progress in plant biology in recent years, such brevity did not come easily, and I am sure that each author struggled to be as objective as possible in deciding what to include. The resulting commentaries are fascinating taken one at a time, but together they demonstrate just how far plant biology has come in a relatively short while. Three major technological advances stand out as being crucial in accelerating the pace of plant biology in the past 25 years: 1) the development of molecular tools, 2) the development of plant transformation by Agrobacterium tumefaciens and other means, and 3) the widespread adoption of Arabidopsis as a model organism by thousands of biologists. Our series of commentaries begins with an analysis of these three breakthroughs. The remaining articles draw from research in the following areas: whole plant physiology and biochemistry; signal transduction; developmental, cell, molecular biology and genetics; and biotechnology. In the foreword to the first issue of Plant Physiology (10), the Journal's founders noted: “It is evident… that these two lines of investigation, practical and fundamental, must always go hand in hand. There can never be a logical separation of these two aspects of our science. Likewise, there can never be a logical separation of the pure physiologists from the practical physiologists. Our tasks are one and we must learn to march together in their performance.” This anniversary issue, 75 years later, is a testament that this statement is just as true today as it was then! The modern tools of plant biology are not only allowing us to answer important questions in basic biology, but are also proving profitable to the farmer and the marketplace. Plant biologists are making tangible contributions to agricultural productivity. Although history teaches us that science is extremely unpredictable, there can be little doubt that the next 25 years will witness a revolution in plant biology of unprecedented scope that will dramatically impact both basic and applied research. The interconnection between biology and various disciplines such as applied mathematics, physics, and chemistry will be crucial in the next decade. New experimental tools that aid in the investigation of gene function at the subcellular, cellular, organ, organismal, and ecosystem levels and new bioinformatics tools for analyzing and extracting meanings from system-based databases will be developed. These technologies will not come cheaply, but they promise to pay great dividends. Funding of plant biology by governments and private sources has increased steadily in the past 25 years and has been critical to the spectacular achievements of the last quarter century. In the United States, the ongoing support of plant biology by NSF and DOE Division of Energy Biosciences was supplemented significantly by the USDA competitive grants program (1978–present) and by innovative programs such as the NSF postdoctoral fellowships in plant biology (1983–1994), the tri-agency (DOE, NSF, and USDA) programs of various kinds (1987, 1992–1994), and the plant genome funding by NSF (1998–present). The development of new and innovative programs by private granting agencies was critical to these research developments. Among the more prominent programs launched by private foundations and corporations were the McKnight Foundation grants program in 1983, the Agrigenetics Corporate Limited Partnership (1981–1988), and the Rockefeller Foundation's worldwide support of rice biology research (1985–2000). The main sources of funding for starting new plant research programs in Europe and in Japan were, respectively, the EC (1990–present) and the Scientific Research on Priority Areas and Basic Research for Innovative Biosciences programs (1987–present). Especially encouraging and innovative was the funding and coordination by various national and international agencies of the multinational Arabidopsis genome research project (1990–present). Given the enormous power of the new tools of molecular biology now at hand, even the substantial increases in funding that we have enjoyed of late are insufficient to fuel the juggernaut of scientific progress. Indeed, we live in a time unprecedented in the history of botanical science. The determination of the Arabidopsis genome sequences laid the groundwork that will make possible phenomenal strides in applied and basic research in the next 10 years (5). We are now at the brink of elucidating the function of all the genes of Arabidopsis and other selected species. Plant scientists now have the technology to conduct basic research that can be rapidly translated into applied gains, such as increased crop yields, more nutritious foods, homegrown energy feedstocks, and life-saving medicines. Plant biologists need to be proactive and vocal in bringing this message to various funding agencies as well as to the public at large. The content of this anniversary issue was thoroughly discussed with many colleagues, and I am extremely grateful for their input and suggestions. Drs. Maarten Chrispeels, Kenneth Keegstra, Hans Kende, Sharon Long, Peter Minorsky, and Chris Somerville deserve particular credit for helping me put this volume together. A project of this size and scope demands a clear image of the big picture and the collaboration of scientists in many diverse fields. We hope that our readers will find that the articles we have selected are representative of this exciting era in plant biology. I would also like to thank the Editorial Board for their exceptional commitment to the science of plant biology and to the Journal. As always, I extend heartfelt thanks to the staff of Plant Physiology: Melissa Junior, Lauren Ransome, Kim Davis, Stephanie Butto, and publications director Nancy Winchester. I am also very grateful to Karen Bird and Darryl Pettway who help me here at the Plant Research Laboratory, Michigan State Univer-sity. The professionalism and enthusiasm of all these people have made this anniversary issue, and indeed every issue, a reality. Isaac Newton once wrote, “If I have seen further, it is by standing on the shoulders of giants.” In the same spirit, I enjoy the honor of being the Editor-in-Chief of the preeminent journal in plant physiology because I, too, stand upon the shoulders of giants. The six previous Editors-in-Chief of Plant Physiology, Charles A. Shull (University of Chicago, 1926–1945), Walter F. Loehwing (State University of Iowa, 1945–1953), David A. Goddard (University of Pennsylvania, 1953–1958), Allan H. Brown (University of Minnesota, 1958–1963), Martin Gibbs (Brandeis University, 1963–1991), and Maarten Chrispeels (University of California, 1992–2000), have all been invaluable in giving Plant Physiology the stature it enjoys today. Given the revolution in plant biology, however, we must not be complacent. As the new Editor-in-Chief of Plant Physiology, I aim to make a good journal even better by increasing the impact of what our Journal publishes. It is my hope that 25 years hence, when the editors of Plant Physiology contemplate the 100th anniversary issue, they will thumb through back issues ofPlant Physiology and marvel at the many truly novel mechanistic and conceptual insights that our Journal will have published since our 75th anniversary. LITERATURE CITED 1 Adams MD Celniker SE Holt RA Evans CA Gocayne JD Amanatides PG Scherer SE Li PW Hoskins RA Galle RF George RA Lewis SE Richards S Ashburner M Henderson SN Sutton GG Wortman JR Yandell MD Zhang Q Chen LX Brandon RC Rogers YH Blazej RG Champe M Pfeiffer BD Wan KH Doyle C Baxter EG Helt G Nelson CR Gabor Miklos GL Abril JF Agbayani A An HJ Andrews-Pfannkoch C Baldwin D Ballew RM Basu A Baxendale J Bayraktaroglu L Beasley EM Beeson KY Benos PV Berman BP Bhandari D Bolshakov S Borkova D Botchan MR Bouck J Brokstein P Brottier P Burtis KC Busam DA Butler H Cadieu E Center A Chandra I Cherry JM Cawley S Dahlke C Davenport LB Davies P de Pablos B Delcher A Deng Z Mays AD Dew I Dietz SM Dodson K Doup LE Downes M Dugan-Rocha S Dunkov BC Dunn P Durbin KJ Evangelista CC Ferraz C Ferriera S Fleischmann W Fosler C Gabrielian AE Garg NS Gelbart WM Glasser K Glodek A Gong F Gorrell JH Gu Z Guan P Harris M Harris NL Harvey D Heiman TJ Hernandez JR Houck J Hostin D Houston KA Howland TJ Wei MH Science 287 2000 2185 2195 Crossref Search ADS PubMed 2 Beevers H Plant Physiol 54 1974 437 442 Crossref Search ADS PubMed 3 Buchanan BB Gruissem W Jones RL Biochemistry and Molecular Biology of Plants. 2000 American Society of Plant Physiologists Rockville, MD 4 Burris RH Plant Physiol 54 1974 443 449 Crossref Search ADS PubMed 5 Chory J Ecker JR Briggs S Caboche M Coruzzi GM Cook D Dangl J Grant S Guerinot ML Henikoff S Martienssen R Okada K Raikhel NV Somerville CR Weigel D Plant Physiol 123 2000 423 426 Crossref Search ADS PubMed 6 Galston AW Plant Physiol 54 1974 427 436 Crossref Search ADS PubMed 7 Higinbotham N Plant Physiol 54 1974 454 462 Crossref Search ADS PubMed 8 Kramer PJ Plant Physiol 54 1974 463 471 Crossref Search ADS PubMed 9 Myers J Plant Physiol 54 1974 420 426 Crossref Search ADS PubMed 10 Shull CA Lipman CB Livingston BE Ball CR Lloyd FE Plant Physiol 1 1926 1 2 11 Somerville C Cell 100 2000 13 25 Crossref Search ADS PubMed 12 The Arabidopsis Genome Initiative Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408 2000 796 815 Crossref Search ADS PubMed WorldCat 13 The C. elegans Sequencing Consortium Science 282 1998 2012 2018 Crossref Search ADS PubMed 14 Thimann KV Plant Physiol 54 1974 450 453 Crossref Search ADS PubMed 15 Zimmermann MH Plant Physiol 54 1974 472 479 Crossref Search ADS PubMed Copyright © 2001 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Goldberg, Robert B.

doi: 10.1104/pp.125.1.4pmid: 11154284

It is difficult to imagine carrying out plant research without personal computers, the Internet, GenBank, e-mail, cell phones, gene cloning, microchips, whole genomic sequences, expressed sequence tags, RFLPs, PCR, knock-outs, Arabidopsis, reverse genetics, transgenic plants, and molecular biology “kits” that are ready-made to carry out almost any type of DNA manipulation experiment imaginable. The plant world in 1975 was vastly different from the one in which we, as plant scientists, operate in today. The International Society of Plant Molecular Biologists did not exist. International forums such as the Plant Molecular Biology Gordon Conference, the Plant-Oriented Keystone Symposia, and the Plant Molecular Biology Congress had not been established. One of the largest gatherings of plant scientists occurred at the annual meetings of the American Society of Plant Physiologists and seldom more than 20 or 30 scientists attended the nucleic acids section in which the most exciting plant molecular biology results were presented. The “real world” was different as well. The Vietnam War had just ended, the Cold War with the Soviet Union raged on, the Berlin Wall split Europe into East and West, and the world economy was in an inflationary spiral due to the emergence of the oil cartel that sent the prices of gasoline skyrocketing. Genetic engineering had been “invented” by Stanley Cohen and Herbert Boyer 2 years earlier (11) and was still limited to an elite number of labs that understood bacterial genetics, had the plasmid vectors for DNA cloning, and had access to the enzymes that we purchase in cloning “kits” today. Procedures for cDNA cloning, creating libraries of large eukaryotic genomes, and isolating structural genes had not yet been published. Genetic engineering was as controversial then as genetically modified organisms are today. The Asilomar Conference took place in 1975, and scientists who wanted to use the emerging tools of genetic engineering were required to follow strict, self-imposed guidelines that specified the conditions under which DNA manipulations could be carried out in the laboratory. Demonstrations occurred across the globe forecasting that “monsters” would be created by the new gene splicing techniques and one city (Cambridge, MA) attempted to ban genetic engineering altogether. Nevertheless, it was a magical time to be studying basic plant processes. For the first time, there was a dream that one could finally “see” a plant gene and begin to unravel the complexity of plant processes at the genome level. THE PRINCIPLES OF PLANT GENOME ORGANIZATION AND GENE REGULATION WERE LAID DOWN IN THE PRECLONING ERA Plant genomes were investigated in the mid- to late-1970s by quantitative DNA reassociation tools (i.e. Cot curves) that had their origins in the 1960s when the principles of DNA denaturation and renaturation were pioneered at the Carnegie Institution of Washington by Roy Britten and his associates (7, 8)—principles that are still used today each time a gel blot or microchip experiment is carried out, a primer Tm is calculated, or PCR conditions are punched into a thermocycler. Plant genomes had been shown to contain repetitive DNA sequences in the mid-1960s and were, therefore, considered to be “eukaryotic-like” and similar to animal genomes in that respect (7, 8). In 1975, genome organization was the “code word” for those of us who studied “genomics” and it was determined that plant genomes had many families of repetitive sequences and that these repeats varied in copy number and arrangement in the genome (17, 20). These repeats were shown to be both scattered around the genome and localized in long clusters and they were also shown to be flanked by complex single-copy sequences (17, 20). Neither these repeats nor any flanking single-copy DNA had been cloned or sequenced at this time. In fact, DNA sequencing procedures (29, 33) had not yet been invented and plant DNA sequences had not yet been cloned (3). However, the general concepts of plant genome organization that were derived from DNA reassociation studies have stood the test of time and have been illuminated in great detail by a knowledge of the actual DNA sequences that span each Arabidopsis chromosome (5). During this same period, important principles of plant gene activity were being established in global terms by the use of RNA-excess/DNA-RNA hybridization techniques (i.e. Rot curves) with either cDNA or genomic single-copy DNA probes (21, 22, 24, 25). The technique of subtraction hybridization (or cascade hybridization as it was first referred to in the literature) was established by Bill Timberlake in this era using kinetically fractionated cDNA populations (36). Both cDNA and genomic single-copy DNA subtraction procedures were used by many of us to investigate developmental changes in plant mRNA populations (21, 22, 24, 25). Several important concepts emerged about higher plant cells in this precloning population hybridization era. First, it became clear that plants contained a complex set of nuclear RNAs and that only about 25% of this complexity was represented in the corresponding mRNA population (21). Today, we know that the additional complexity in the nuclear RNA represents primarily unprocessed introns in primary transcripts. However, this was not understood at the time because plant genes had not yet been cloned and sequenced, and introns had not yet been discovered in any eukaryotic gene. Second, it became clear that a large number of genes were active in plant cells and that these genes were highly regulated in the plant life cycle (21, 24, 25). Each plant organ system was shown to have a unique set of active genes and it was estimated that approximately 60,000 genes were required to program and maintain the entire life cycle of the tobacco plant (24). This estimate of the number of tobacco genes has stood the test of time for plants with large genomes (i.e. corn) and, considering the “bluntness” of the tools used and assumptions that had to be made (e.g. average mRNA size), is not that far off from the 25,000 genes that has been shown by sequencing to be present in the small Arabidopsis genome (5). Finally, it was established that mRNA populations contained sequences with varying degrees of prevalence and that both transcriptional and posttranscriptional processes established the mRNA sequence sets present in various plant organs and tissue types. By the end of the Cot and Rot curve era (mid-1980s), it was clear that plant cells resembled animal cells with respect to the number of genes and the complexity of gene regulatory processes. It was not known, however, how any individual gene was regulated or how sets of genes were co-expressed in space and time. PLANT GENES CAN BE CLONED! By the end of the 1970s, exciting new procedures were developed by Tom Maniatis and others to construct cDNA clones of specific eukaryotic mRNAs and isolate the corresponding genes from the genome (26, 27). In addition, techniques were devised to sequence DNA segments (29, 33), visualize genes directly in the electron microscope in association with their RNAs (i.e. R loops; 38), and detect specific DNA fragments and mRNAs using DNA and RNA gel blots, respectively (1, 34). These procedures established a new revolution in molecular biology because, for the first time, the structures of individual genes could be studied and their expression patterns, mechanisms of regulation, and evolutionary origins analyzed. This was an exciting period and the most surprising and startling observation made with the new DNA cloning techniques was that the coding regions of eukaryotic genes were interrupted by non-coding sequences (23)! New words, intron and exon, were introduced into the molecular biology lexicon (19) and posttranscriptional splicing mechanisms were hypothesized and studied (23). Only a few plant scientists at that time had any experience with bacterial genetics, the new recombinant DNA techniques, or access to enzymes required for DNA cloning and manipulation. In fact, most of us did not know a restriction enzyme from a ligase and had to learn from “scratch” how to streak and grow bacterial cells in order to attempt to clone plant DNA sequences! In the 1970s and 1980s (as well as today) plant scientists were playing “catch-up” with their animal counterparts and were competing for a meager pot of money. It was during this time that Joe Key played a huge role in establishing the U.S. Department of Agriculture Competitive Research Grants Program after many years of fighting the U.S. Department of Agriculture bureaucracy and Congress. This Program has made a major impact over the past 25 years in keeping plant sciences in the forefront of pioneering research. Rumors began to circulate in the late 1970s that plant DNA could not be cloned. One well-known plant molecular biologist (who will remain anonymous) went from meeting to meeting like Paul Revere declaring that plant DNA was “different” from animal or bacterial DNA and that it could not be cloned! John Bedbrook and colleagues in Dick Flavell's lab in Cambridge, England soon showed that this was not the case and demonstrated directly that plant DNA could be cloned and replicated in bacteria just like the DNA from other organisms (3). They reported their results in 1979 at a meeting in Minneapolis and the era of plant gene cloning began with the successful cloning of ribosomal DNA and telomeric repeated sequences from wheat (3). A pioneering principle was established—plant DNA was similar to that of all other organisms and could be manipulated using the same enzymes, cells, and vector systems. Soon thereafter, libraries of many plant genomes were constructed and, in the early 1980s, were made available to plant scientists around the world (16, 35). In addition, the first plant structural genes were cloned, sequenced, and visualized in the electron microscope (16, 35). These genes, encoding seed storage proteins (16, 35) and the small subunit of ribulose bisphosphate carboxylase (4), were shown to contain introns similar to those in animal genes, which supported the notion that plant cells had genetic processes similar to those in animals. It was also demonstrated that plant genes were located relatively close to each other on plant chromosomes (approximately every 4–6 kb) and that genes with different expression patterns were interspersed among each other, implying that each functioned as an independent unit (16)—a suggestion that was verified during the post-transformation era (9, 31, 32). During the same period, cDNA libraries were constructed for almost every imaginable plant organ system and developmental state, and cDNA clones representing prevalent plant mRNAs, such as those encoding seed proteins, light-regulated proteins, hormone-induced proteins, and cell wall proteins were identified. These cDNA clones were used to demonstrate directly that both transcriptional and posttranscriptional processes played a role in controlling plant gene expression, but that the primary control for most plant genes was at the level of transcription. In addition, the vast array of cDNAs that became available and were sequenced and studied in the 1980s began to illuminate a range of plant developmental, metabolic, and biochemical processes. The age of understanding “how to make a plant” had begun. PICKING APART PLANT GENES As the new era of plant gene cloning began, another revolution was occurring in several labs that were engaged in a fierce competitive battle to be the first to transform plant cells. The laboratories of Jeff Schell and Marc Van Montagu (Gent, Belgium), Rob Schilperoort (Leiden, The Netherlands), Mary-Dell Chilton and Michael Bevan (Washington University, St. Louis; Cambridge University, UK), and Rob Fraley, Steve Rogers, and Rob Horsch (Monsanto, St. Louis) were utilizing the new recombinant DNA techniques to constructAgrobacterium tumafaciens T-DNA vectors that could be used to introduce new genes into plant cells. In the mid-1970s, Mary-Dell Chilton had shown that A. tumafaciens T-DNA was integrated into the chromosomes of plant cells (10), setting the stage for the revolution in plant genetic engineering that continues to this day. In 1983, the Gent, Monsanto, and Washington/Cambridge groups showed independently that T-DNA vectors could be used to transfer bacterial antibiotic resistance genes into plant cells and that these genes could be expressed if engineered with the correct promoters (6, 12, 18). Much to the surprise of everyone in the plant research world, a different group, headed by Tim Hall, demonstrated that the phaseolin seed storage protein gene from french beans could be transferred to sunflower cells and be expressed (31). This now-famous (or infamous) “sunbean” plant made the front page of the New York Times and was proclaimed in Time to be a “glowing achievement… the first time a gene from one plant had been inserted into the chromosomes of an unrelated species and made to express itself.” The sunbean experiment was reported initially at the first University of California (Los Angeles) Keystone Meeting on Plant Molecular Biology that I organized in April of 1983 and was greeted at the time by a now-famous plant cell biologist (who I will not name) as “nonsense!” Nevertheless, it showed for the first time that gene cloning andA. tumafaciens transformation techniques could be combined to transfer foreign genes into plant cells and study their function. The age of plant genetic engineering and gene manipulation had begun! FROM PHENOTYPE TO GENE Throughout the 1980s and 1990s, many plant genes were cloned and investigated in transformed cells in order to understand the mechanisms regulating their expression. Numerous plant promoters were characterized and DNA sequence elements programming transcription in specific developmental states were uncovered. The prediction of earlier experiments on the structure and organization of plant genes proved correct and a major new concept emerged—plant genes functioned as independent units and contained regulatory regions that could program their correct expression in foreign cell environments. These experiments set the stage for engineering new crops with novel traits that are produced at specific times during the plant life cycle (28). A major switch in plant gene cloning occurred in the beginning of the late 1980s and early 1990s. Many interesting genes that produced novel phenotypes were being uncovered in corn and Arabidopsis using genetic approaches that were being adopted rapidly by plant scientists. Because their products were unknown and/or very rare, it was not possible to use conventional cloning methods to isolate these genes. Several pioneering procedures were invented that circumvented this problem and enabled a wide range of plant genes to be cloned. First, T-DNA was shown to act as a mutagen in plant cells and, as such, could be used as a tag to identify and clone genes that specified novel phenotypes (15). Ken Feldmann and his colleagues established a novel seed transformation method to obtain large numbers of T-DNA transformed Arabidopsis lines and this method was used to identify important plant genes, such as those involved in the control of floral organ identity and hormone perception (14, 15). In my opinion, this was one of the most important advances in plant biology in the past 25 years because it allowed, for the first time, a relatively simple way to clone plant genes associated with fascinating mutant phenotypes. The availability of Ken Feldmann's T-DNA lines caused numerous investigators (including myself) to adopt Arabidopsis as a model system and opened up many new problems in plant biology to investigation. It also paved the way to the reverse genetics approaches in use today—identifying mutant lines associated with randomly sequenced genes (30). A second approach to cloning plant genes was also being developed at the same time. During the 1980s, Nina Fedoroff and Sue Wessler cloned the corn Ac and Ds transposable elements (13). This pioneering experiment paved the way for using transposons to tag and capture novel plant genes for which only a phenotype could be identified. The transposon tagging and gene cloning procedure complemented the T-DNA approach and led to the identification of many important new genes in several plants including corn, snapdragon, and Arabidopsis (37). It also became possible in the 1990s to use map-based cloning strategies to identify and clone plant genes—particularly in Arabidopsis because of its small genome size (2). With the completion of the Arabidopsis Genome Project last fall, and the identification of 30,000 single nucleotide polymorphisms in the Arabidopsis genome, map-based cloning of plant genes should permit the identification of any gene for which there is a mutant phenotype—even those induced by chemical mutagens such as ethyl methane sulfonate. BACK TO THE FUTURE Looking back, in 1975 plant molecular biologists were asking questions about the number of genes in plant chromosomes and how these genes are regulated in development. We were using precloning tools of DNA and RNA hybridization that gave precise answers, but which could not focus in on specific genes. The questions addressed then are being addressed once again today in the genomics age. In a sense, we have come full circle in trying to understand how plant chromosomes are constructed and how populations of genes are expressed in various cells, tissues, and organs. We progressed from studying populations of genes and mRNAs to investigating individual cloned genes and mRNAs to using high throughput experiments with arrays of thousands of specific genes in order to uncover the secrets of plant cells. Twenty years after the cloning of the first plant DNA segments (3), the genomes of Arabidopsis and rice have been sequenced and numerous expressed sequence tag sequencing projects have uncovered tens of thousands of mRNAs in a wide range of plants (5). It is remarkable that the era of gene cloning is coming to an end. Nevertheless, the challenges are no less daunting and are even more complex: What are the functions of all plant genes and how is the information in plant genomes utilized in order to program plant development from fertilization to seed dormancy? LITERATURE CITED 1 Alwine JC Kemp DJ Parker BA Reiser J Renart J Stark GR Wahl GM Methods Enzymol 68 1979 220 242 Crossref Search ADS PubMed 2 Arondel V Lemieux B Hwang I Gibson S Goodman H Somerville CR Science 258 1992 1353 1355 Crossref Search ADS PubMed 3 Bedbrook J Gerlach W Thompson R Flavell RB Emergent Techniques. Rubenstein I Gengenbach B Phillips R Green CE 1980 93 113 University of Minnestoa Press Minneapolis 4 Berry-Lowe SL McKnight TD Shah DM Meagher RB J Mol Appl Genet 1 1982 483 498 PubMed 5 Bevan M Bancroft I Bent E Love K Goodman H Dean C Bergkamp R Dirkse W Van Staveren M Stiekema W Nature 391 1998 485 488 PubMed 6 Bevan MW Flavell RB Chilton M Nature 304 1983 184 187 Crossref Search ADS 7 Bolton ET Britten RJ Cowie DB Roberts RB Szafranski P Waring MJ Carnegie Inst Wash Year Book 64 1965 314 333 8 Britten RJ Kohne DE Science 161 1968 529 540 Crossref Search ADS PubMed 9 Broglie R Coruzzi G Fraley RT Rogers SG Horsch RB Niedermeyer JG Fink CL Flick JS Chua NH Science 224 1984 838 843 Crossref Search ADS PubMed 10 Chilton MD Drummond MH Merio DJ Sciaky D Montoya AL Gordon MP Nester EW Cell 11 1977 263 271 Crossref Search ADS PubMed 11 Cohen SN Chang AC Boyer HW Helling RB Proc Natl Acad Sci USA 70 1973 3240 3244 Crossref Search ADS PubMed 12 Estrella-Herrera L Depicker A Van Montagu M Schell J Nature 303 1983 209 213 Crossref Search ADS 13 Fedoroff N Wessler S Shure M Cell 35 1983 235 242 Crossref Search ADS PubMed 14 Feldmann KA T-DNA insertion mutagenesis in Arabidopsis. Plant J 1 1991 71 82 Google Scholar Crossref Search ADS WorldCat 15 Feldmann KA Marks MD Mol Gen Genet 208 1987 1 9 Crossref Search ADS 16 Fischer RL Goldberg RB Cell 29 1982 651 660 Crossref Search ADS PubMed 17 Flavell RB Bennett MD Smith JB Smith DB Biochem Genet 12 1974 257 269 Crossref Search ADS PubMed 18 Fraley RT Rogers SG Horsch RB Sanders PR Flick JS Adams SP Bittner ML Brand LA Fink CL Fry JS Galluppi GR Goldberg SB Hoffmann NL Woo SC Proc Natl Acad Sci USA 80 1983 4803 4807 Crossref Search ADS PubMed 19 Gilbert W Nature 271 1978 501 Crossref Search ADS PubMed 20 Goldberg RB Biochem Genet 16 1978 45 68 Crossref Search ADS PubMed 21 Goldberg RB Hoschek G Kamalay JC Timberlake WE Cell 14 1978 123 131 Crossref Search ADS PubMed 22 Goldberg RB Hoschek G Tam SH Ditta GS Breidenbach RW Dev Biol 83 1981 201 217 Crossref Search ADS PubMed 23 Jeffreys AJ Flavell RA Cell 12 1977 1097 1108 Crossref Search ADS PubMed 24 Kamalay JC Goldberg RB Cell 19 1980 935 946 Crossref Search ADS PubMed 25 Kamalay JC Goldberg RB Proc Natl Acad Sci USA 81 1984 2801 2805 Crossref Search ADS PubMed 26 Maniatis T Hardison RC Lacy E Lauer J O'Connell C Quon D Sim GK Efstratiadis A Cell 15 1978 687 701 Crossref Search ADS PubMed 27 Maniatis T Kee SG Efstratiadis A Kafatos FC Cell 8 1976 163 182 Crossref Search ADS PubMed 28 Mariani C Debeuckeleer M Truettner J Leemans J Goldberg RB Nature 347 1990 737 741 Crossref Search ADS 29 Maxam AM Gilbert W Proc Natl Acad Sci USA 74 1977 560 564 Crossref Search ADS PubMed 30 McKinney EC Ali N Traut A Feldmann KA Belostotsky DA McDowell JM Meagher RB Plant J 8 1995 613 622 Crossref Search ADS PubMed 31 Murai N Sutton DW Murray MG Slightom JL Merlo DJ Reichert NA Sengupta-Gopalan C Stock CA Barker RF Kemp JD Hall TC Science 222 1983 476 482 Crossref Search ADS PubMed 32 Okamuro JK Jofuku KD Goldberg RB Proc Natl Acad Sci USA 83 1986 8240 8244 Crossref Search ADS PubMed 33 Sanger F Nicklen S Coulson AR Proc Natl Acad Sci USA 74 1977 5463 5467 Crossref Search ADS PubMed 34 Southern EM J Mol Biol 98 1975 503 517 Crossref Search ADS PubMed 35 Sun SM Slightom JL Hall TC Nature 289 1981 37 41 Crossref Search ADS 36 Timberlake WE Dev Biol 78 1980 497 510 Crossref Search ADS PubMed 37 Walbot V Annu Rev Plant Phys Plant Mol Biol 43 1992 49 82 Crossref Search ADS 38 White RL Hogness DS Cell 10 1977 177 192 Crossref Search ADS PubMed Author notes * E-mail [email protected]; fax 310–825–8201. Copyright © 2001 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Chilton, Mary-Dell

doi: 10.1104/pp.125.1.9pmid: 11154285

This little memoir is not a review; the reader is directed to current authoritativeAgrobacterium reviews with genetic (23) or cell biology emphasis (24). Likewise, this is not an update on recent advances in plant genetic engineering, which are the subject of a recent book (13). Rather, I invite you to join me on a foray through the story of Agrobacterium transformation of plant cells. Our journey will take us back in time about 30 years, and we will note early contributions from laboratories around the globe, including Belgium, the Netherlands, France, Australia, and several in the United States. The scientists in our story represented many disciplines, from traditional ones such as plant pathology, microbiology, and chemistry to younger fields such as molecular biology, plant tissue culture, and plant metabolic chemistry. Many in the course of investigatingAgrobacterium found intellectual haven in the newly emerging field of plant molecular biology. Beginning at a time when bulk DNA was analyzed as a macromolecule, our story spans the birthing and growth of recombinant DNA technology. Lest the experiments we revisit seem simple when viewed from the 21st century, our first stop will be a museum of molecular biology research in the time about which I will write, circa 1970. The catalog of restriction endonucleases was unrecognizably thin. What few enzymes were available often were tainted. Kits were unknown. Procedures often did not work. We sized DNA and determined its percentage of G and C in the model E ultracentrifuge. We measured small volumes with 5-, 20-, 50-, or 100-μL glass capillaries. We cultured our plant calli in jelly jars and fleakers. Instead of laminar flow hoods we worked in still air hoods. A few years later when the plasmid came into our lives, we taught ourselves how to do gel electrophoresis, and we designed and built our own gel rigs. (The one with the agarose wicks was known, of course, as the wicked gel.) We made combs from square aluminum rod, using double-stick tape to mount teeth that were pieces of glass cut with great difficulty from microscope slides. Each of us hoarded his or her own collection of glass teeth, and it was not uncommon to hear an anguished voice cry “Who took my teeth?” Research in this period presented unique challenges. The first cloning of DNA was out of sight, just over the horizon, and of course PCR was not yet conceived. With this setting in mind, then, let us turn our attention to the crown gall problem and consider what was known at the beginning of the 1970s. An Idea Born Before Its Time Dr. Armin Braun of the Rockefeller University (New York), whom many regard as the godfather of the crown gall story, first demonstrated that tumor cells are transformed, i.e. they can be freed from Agrobacteria and grown in vitro without the supplemental auxin and cytokinin required by normal plant cells in vitro (5). Braun kept tumor lines growing on hormone-free medium quite literally for decades. He reasoned that Agrobacterium must give these cells something, and he proposed that this gift must replicate because it is never lost by dilution. He proposed for it the term TIP (tumor inducing principle). Georges Morel of the Institut National Recherche Agronomique on the grounds of the Palais de Versailles in France discovered copious amounts of new metabolites—octopine and nopaline—in cultured crown gall tumor cells that were free from bacteria (18). Morel's group showed that the Agrobacterium strain, not the plant, determines the opine made by the tumor. Furthermore, eachAgrobacterium strain can grow on its own particular opine but not on a different one. He thought Braun's TIP must be or include a gene responsible for opine synthesis in the plant. He proposed that a single enzyme catalyzed opine synthesis in the plant and opine breakdown in Agrobacterium, in order to account for the strain specificity of opine catabolism. We now know that part of Morel's model was not correct (the bacteria use a different enzyme for catabolism), but he was certainly on the right track about opine synthesis in tumors. However, the scientific community in 1970 was far from ready to accept the notion of a bacterial gene getting into a plant cell and functioning there. More direct evidence would be needed to support such a radical idea. Bacterial DNA in Crown Gall Tumors? Rob Schilperoort at the State University of Leiden, the Netherlands, as part of his Ph.D. research, prepared DNA filters with crown gall DNA and found that they bound radiolabeledAgrobacterium DNA amazingly well. The thesis and other publications of Schilperoort (see citations in 6) were an important factor in the founding of our Seattle Crown Gall Group. Microbiologist Gene Nester, plant viral RNA biochemist Milt Gordon, and I, an organic-chemist-turned-DNA-hybridizer, all were intrigued by the idea of gene transfer to plants. We realized that we three might collaboratively do a much more definitive type of experiment to identify bacterial DNA in tumors—if it was really there! In 1971 we began our collaboration. Tom Currier, Nester's graduate student, set about giving cancer to tobacco plants using Agrobacterium tumefaciens strains from the American Type Culture Collection (Manassas, VA). He inoculated the bacteria into wound sites in the stems of young plants and observed the development of crown gall tumors that were to make biological history. The first contribution of our Seattle Crown Gall Group to the problem was a negative one that showed how large a challenge lay ahead. We found that the DNA-filter results reported by Schilperoort were caused by impurities (polysaccharides) in the DNA extracted from tumor cells, and that this technique did not have the sensitivity to detect 1% bacterial DNA in model mixtures (6). (One bacterial genome per plant cell would constitute approximately 0.1%.) We next employed DNA renaturation kinetic analysis, which tested whether a high concentration of tumor DNA (“driver DNA”) could make labeledAgrobacterium DNA (“labeled probe”) renature faster. We showed that this method was sensitive enough to detect one copy of the bacterial genome per three tumor cells, but tumor DNA did not drive our labeled probe (6). It was a clear negative result. We recognized that this method could only detect DNA corresponding to a significant fraction of our labeled probe. The bacterial genome contains perhaps a few thousand genes, so the acceleration of renaturation by even 10 specific bacterial genes in the tumor cells (a fraction of 1% of total bacterial DNA probe) would be below the limit of detection. Tumor-Inducing Genes Are on an Extra-Chromosomal Element Indirect genetic evidence that Agrobacteriummight carry a virus or plasmid with tumor-inducing genes emerged from two kinds of experiments published in 1971. Hamilton and Fall at the University of Pennsylvania (Philadelphia) discovered that strain C58, when grown at 37° (28° is optimal), lost virulence irreversibly. They proposed that tumor induction must be a plasmid- or virus-born trait because of its susceptibility to “curing” (12). At the same time, plant pathologist Allen Kerr at the Waite Institute in Adelaide, South Australia, was attempting to develop a biocontrol microbe to protect plants against crown gall disease. He co-inoculated avirulent and virulent Agrobacteria into the same sunflower plant. When he re-isolated the “avirulent” strain from the gall, it had become virulent! This transfer of virulence suggested to Kerr the existence of an extrachromosomal element as vector for tumor induction (16). Back in Seattle, Gene Nester read these reports and became convinced that there must be a plasmid in Agrobacterium. He and Alice Montoya reproduced the transfer of virulence with our own strains. Bruce Watson, a student in Milt Gordon's lab, reproduced the C58 curing experiment also. (The reproduction of published claims was clearly an important activity for our group, cast as we found ourselves in the role of iconoclasts. It was essential to know what could be believed.) Nevertheless, Bruce Watson repeatedly had no luck when he looked for plasmids in Agrobacterium using established methods (i.e. methods that were established for small plasmids). Key Discovery in Ghent: Ti Plasmid Is Gigantic In 1974, Ivo Zaenen (Fig. 1) at the University of Ghent (Belgium) cracked the crown gall problem wide open for everyone. Working in the laboratory of Jeff Schell and Marc van Montagu, Ivo Zaenen was the first to lay eyes on the megaplasmids of Agrobacterium. I asked him recently how he had succeeded where others had failed. He replied that at first he did not recognize what he had found. He was using alkaline Suc gradients to look for something else: a replicating form of anAgrobacterium phage called PS8 (whose DNA was once claimed to be in tumor DNA). He eventually found plasmids ranging from 96 × 106 to 156 × 106 M r in 11 virulent strains and not in eight avirulent strains (22). His publication in the prestigiousJournal of Molecular Biology is a landmark. Fig. 1. Open in new tabDownload slide Photograph of Ivo Zaenen, who discovered the Ti plasmid of Agrobacterium. Fig. 1. Open in new tabDownload slide Photograph of Ivo Zaenen, who discovered the Ti plasmid of Agrobacterium. When news of this discovery came to us in Seattle, it set off a flurry of experiments and launched a vigorous competition between the Seattle and Ghent groups. We quickly isolated plasmid DNA from severalAgrobacterium strains by Zaenen's method. Both groups found that strain C58 lost a mega-plasmid when grown at 37°. Transfer of virulence was mediated by transfer of a plasmid. It quickly emerged that the genes for catabolism of octopine and nopaline were located on their respective giant plasmids, which the Ghent group christened Ti (tumor-inducing) plasmid. Is the Ti Plasmid DNA in Tumor Cells? At last with the Ti plasmid of our Agrobacterium strain in hand, we felt confident that we had the right probe to look for TIP in crown gall tumors. But when we performed renaturation kinetic analysis with the whole plasmid as probe, we got the by now too familiar result: It was not there. Our experiment ruled out the presence of the entire plasmid, but just as before, we recognized that a few genes could be there without our noticing any kinetic change. In order to settle the issue, we decided to cut the Ti plasmid into specific fragments and test each piece by renaturation kinetic analysis. It was a brute force experiment involving everyone in the lab (Fig.2). In order to label our probe to maximum specific activity, Martin Drummond seized the fresh32P-dCTP the moment we received it from New England Nuclear and labeled our plasmid DNA by nick translation. Daniela Sciaky digested the labeled DNA with SmaI (purified by Alice Montoya—the enzyme was not for sale). Daniela and I ran the preparative gel, made an autoradiogram, and decided whether the fragments looked good enough. (Too much nicking in the nick translation reaction could lead to breakage of the largest SmaI fragments, which then ran too fast during electrophoresis and contaminated the smaller fragments.) If the autoradiogram looked good, we all canceled plans for the weekend: the experiment had to be completed within 48 h, before radiation damage to the DNA began to affect the kinetics. I excised the 15 resolvable plasmid bands, which were passed to Don Merlo for electroelution of DNA from the gel slices. (He used a device involving many small dialysis bags that he designed for the purpose. He called it “The Cow” for reasons that I will leave to the reader's imagination.) We set up 75 renaturation kinetic assays (5 unlabeled “driver” DNAs × 15 labeled probes) and worked around the clock to sample the reactions and assay the percentage of renatured probe DNA in 525 (7 × 75) samples. Milt held the stopwatch and called out time points. We all did whatever had to be done next. I have never experienced such completely committed teamwork in my entire career, before or since. Although it is now nearly 25 years ago, I can clearly remember the moment of truth. While calculating and plotting the results amid a sprawl of printer tape from the scintillation counter, I suddenly saw that the T-DNA (as it would soon be known) was there in the tumor cells. Labeled probes of band 3AB, and later on triplet band 10ABC, renatured faster in the presence of tumor DNA, and no other part of the plasmid did. Fig. 2. Open in new tabDownload slide Photograph of collaborators in the “brute force” experiments that first demonstrated the presence of T-DNA in crown gall tumor DNA. Left to right: Don Merlo, Martin Drummond, Gene Nester, Daniela Sciaky, Mary-Dell Chilton (author of this article), Alice Montoya (deceased 1990), and Milt Gordon. Fig. 2. Open in new tabDownload slide Photograph of collaborators in the “brute force” experiments that first demonstrated the presence of T-DNA in crown gall tumor DNA. Left to right: Don Merlo, Martin Drummond, Gene Nester, Daniela Sciaky, Mary-Dell Chilton (author of this article), Alice Montoya (deceased 1990), and Milt Gordon. A reviewer of the manuscript describing our findings required that we separate the doublet 3AB and determine which fragment was in the tumor. Although initial cloning experiments were just beginning in our group, we had no idea how to clone these blunt-ended SmaI fragments, and we found no enzyme that would cut one member of the doublet and spare the other. In desperation I finally managed to separate fragment 3A from 3B by a heroic serial electrophoresis of 4 d duration. We found that 3B was the fragment in the plant cells, and the paper was accepted (7). Resolution of the band 10 triplet showed us that 10C was the member in T-DNA, and when we subsequently determined the fragment map of our Ti plasmid, fragments 3B and 10C were contiguous, showing that T-DNA was a single segment of the Ti plasmid. Where Is T-DNA and What Defines It? By this time, genomic Southern blots had been developed and were clean enough to show T-DNA bands; renaturation kinetic analysis was a dying art that nobody mourned. The Southern blots showed recognizable intact Ti plasmid fragments and in addition “border fragments” that were different in different tumor lines, suggesting attachment of T-DNA to plant genomic DNA. By analysis of Southerns of nuclear DNA, chloroplast DNA and mitochondrial DNA, the T-DNA of several tumor lines was proven to be located in the nuclear fraction (8, 21). In 1979, I moved from the University of Washington to Washington University in St. Louis, and focussed on nopaline Ti plasmids, while the founding group in Seattle continued with the octopine strain. My new group at Washington University, the Seattle group, and Patti Zambryski in the Ghent group (Fig. 3) all succeeded in cloning T-DNA fragments from tumor DNA. When we sequenced through the junctions of T-DNA and plant DNA, comparing plasmid DNA with T-DNA, we found a 25-bp imperfect direct repeat on the Ti plasmid at the edges of what is incorporated into the plant genome. These border sequences define T-DNA on the plasmid but not in the plant: they are not transferred intact to the plant cell (reviewed in 4). Fig. 3. Open in new tabDownload slide Photograph of research group in Ghent, Belgium, 1984. Left to right: Jeff Schell, a visiting scientist from China, Marc van Montagu, Patricia Zambryski, and Ken Wang (a student). Fig. 3. Open in new tabDownload slide Photograph of research group in Ghent, Belgium, 1984. Left to right: Jeff Schell, a visiting scientist from China, Marc van Montagu, Patricia Zambryski, and Ken Wang (a student). Genetic Picture of the Ti Plasmid vir Genes Transposon mutagenesis of the Ti plasmid in Leiden, in Seattle, and in Ghent showed that all mutations affecting tumor induction mapped to a sector of approximately 42 kb, separate from T-DNA, called the virulence (vir) region. The vir genes constitute a regulon inducible by acetosyringone and other phenolics that are found in plant wound juice (19). These compounds, directly or indirectly, affect the “antenna” protein VirA, which autophosphorylates, then phosphorylates VirG, a transcriptional activator for all of the vir genes. T-DNA is excised from the Ti plasmid by endonuclease VirD2, with facilitation by VirD1 and VirC1. VirD2 nicks the bottom strand of the right border sequence after the third base and attaches to the 5′ end at the nick, forming the “leading” end of the T-strand to be delivered to the plant. The details of left border scission are not clear, but VirD2 produces a similar nick there. The vir E2 gene encodes a single-strand binding protein essential for tumor induction, that can alternatively be expressed in the plant with equal effect. The VirB operon consists of 11 open reading frames, which encode the T-DNA conduit from bacterium to plant. The structural and functional similarity of many of these to proteins involved in plasmid transfer to other bacteria has led to the view that T-DNA transfer has evolved from plasmid conjugation (reviewed in 23 and 24). T-DNA Genes Transposon hits in T-DNA were found to eliminate opine production or to alter tumor morphology or to have no recognizable effect at all. The morphology mutations were eventually shown to eliminate cytokinin autonomy (“rooty” tumors) or auxin autonomy (“shooty” tumors). T-DNA genes were shown to encode a two-step pathway to the plant auxin indoleacetic acid and an enzyme producing the cytokinin isopentenyladenosine 5′monophosphate (reviewed in 4). Most importantly, no mutation in T-DNA blocked T-DNA transfer. All of the genes affecting the process of T-DNA export to the plant cell mapped in the vir region. This fact would greatly simplify the disarming of T-DNA and construction of virregion-containing helper plasmids lacking any T-DNA. From Pathogen to Gene Vector In order to use the Ti plasmid as a vector, we needed a method of putting genes into T-DNA (and knocking some out, as well). In Ghent and in St. Louis, methods were developed for inserting DNA into any specific part of the Ti plasmid. The DNA to be inserted was cloned between pieces of T-DNA on a plasmid, introduced into the bacterium by conjugation or by transformation, and subjected to “forced recombination” (17, 20). A simpler approach to engineering T-DNA was to make a small separate T-DNA plasmid that could be manipulated directly. Although Agrobacterium, in nature, keepsvir genes and T-DNA on the same replicon, there is no requirement for this arrangement. If you place T-DNA on a separate replicon in Agrobacterium (a binary vector, as it is now called), the process of T-DNA transfer to the plant cell still occurs with good efficiency (9, 15). Thus, the T-DNA of a binary vector could be engineered directly in Escherichia coli and then transformed into Agrobacterim. Another problem for the genetic engineer was plant regeneration. All efforts to regenerate a plant from transformed cells were initially rewarded with only rare deletion mutants that had lost practically all of their T-DNA, a strong indication that at least part of T-DNA was inimical to plant regeneration. We discovered the critical part almost serendipitously. Tony Matzke and Ken Barton, post-docs in my group, introduced a yeast gene into T-DNA in a position that we thought might hit an oncogene (17). It turned out indeed to inactivate the cytokinin production gene. In collaboration with Andrew Binns at the University of Pennsylvania, we discovered that this single insertion event produced an engineered T-DNA that was completely disarmed. It produced transformants that synthesized nopaline but that could not grow autonomously without hormones. Binns identified the transformed plant cells by screening for nopaline production. In contrast to crown gall tumor cells, the tobacco cells transformed by multiple copies of this T-DNA were able to regenerate into normal plants that passed the T-DNA copies to progeny plants as Mendelian traits (1). By 1982 we had the first evidence that foreign DNA engineered between T-DNA borders and transformed into the plant nuclear DNA could be stably maintained in the plant genome and passed intact to progeny. Starting in about 1980, a formidable new group was assembled by Ernie Jaworski at Monsanto (our neighbor in St. Louis) to harness the T-DNA transfer technology for crop improvement. At this time Michael Bevan in my laboratory found himself in a race with Patti Zambryski's team in an effort to sequence the nopaline synthase (nos) gene and map its promoter and terminator by S1 nuclease protection (2, 10). Then a second race ensued amongst Bevan, Zambryski and her Ghent collaborators, and the Monsanto group to isolate the nosgene promoter and splice it to a kanamycin resistance coding region in order to create a selectable marker that might work in plant cells. If this scheme worked, then one would no longer have to screen for nopaline production to find transformed plant cells: one could select the cells with T-DNA inserts on kanamycin agar. The symbolic coming of age of genetic engineering occurred at the Miami Winter Symposium, January 18, 1983. During one session, Jeff Schell, Rob Horsch from Monsanto, and I all gave talks aboutAgrobacterium and its adaptation as a gene vector for plants. All three of us reported success with chimeric kanamycin resistance genes as a selectable marker for plant cells (3, 11, 14). I described initial success in transforming tobacco cells with binary vectors (which we called MiniTi at that time). In addition, I described our tobacco plants engineered with a disarmed Ti plasmid, and Southern blots proving that they passed their T-DNA insert to progeny intact. It was clear from the progress in all three groups that crop improvement by genetic engineering would become a reality. Reflections from 2000 Finding that T-DNA can integrate into a plant genome without benefit of homology was a real intellectual shock to me. The bacterial transformation studies I had made as a student and again as postdoc taught me the absolute need for good homology in those systems. Now illegitimate recombination seems the rule not only for T-DNA but also for foreign DNA integration in animal cells and indeed naked DNA delivery to plant protoplasts or bombardment of plant cells with DNA-coated microprojectiles. Incorporation of foreign DNA is clearly a process that cells carry out efficiently, perhaps in the course of repairing genomic damage. It is not a tradesecret ofAgrobacterium, although we may yet discover some secret details of the process. Agrobacterium acted as an inspiration to others who have developed various means of DNA delivery. It gave us our first selectable marker (tumor induction). It gave us the promoters and terminators for the next generation of selectable markers (octopine/nopaline synthase promoters and nosterminator, bounded as they were by T-DNA borders and neighboring genes in this highly compact T-DNA). The wide host range ofAgrobacterium (even wider now that monocot transformation is facile) inspired the idea that DNA incorporation may be a universal phenomenon. But perhaps the most important legacy fromAgrobacterium has been its inspiration of confidence that foreign gene integration, even though DNA is sometimes delivered artificially, is a perfectly natural process. My most fervent wish is that well-meaning environmental proponents will come to recognize this and embrace the technology based on it. LITERATURE CITED 1 Barton KA Binns AN Matzke AJM Chilton M-D Cell 32 1983 1033 1043 Crossref Search ADS PubMed 2 Bevan MW Barnes WM Chilton M-D Nucleic Acids Res 11 1983 369 385 Crossref Search ADS PubMed 3 Bevan MW Flavell RB Chilton M-D Nature 304 1983 184 187 Crossref Search ADS 4 Binns AN Thomashow MF Ann Rev Microbiol 42 1988 575 606 Crossref Search ADS 5 Braun AC Proc Natl Acad Sci USA 44 1958 344 459 Crossref Search ADS PubMed 6 Chilton M-D Currier TC Farrand SK Bendich AJ Gordon MP Nester EW Proc Natl Acad Sci USA 71 1974 3672 3676 Crossref Search ADS PubMed 7 Chilton M-D Drummond MH Merlo DJ Sciaky D Montoya AL Gordon MP Nester EW Cell 11 1977 263 271 Crossref Search ADS PubMed 8 Chilton M-D Saiki RK Yadav N Gordon MP Quetier F Proc Natl Acad Sci USA 77 1980 4060 4064 Crossref Search ADS PubMed 9 De Framond AJ Barton KA Chilton M-D Bio-Technology 1 1983 262 269 10 Depicker A Stachel S Dhaese P Zambryski P Goodman HM J Mol Appl Genet 1 1982 561 573 PubMed 11 Fraley RT Rogers SG Horsch RB Sanders PR Flick JS Adams SP Bittner ML Brand LA Fink CL Fry JS Galluppi GR Goldberg SB Hoffmann NL Woo SC Proc Natl Acad Sci USA 80 1983 4803 4807 Crossref Search ADS PubMed 12 Hamilton RH Fall MZ Experentia 27 1971 229 230 Crossref Search ADS 13 Hammond J McGarvey P Yusibov V Plant Biotechnology. 1999 Springer Verlag Berlin 14 Herrera-Estrella L De Block M Messens E Hernalsteens J-P Van Montagu M Schell J EMBO J 2 1983 987 995 Crossref Search ADS PubMed 15 Hoekema A Hirsch PR Hooykaas PJJ Schilperoort RA Nature 303 1983 179 180 Crossref Search ADS 16 Kerr A Physiol Plant Pathol 1 1971 241 246 Crossref Search ADS 17 Matzke AJ Chilton M-D J Mol Appl Genet 1 1981 39 49 PubMed 18 Petit A Delhaye S Tempé J Morel G Physiol Veg 8 1970 205 213 19 Stachel SE Messens E Van Montagu M Zambryski P Nature 318 1985 624 629 Crossref Search ADS 20 Van Haute E Joos H Maes M Warren G Van Montagu M Schell J EMBO J 2 1983 411 417 Crossref Search ADS PubMed 21 Willmitzer L De Beuckeleer M Lemmers M Van Montagu M Schell J Nature 287 1980 359 361 Crossref Search ADS 22 Zaenen I Van Larebeke N Teuchy H Van Montagu M Schell J J Mol Biol 86 1974 109 127 Crossref Search ADS PubMed 23 Zhu J Oger PM Schrammeijer B Hooykaas PJJ Farrand SK Winans SC J Bacteriol 182 2000 3885 3895 Crossref Search ADS PubMed 24 Zupan J Muth TR Draper O Zambryski P Plant J 23 2000 11 28 Crossref Search ADS PubMed Author notes * E-mail [email protected]; fax 919–541–8585. Copyright © 2001 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Meyerowitz, Elliot M.

doi: 10.1104/pp.125.1.15pmid: 11154286

The earliest non-taxonomic appearance of Arabidopsis in the literature of botany appears to be a paper by Alexander Braun in 1873, describing a mutant plant found in a field near Berlin (7). The mutation was almost certainly in theAGAMOUS gene, now well known as one of the floral ABC regulators and cloned in 1990 (54). The next notable appearance of Arabidopsis in the experimental literature was in 1907, when Friedrich Laibach (1885–1967), a student in Strasburger's laboratory in Bonn, published an account of the chromosome number of several plants. He was attempting to find a plant with a small number of large chromosomes to be used in experiments to determine the individuality of chromosomes (23). Arabidopsis was not such a plant: the chromosomes are very small. The next relevant appearance of Arabidopsis was in a 1935 paper that resulted from a Russian expedition to find a plant that could be used in genetics and cytogenetics, as Drosophila was then used (15, 51). Although the small chromosome number (incorrectly stated by Titova to be a haploid no. of three; Laibach had correctly counted five in 1907) and rapid time to flowering were considered useful features, the small size of the plant and its parts were considered a disadvantage, as was the inability to distinguish different chromosome pairs. It does not appear that Arabidopsis was ever used in the laboratory by Titova and her colleagues. Arabidopsis crops up again as a subject for laboratory investigation in 1943 when Laibach described the early results of studies in which he showed the short generation time, fecundity, ease of crosses, and the possibility of mutagenesis, and on this basis proposed adoption of Arabidopsis as a genetic model organism (24). The detailed results of the Laibach laboratory's studies on x-ray mutagenesis, which led to the first collection of Arabidopsis mutants, were published as a Ph.D. thesis by Laibach's student Erna Reinholz. The full publication of her 1945 thesis was, in fact, by the U.S. military: it seems that the thesis, with the word “Röntgen-Mutationen” in the title, came to the attention of those looking for a German atomic bomb program. It was published in 1947 as an unclassified captured document of the Joint Intelligence Objectives Agency (46). There are reports through the 1950s and 1960s of the creation of mutants (25) and mutant collections (34, 35), of methods for generation of embryo lethals, and use of such methods to assess mutagenicity of chemicals (40, 44), and of use of the plant for controlled-environment studies and quantitative genetics (26, 27), but surprisingly little use was made of what is now such a central organism for laboratory work on flowering plants. There were the first stirrings of organization: A newsletter called Arabidopsis Information Service was founded in 1964 (publication continued until 1990). The original advisory board was F. Laibach, A. Müller, G. Rédei, and J. Veleminsky, with G. Röbbelen of the University of Göttingen serving as editor. Starting with the 1974 issue, the position of editor was taken by Albert Kranz of the University of Frankfurt. Two International Congresses of Arabidopsis were held before the molecular biology era: the first in Göttingen in April, 1965 (Fig.1) and the second in Frankfurt am Main in September of 1976 (Fig. 2). Laibach and his students continued their Arabidopsis work by collecting a large number of ecotypes, which after their organization by Albert Kranz, formed the basis for the current ecotype collection (22). Fig. 1. Open in new tabDownload slide First International Symposium on Arabidopsis Research in Göttingen, April 21–24, 1965 (after the International Congress of Genetics in Scheweningen, and in honor of Laibach's 80th birthday). Left to right, first row: G. Röbbelen, S. Walles, I. Barthelmess, J. Veleminsky, ?, ?, A.D. McKelvie, ?, ?, and J. Bouharmont. Second row: G.P. Rédei, J.A.M. Brown, F. Laibach, E. Reinholz, T. Gichner, ?, B. Berger, and K. Napp-Zinn. Third row, J. Langridge, J. H.van der Veen, A. Müller, A.R. Kranz, ?, M. Jacobs, ?, ?, W.J. Feenstra, F. Schwanitz, ?, and F.J. Kribben. A ? indicates that the name of the individual is unknown. Copies of this photo courtesy of G.P. Rédei and A.R. Kranz. Fig. 1. Open in new tabDownload slide First International Symposium on Arabidopsis Research in Göttingen, April 21–24, 1965 (after the International Congress of Genetics in Scheweningen, and in honor of Laibach's 80th birthday). Left to right, first row: G. Röbbelen, S. Walles, I. Barthelmess, J. Veleminsky, ?, ?, A.D. McKelvie, ?, ?, and J. Bouharmont. Second row: G.P. Rédei, J.A.M. Brown, F. Laibach, E. Reinholz, T. Gichner, ?, B. Berger, and K. Napp-Zinn. Third row, J. Langridge, J. H.van der Veen, A. Müller, A.R. Kranz, ?, M. Jacobs, ?, ?, W.J. Feenstra, F. Schwanitz, ?, and F.J. Kribben. A ? indicates that the name of the individual is unknown. Copies of this photo courtesy of G.P. Rédei and A.R. Kranz. Fig. 2. Open in new tabDownload slide Second International Symposium on Arabidopsis Research in Frankfurt am Main, September 13–15, 1976. From left to right: front row, Gräf, Acedo, Venketeswaran, and Kranz. Second row, Demchenko, Scheidemann, Doddema, and Gresshof. Third row, Schweizer, Corcos, Negrutiu, D'Souza, and Sopory. Back row, Ledoux, Ratcliffe, Ambros, Maliga, Matigne, Jacobs, Feenstra, Rédei, Napp-Zinn, and Gomez-Campo. Copies of this photo courtesy of Ioan Negrutiu and Albert R. Kranz. Fig. 2. Open in new tabDownload slide Second International Symposium on Arabidopsis Research in Frankfurt am Main, September 13–15, 1976. From left to right: front row, Gräf, Acedo, Venketeswaran, and Kranz. Second row, Demchenko, Scheidemann, Doddema, and Gresshof. Third row, Schweizer, Corcos, Negrutiu, D'Souza, and Sopory. Back row, Ledoux, Ratcliffe, Ambros, Maliga, Matigne, Jacobs, Feenstra, Rédei, Napp-Zinn, and Gomez-Campo. Copies of this photo courtesy of Ioan Negrutiu and Albert R. Kranz. The widespread adoption of Arabidopsis as a model plant, followed by the current revolution in plant genetics, physiology, and molecular genetics, occurred in the 1980s (Fig. 3). The idea that plant biologists should concentrate on a model organism was then under intense discussion, and a number of proposals were made such as using petunia because of its ease of transformation and the availability of haploid lines, or using tomato because of the availability of mutants (e.g. 42). Use of Arabidopsis for genetic experiments in plant physiology, in particular for finding auxotrophic mutations, had been proposed by George Rédei in 1975, in an article in the Annual Review of Genetics that brought Arabidopsis to the attention of many young geneticists and soon-to-be molecular cloners (45). What swung the balance in favor of Arabidopsis is not certain, though several contributions can be pointed out. One was the demonstration that mutational analysis can be done to saturation in laboratory conditions, and therefore that informative mutations in any gene could be obtained in screens of a practicable size (48, 49). Another was the demonstration that Arabidopsis has a very small genome and is therefore convenient for gene cloning, which at that time was difficult for large-genome organisms (28, 43); yet another was the demonstration that Arabidopsis could be transformed with exogenous DNA (1, 29). These discoveries followed the publication of the first complete linkage map of Arabidopsis, which, along with the genome size data, showed that the relation between centimorgans and kilobases would permit straightforward map-based cloning, and showed clearly that morphological, life cycle, and hormone mutations were easily obtained (21). In addition, it was clear from even earlier work that embryo lethals could be produced and studied in detail (40, 36), and that Arabidopsis could be used as a model system for genetic analysis of plant embryo development (36). Fig. 3. Open in new tabDownload slide Arabidopsis molecular biologists at Keystone, Colorado, 1985. Left to right, S. Somerville, C. Somerville, E. Meyerowitz, D. Meinke, M. Crouch, and M. Koornneef (see 41). Photo courtesy of Maarten Koornneef. Fig. 3. Open in new tabDownload slide Arabidopsis molecular biologists at Keystone, Colorado, 1985. Left to right, S. Somerville, C. Somerville, E. Meyerowitz, D. Meinke, M. Crouch, and M. Koornneef (see 41). Photo courtesy of Maarten Koornneef. A summary of the reasons to adopt Arabidopsis as a model system for plant development, physiology, and molecular genetics was published inScience in 1985 (38), another adding the possibility for complete mutant screens in Trends in Genetics in 1986 (14), and another with more emphasis on developmental mutations in 1987 (37). The first gene sequences were published in 1986 (10), and T-DNA-mediated transformation of Arabidopsis was also first established in 1986 (1, 29). This was followed by the first restriction fragment-length polymorphism map in 1988 (8), T-DNA insertional cloning (16, 30), map-based cloning (18, 2), and the extremely efficient vacuum infiltration method of transformation (5). Each method was developed to solve specific biological problems, and each added to the reasons to use Arabidopsis in the laboratory. The list of reasons to use Arabidopsis thus grew from the intrinsic properties of the plant such as small size, large seed number, and small genome to include experimentally derived properties such as ease of mutagenesis and transformation. Complete and free sharing of experimental protocols and material was established as the norm, further motivating researchers to use the organism. The widespread adoption of Arabidopsis as a laboratory model system in plant biology has led to additional meetings; the 11th International Conference on Arabidopsis Research was held this summer, and the now-annual meetings have an attendance of nearly 1,000. These meetings, in addition to stock centers from which wild-type and mutant seed, as well as specific cDNA clones, genomic clones, DNA, and seed of T-DNA mutagenized pools are freely available, and a public internet-based database of sequence, clone, and mutant data add to the derived experimental properties, a set of derived social properties of the plant that further increases its value as an experimental system. Concentration on the Arabidopsis model genetic system has brought to plant biology a fusion of classical and molecular genetics with plant development, plant physiology, and plant pathology. This has in turn led to our first mechanistic understanding of the information transfer and cellular processes that regulate plant life—a first glimpse at how plants really work at the molecular level. Some (among many) areas where application of Arabidopsis genetics to the problems of plant biology has led to answers to longstanding questions include cell morphogenesis (19), root development (53), floral induction (3), flower and fruit development (47, 17), plant light perception (20), plant disease resistance (32, 13), plant cold and freezing resistance (50), and plant hormone action (31). One comparison that helps to explain the revolution in plant biology stemming from Arabidopsis research is a comparison of the genetic versus the physiological ways of thinking. Prior to the fusion of genetics via Arabidopsis with plant physiology, plant physiologists were concerned with the flow and movement of substances in plants. Although this is still a fundamental concern in physiology (just think water), genetics added to this the view of organisms as flows of information as well as substances. The original concern of genetics was the flow of the information for development from one generation to the next. In the last 50 years, however, the informational view of life has expanded to include flow of information into cells (via ligands for receptors), flow from the cell surface to the nucleus via signal transduction cascades, and flow from the nucleus to the cytoplasm via mRNA and nuclear protein transit. Before Arabidopsis genetics and molecular genetics was applied, for example, to understanding ethylene as a plant hormone, the experiments in this field were largely on the effects of ethylene treatment on plants and cells, and on how and under what conditions ethylene is synthesized. Application of genetic methods to find Arabidopsis mutations that blocked information flow via ethylene (6) led to the other half of the field as we now know it—the nature of the receptors (9), the molecules in the signal transduction pathway, and the nuclear transcription factors that interact with the genes activated by ethylene in different cells (33). We now think of the hormone as a carrier of information that transmutes through a series of different biochemical forms, from a gas to a series of phosphorylated cytoplasmic proteins to nuclear DNA-binding proteins—a rather different view than that before genetics came to plant physiology. A similar comparison of plant development before and after Arabidopsis can be made by reference to studies in plant responses to light. A recent history of this field makes exactly the point that Arabidopsis genetics has allowed a transition from studies of physiological response to light, to a mechanistic model of information transfer, described in terms of regulatory pathways (52). Another example of the change in viewpoint from physiological to physiological and genetic is in consideration of plant cell biology; this shift and the central role of Arabidopsis genetics in it has also been reviewed recently (11). It is worth emphasizing that the change in plant biology brought by research on Arabidopsis has been conceptual as well as methodological. Flower development and its mechanisms were under study, and floral development mutants were available for more than a century before Arabidopsis came into the field. However, until genetical thinking came to plant biology, no double mutants of floral development genes were made. The experiments that led to our present models of flower development (12) could have been completed with Antirrhinummutants available in the 1930s (39). The methods to do the work were not lacking in the 1930s, but the concepts of developmental genetics, of plant and animal life as a process of information flow from the genome that results in cellular differentiation, were not developed and applied to plants until much later. Thus experiments that now seem obvious were not done. The most recent methodological breakthrough, and perhaps a precursor to the next stage in the evolution of our concept of plants, is the completion of the DNA sequencing of the Arabidopsis genome. We now know much of the information content of a plant cell, though in a highly encoded fashion. The information that is immediately accessible is the estimated sequence of 25,000 proteins. These include not only the functional recipes for plant life, but also important aspects of evolutionary history, thus forming a resource for future analysis. As almost one-half of the proteins indicated so far are unrelated to any protein with a known function, we can for the first time quantify our ignorance and browse a list of what we don't know. This in itself is a grand stimulus to curiosity-driven research. Additional structural information may soon follow: Electron tomography methods are approaching the resolution where entire cells may soon be described at the atomic level (4), expression data on each of the genes will no doubt accumulate, and the existing large collections of gene knockouts will eventually allow us to know the phenotypes of loss- and gain-of-function of all of the genes. To know what experiments to do next will not come automatically, however, our concept of plant life must continue to evolve. To use the new information productively we have to continue using specific tests of specific hypotheses to address such fundamental questions as how plants grow, how plant cells function and communicate with their neighbors, how plants sense and respond to their environments, and how plants change over evolutionary time. ACKNOWLEDGMENTS I would like to thank Profs. A.R. Kranz, G. P. Rédei, and I. Negrutiu for sharing their photographs and recollections of the premolecular biology of Arabidopsis, and Prof. Kranz for detailed information on Laibach's work and career. Thanks also to Prof. L. Nover, who provided additional information on F. Laibach. My laboratory's work on Arabidopsis has been funded by the U.S. National Science Foundation, by the National Institutes of Health, by the U.S. Department of Energy, by the U.S. Department of Agriculture, and by the Human Frontiers Science Program. LITERATURE CITED 1 An G Watson BD Chiang CC Plant Physiol 81 1986 301 305 Crossref Search ADS PubMed 2 Arondel V Lemieux B Hwang I Gibson S Goodman HM Somerville CR Science 258 1992 1353 1355 Crossref Search ADS PubMed 3 Aukerman MJ Amasino RM Semin Cell Dev Biol 7 1996 427 433 Crossref Search ADS 4 Baumeister W Grimm R Walz J Trends Cell Biol 9 1999 81 85 Crossref Search ADS PubMed 5 Bechtold N Ellis J Pelletier G CR Acad Sci III-Vie 316 1993 1194 1199 6 Bleecker AB Estelle MA Somerville C Kende H Science 241 1988 1086 1089 Crossref Search ADS PubMed 7 Braun A Freunde z Berlin 1873 75 8 Chang C Bowman JL DeJohn AW Lander ES Meyerowitz EM Proc Natl Acad Sci USA 85 1988 6856 6860 Crossref Search ADS PubMed 9 Chang C Kwok SF Bleecker AB Meyerowitz EM Science 262 1993 539 544 Crossref Search ADS PubMed 10 Chang C Meyerowitz EM Proc Natl Acad Sci USA 83 1986 1408 1412 Crossref Search ADS PubMed 11 Chrispeels MJ Holuigue L Latorre R Luan S Orellana A Peña-Cortés H Raikhel NV Ronald PC Trewavas A Biol Res 32 1999 35 60 PubMed 12 Coen ES Meyerowitz EM Nature 353 1991 31 37 Crossref Search ADS PubMed 13 Delaney TP Trends Plant Sci 5 2000 49 51 Crossref Search ADS PubMed 14 Estelle MA Somerville CR Trends Genet 2 1986 89 93 Crossref Search ADS 15 Ezhova TA Russ J Genet 35 1999 1312 1325 16 Feldmann KA Marks MD Christianson ML Quatrano RS Science 243 1989 1351 1354 Crossref Search ADS PubMed 17 Ferrandiz C Pelaz S Yanofsky MF Annu Rev Biochem 68 1999 321 354 Crossref Search ADS PubMed 18 Giraudat J Hauge BM Valon C Smalle J Parcy F Goodman HM Plant Cell 4 1992 1251 1261 PubMed 19 Hulskamp M Folkers U Grini PE Bioessays 20 1998 20 29 Crossref Search ADS PubMed 20 Khurana JP Kochhar A Tyagi AK Crit Rev Plant Sci 17 1998 465 539 Crossref Search ADS 21 Koornneef M Van Eden J Hanhart CJ Stam P Braaksma FJ Feenstra WJ J Hered 74 1983 265 272 Crossref Search ADS 22 Kranz AR Kirchheim B Arab Inf Serv 3.1 1987 1 167 23 Laibach F Bot Centbl Beihefte (I) 22 1907 191 210 24 Laibach F Bot Archiv 44 1943 439 455 25 Langridge J Nature 176 1955 260 Crossref Search ADS PubMed 26 Langridge J Aust J Biol Sci 11 1958 457 470 Crossref Search ADS 27 Langridge J Griffing B Aust J Biol Sci 12 1959 117 135 Crossref Search ADS 28 Leutwiler LS Hough-Evans BR Meyerowitz EM Mol Gen Genet 194 1984 15 23 Crossref Search ADS 29 Lloyd AM Barnason AR Rogers SG Byrne MC Fraley RT Horsch RB Science 234 1986 464 466 Crossref Search ADS PubMed 30 Marks MD Feldmann KA Plant Cell 1 1989 1043 1050 Crossref Search ADS PubMed 31 McCourt P Annu Rev Plant Physiol Plant Mol Biol 50 1999 219 243 Crossref Search ADS PubMed 32 McDowell JM Dangl JL Trends Biochem Sci 25 2000 79 82 Crossref Search ADS PubMed 33 McGrath RB Ecker JR Plant Physiol Biochem 36 1998 103 113 Crossref Search ADS 34 McKelvie AD Rad Bot 1 1962 233 241 Crossref Search ADS 35 McKelvie AD Rad Bot 3 1963 105 123 Crossref Search ADS 36 Meinke DW Sussex IM Dev Biol 72 1979 50 61 Crossref Search ADS PubMed 37 Meyerowitz EM Annu Rev Genet 21 1987 93 111 Crossref Search ADS PubMed 38 Meyerowitz EM Pruitt RE Science 229 1985 1214 1218 Crossref Search ADS PubMed 39 Meyerowitz EM Smyth DR Bowman JL Development 106 1989 209 217 Crossref Search ADS 40 Müller AJ Biol Zentbl 82 1963 133 163 41 North G Nature 315 1985 366 367 42 International Society for Plant Molecular Biology Plant Mol Biol Newsl 1 1980 4 43 Pruitt RE Meyerowitz EM J Mol Biol 187 1986 169 183 Crossref Search ADS PubMed 44 Rédei GP Bibliogr Genet 20 1970 1 151 45 Rédei GP Annu Rev Genet 9 1975 111 127 Crossref Search ADS PubMed 46 Reinholz E (1947) Field Inf Agency Technical Report No. 1006: 1–70 47 Sessions A Yanofsky MF Weigel D Semin Cell Dev Biol 9 1998 221 226 Crossref Search ADS PubMed 48 Somerville CR Ogren WL Nature 280 1979 833 836 Crossref Search ADS 49 Somerville CR Ogren WL Methods in Chloroplast Molecular Biology Edelman M Hallick RB Chua NH 1982 Elsevier Biomedical Press Amsterdam , 129–138 50 Thomashow MF Annu Rev Plant Physiol 50 1999 571 599 Crossref Search ADS 51 Titova NN Sovietskaia Botanika 2 1935 61 67 52 Tong Z Zhao YJ Wang T Li NH Yarmamat M Acta Bot Sin 42 2000 111 115 53 Wysocka-Diller JW Benfey PN Bioessays 19 1997 959 965 Crossref Search ADS PubMed 54 Yanofsky MF Ma H Bowman JL Drews GN Feldmann KA Meyerowitz EM Nature 346 1990 35 39 Crossref Search ADS PubMed Author notes * E-mail [email protected]; fax 626–449–0756. Copyright © 2001 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Somerville, Chris R.

doi: 10.1104/pp.125.1.20pmid: 11154287

One of the great discoveries of 20th century biology was the elucidation of the pathway of photosynthetic CO2 fixation by Calvin, Benson, and colleagues (18). Among the many loose ends that remained after the photosynthetic carbon reduction cycle had been defined was a series of observations showing that when14CO2 was supplied to higher plants in the light, glycolate, Gly, Ser, and several other metabolites that could not be placed in the cycle were also rapidly labeled. By the late 1960s Ed Tolbert, Israel Zelitch, and others had identified the steps of a metabolic pathway in which two molecules of glycolate were converted in a series of enzymatic reactions through glyoxylate, Gly, Ser, and hydroxypyruvate to one molecule each of CO2 and phosphoglycerate (Fig.1; 23, 24). It had also been established that the CO2 released from glycolate metabolism was the source of at least some of the CO2released during a process that had become known as photorespiration (4, 25). However, there was no generally accepted explanation for the biosynthetic origin of glycolate or why it was rapidly labeled by14CO2. Fig. 1. Open in new tabDownload slide An abbreviated scheme of the photorespiratory pathway. Phosphoglycolate produced by ribulose bisphosphate (RuBP) oxygenase activity is converted to glycolate by phosphoglycolate phosphatase in the chloroplast. Glycolate enters peroxisomes and is converted to glyoxylate by glycolate oxidase. Glyoxylate is transaminated to Gly by either Ser:glyoxylate aminotransferase or Glu:glyoxylate aminotransferase. In mitochondria, Gly is converted to CO2, ammonia and the methylene group of methylene tetrahydrofolate (C1-THF). Gly and C1-THF condense to produce Ser. Peroxisomal Ser is deaminated to hydroxypyruvate, which is reduced to glycerate by hydroxypyruvate reductase. Glycerate enters the chloroplast and is phosphorylated to 3-phosphoglycerate, an intermediate of the Calvin cycle. Ammonia released during Gly decarboxylation is used by Gln synthetase to produce Gln. Glu synthase condenses 2-oxoglutarate (2-OG) and Gln to produce two molecules of Glu. A dicarboxylate transporter in the chloroplast envelope transfers oxoglutarate, Glu, and Gln across the chloroplast envelope. Overall, two molecules of phosphoglycolate are converted to one molecule of phosphoglycerate and one molecule of CO2. Fig. 1. Open in new tabDownload slide An abbreviated scheme of the photorespiratory pathway. Phosphoglycolate produced by ribulose bisphosphate (RuBP) oxygenase activity is converted to glycolate by phosphoglycolate phosphatase in the chloroplast. Glycolate enters peroxisomes and is converted to glyoxylate by glycolate oxidase. Glyoxylate is transaminated to Gly by either Ser:glyoxylate aminotransferase or Glu:glyoxylate aminotransferase. In mitochondria, Gly is converted to CO2, ammonia and the methylene group of methylene tetrahydrofolate (C1-THF). Gly and C1-THF condense to produce Ser. Peroxisomal Ser is deaminated to hydroxypyruvate, which is reduced to glycerate by hydroxypyruvate reductase. Glycerate enters the chloroplast and is phosphorylated to 3-phosphoglycerate, an intermediate of the Calvin cycle. Ammonia released during Gly decarboxylation is used by Gln synthetase to produce Gln. Glu synthase condenses 2-oxoglutarate (2-OG) and Gln to produce two molecules of Glu. A dicarboxylate transporter in the chloroplast envelope transfers oxoglutarate, Glu, and Gln across the chloroplast envelope. Overall, two molecules of phosphoglycolate are converted to one molecule of phosphoglycerate and one molecule of CO2. Photorespiration was discovered shortly after the first infrared gas analyzers became available in the mid 20th century (6). The phenomenon was described as the light-dependent release of CO2, a difficult process to measure against a background of concurrent photosynthetic CO2 fixation and mitochondrial or “dark” respiration. To accurately measure the magnitude of photorespiration it was necessary to use elaborate pulse-chase isotope labeling methods that could distinguish recently fixed carbon from carbon fixed during an earlier time period (2). The best estimates suggested that under normal circumstances, a C3 plant could photorespire as much as 25% of the carbon fixed by photosynthesis. Thus, photorespiration was considered a potentially wasteful process that was limiting plant productivity. My interest in the problem was stimulated by a theory advanced by Bill Ogren and George Bowes that was the equivalent of the Grand Unified Theory of Photosynthesis and Photorespiration. In the late 1960s, Ogren had been intrigued by the observation that photosynthetic CO2 fixation is strongly inhibited by oxygen. This is simply demonstrated: Plants grown in 350 μL L−1 CO2 and 2% (v/v) O2 have much higher rates of CO2 fixation than plants grown in 350 μL L−1 CO2 and 21% (v/v) O2. Higher levels of CO2, however, suppress the negative effect of O2. These effects were exhaustively measured in a series of carefully executed experiments on photosynthetic gas exchange that became a scientific touchstone for Ogren (7). He resolved to try to find a mechanistic model of photosynthetic CO2fixation that would explain the inhibitory effect of O2 and the salutatory effect of CO2. The recognition that O2 and CO2 had mutually competitive effects on photosynthesis led him invariably to the conclusion that O2 must compete with CO2 as a substrate for the enzyme responsible for photosynthetic CO2 fixation, RuBP carboxylase. I consider this to be one of the most brilliant examples of deductive reasoning in 20th century plant biology. On the basis of this theory, Ogren's postdoc, George Bowes, carried out a protracted search for RuBP oxygenase activity. After more than a year of many failed attempts, RuBP oxygenase activity was at last detected and determined to be a property of RuBP carboxylase (1, 14). The enzyme was subsequently renamed RuBP carboxylase/oxygenase, or Rubisco. I think about this experiment frequently when something is not working in my lab—one of the great challenges of experimental science is to decide when to abandon a line of experimental work that is not progressing and when to keep trying. If there is a lesson from the RuBP oxygenase example I think it is that nothing substitutes for a good theory (and tenacity). It was not just George Bowes who initially had trouble demonstrating RuBP oxygenase activity. About a year after the oxygenase paper was published, George Lorimer, a student of Ed Tolbert's at the time, reportedly burst into Ogren's office with an armful of O2 electrode tracings from failed attempts to measure RuBP oxygenase activity and dumped them on Ogren's desk with the words “It doesn't work.” Lorimer was so inflamed with the idea that Bowes' and Ogren's paper was erroneous, and that he had wasted time testing their idea, that he had driven all the way from Lansing to Urbana to deliver the message in person. Of course it did work and Lorimer went on to show why with an elegant series of papers on the mechanistic basis of catalysis by the enzyme (5). The product of RuBP oxygenase activity is phosphoglycolate (14). Thus, the discovery of oxygenase activity provided a credible explanation for the origin of glycolate. Because CO2 and O2 are mutually competitive substrates of RuBP carboxylase/oxygenase, the discovery of oxygenase activity also explained the effects of CO2 and O2 concentration on photosynthesis and photorespiration. Ogren went one step further to suggest that photorespiration was not biologically necessary—that it had evolved only to recycle carbon from phosphoglycolate back into the Calvin cycle and that the CO2 loss was the cost of recycling the other three carbons back into the Calvin cycle. The evidence for this was that plants grown in low levels of O2 or high levels of CO2 were more productive than plants grown in air despite strongly reduced levels of flux through the photorespiratory pathway. Thus, the implication was clear: Plant productivity could be strongly enhanced by identifying mutants with reduced amounts of photorespiration. However, in a precient analysis of Rubisco's probable catalytic mechanism, Lorimer and John Andrews hypothesized that RuBP carboxylation and oxygenation could not be uncoupled because oxygenase activity is due to autoxidation of an obligatory intermediate in the carboxylation reaction (12). Except for Tolbert and colleagues, who had verified for themselves the existence of RuBP oxygenase activity, the RuBP oxygenase theory of photorespiration gained acceptance rather slowly. I arrived in Ogren's lab as a postdoc about 7 years after the first paper on RuBP oxygenase had been published and the topic was still a subject of heated debate; people on opposite sides of the issue were literally shouting at each other during long public arguments at scientific meetings. I have never witnessed any public arguments comparable with those that dominated the 1978 Gordon Conference on photosynthesis. The opposition was led by Israel Zelitch who was of the opinion that the RuBP oxygenase-based mechanism of glycolate synthesis was inconsistent with many miscellaneous observations that had been made during the long search for the source of photorespiratory glycolate (25). Zelitch and others also argued that phosphoglycolate could not be an important precursor of photorespiratory glycolate because measurements of flux through phosphoglycolate were much too low to account for the magnitude of photorespiratory CO2 metabolism (25). Zelitch further claimed that he had been able to reduce glycolate synthesis and photorespiration by treating plant tissues with glycidate, that blocking glycolate oxidation inhibited photorespiration and increased photosynthesis, that Gly oxidation could not account for most photorespiratory CO2 release, and that there was substantial genetic variation in the ratio of photosynthesis to photorespiration. These and many related observations seemed at variance not only with Ogren's theory but also with some aspects of the scheme for glycolate metabolism developed by Tolbert and colleagues (23). In retrospect, it seems to me that Zelitch was misled by reliance on a number of technically flawed attempts to quantitatively measure photorespiration and metabolite flux. Shauna Somerville and I arrived in Bill Ogren's lab for graduate and postdoctoral work, respectively, in 1978 with three clear ideas. The first was that plant biology as a field needed a model organism with good experimental properties. The second was that Arabidopsis was that organism. The third was an idea about how to solve some of the problems that were generating heated debate among plant physiologists and biochemists at that time. We had formulated the idea of developing Arabidopsis as a model organism for plant biology during an extended visit to Paris where we spent our time reading in the library of the Institut Pierre et Marie Curie and doing gedanken experiments in the cafes. The groundbreaking work of Chilton et al. (3), showing thatAgrobacterium tumefaciens transferred a fragment of DNA from the Ti plasmid into plant genomes, led us to conclude that the development of methods for plant transformation were imminent. We realized that this new technology would create a new opportunity to develop the use of genetics and molecular biology as a general approach to problems in plant biology. We had begun using the tools of molecular biology as graduate students and were certain that these tools would be rapidly adapted by plant biologists and that, when that happened, plant biologists would recognize the need for a facile genetic system. Although there was a long history of very sophisticated genetics in plant biology, our impression was that plant genetics was dissociated from mainstream plant biology and the average plant biologist did not understand the power of genetics as a tool for dissecting problems in general biology. I think this may have arisen, in part, because many plant geneticists had a tendency to work on problems that were of interest primarily in the context of genetics rather than using genetic methods to solve problems of interest to physiologists or biochemists. Therefore, we decided that to stimulate the interest of plant biologists in the use of genetic methods, we should focus on solving a problem that was of broad interest and was amenable to a genetic approach. In the cafes of Paris we formulated a “demonstration experiment” in which we envisioned solving a problem in plant biology using the kind of genetic approach that was used in Escherichia coli genetics. We spent our mornings reading the current issues of the major plant journals, and the rest of the day sitting in the cafes talking about the papers we had read that day and trying to envision a genetic approach to the problems they discussed. In the course of our reading we had come upon an article by George Redei extolling the virtues of Arabidopsis as a model system for plant genetics (15). Redei pointed out that Arabidopsis was closely related to many important crop species, small, rapid cycling, diploid, self-fertilizing, easily mutagenized, had the smallest known plant genome, and already had the rudiments of a genetic map. These properties corresponded to those that we were looking for in a model plant and we determined to adopt Arabidopsis as our experimental system. We assumed that routine genetic transformation was imminent and that we should focus our efforts on genetically defining an interesting problem so that we could make use of the molecular tools that were being developed by others. We were attracted to the problem of photorespiration because it seemed important and it was vividly controversial. One need only compare the views expressed in two contemporaneous reviews of the subject to get a clear impression of a major scientific controversy of the period (4, 25). The problem seemed important because, although there was a lot of disagreement about the mechanism of photorespiration, there was broad agreement that if it could be genetically reduced it would lead to a major increase in primary plant productivity. One of the major challenges we faced from the outset was deciding which side of the controversy was most likely to be correct. I had done my graduate work in E. coli genetics and Shauna had done a masters degree in plant breeding so neither of us had any experience with plant physiology or biochemistry. We eventually decided that the proponents of the RuBP oxygenase theory were advocating the most convincing explanation for the biological phenomena. We realized that the competitive actions of O2 and CO2 on the outcomes of the RuBP carboxylase/oxygenase reactions could be used as a basis for mutant selection. We hypothesized that we could isolate plant mutants with defects in photorespiration by growing mutant populations in high concentrations of CO2, where the pathway was suppressed, then scoring for mutations in the pathway by placing them in air. We guessed that mutants with enzymatic defects in the pathway would be viable in high CO2 but would be inviable in air because of the drain of carbon from the Calvin cycle or other effects. After our return from Paris we joined the laboratory of Bill Ogren who agreed to let us test our ideas, and to help us learn plant physiology and biochemistry. Following methods that George Redei and others had developed for mutagenizing Arabidopsis with ethyl methane sulfonate we produced a mutagenized M2 population. We grew the plants in growth chambers in which the atmosphere was held at roughly 1% (v/v) CO2 by pumping air into the chambers at several liters per minute and bleeding inexpensive welding grade CO2 into the air stream. Once the plants became established, we removed any plants that were chlorotic, stunted, or morphologically abnormal, then stopped supplementing the plants with CO2. After several days of illumination in air, we scored the populations for plants that were chlorotic. To our delight, dozens of plants from the first screen turned chlorotic! As anyone who has done a mutant screen knows, the risk of investing a lot of effort for no result was behind us. After verifying that the mutant phenotypes were heritable, we set about trying to determine the biochemical nature of the defects. A wealth of literature existed concerning the labeling of the products of photosynthesis with 14CO2. By labeling the various mutants and then resolving the primary products by a combination of ion-exchange and thin-layer chromatography we were able to group the mutants into various classes based on what metabolites accumulated. We then performed enzymes assays on extracts of the various mutants. The first unambiguous result that we obtained was a mutant that was completely deficient in phosphoglycolate phosphatase (19). I finished the enzyme assays at about midnight and was so excited by the evidence that we had identified a mutant for the enzyme that I phoned Bill Ogren at home to share the news. Bill was characteristically calm but enthusiastic considering the late hour. By similar approaches we identified mutants in Ser:glyoxylate aminotransferase, Gly decarboxylase, and Ser transhydroxymethyltransferase (13, 17) and Glu synthase (20). These mutants were very useful in resolving many of the problems in the area of photorespiration that had been intractable to conventional biochemical approaches (13). For instance, when illuminated in air, the phosphoglycolate phosphatase mutant rapidly accumulated large amounts of phosphoglycolate but failed to accumulate glycolate and essentially lacked photorespiration. This observation largely ended debate about the key issues of whether the amount of RuBP oxygenase activity in vitro was adequate to support photorespiration or whether there were alternate sources of glycolate. In a similar manner, when placed in air the Glu synthase mutants became rapidly depleted of Glu (20), confirming the recently proposed role for the enzyme in recycling photorespiratory nitrogen (9). Shauna also characterized a mutant deficient in the chloroplast dicarboxylate transporter and showed that the mutants were unable to recycle photorespiratory ammonia, demonstrating the operation of a Glu:2-oxoglutarate shuttle in the photorespiratory cycle (22). When provided with exogenous ammonium, the Ser transhydroxymethyltransferase mutant was found to be completely deficient in photorespiratory CO2 release, providing unambiguous evidence that in plants with adequate nitrogen, Gly decarboxylation was normally the sole source of photorespiratory CO2 (17). In short, the mutants provided novel and compelling tests of the various theories that had been proposed based on biochemical or physiological criteria. The results confirmed all of the predictions of the RuBP carboxylase/oxygenase-based theory of photorespiration and also confirmed the role of many of the steps in photorespiratory metabolism that had been proposed by Ed Tolbert and colleagues. The success of the approach generated some of the earliest converts to the utility of Arabidopsis genetics. In addition, Peter Lea, Ben Miflin, Alf Keys, and colleagues at Rothamstead used similar approaches to isolate a rich collection of photorespiratory mutants of barley that have been extensively utilized in continuing studies of photorespiration (8, 10). In addition to the mutants in the photorespiratory pathway, we had isolated several mutants that we could not place in the pathway by isotopic labeling experiments. The mutants were clearly defective in photosynthetic CO2 fixation at low concentrations of atmosphereic CO2 but had relatively normal levels of CO2 fixation at high levels of CO2 (21). In vitro assays with Rubisco showed that, in the mutants, the enzyme was present in an inactive form that could be converted to normal levels of activity by preincubation with high levels of sodium bicarbonate. Bill Laing and Ogren had discovered the activation of Rubisco by bicarbonate some years earlier. This effect had been shown by George Lormier to be due to the formation of a carbamate on a Lys group of the enzyme that was involved in binding a metal ion required for catalysis (11). This led to the idea that the mutants had a defect in RuBP carboxylase/oxygenase activation. Shortly after isolating this mutant I left Ogren's lab to found my own lab at the University of Alberta in Edmonton. Mike Salvucci, a new postdoc in Ogren's lab, inherited the mutant and went on to show with Archie Portis that the mutant was deficient in an enzyme that was specifically required to activate Rubisco (16). This unique enzyme, now called Rubisco activase, is thought to activate Rubisco by removing an inhibitory isomer of RuBP from the active site of Rubisco. The discovery of Rubisco activase provides a satisfying example of the utility of the genetic approach to biological problems. The existence of the enzyme was not even hinted at in the hundreds of papers describing the properties of Rubisco prior to the isolation of the mutant. In retrospect, the photorespiratory mutant work provided a timely example of the use of a directed genetic approach to dissect a complex problem in plant biology and helped pave the way for acceptance of Arabidopsis as a model organism. I think that the success of the project was due, in part at least, to having spent a lot of time thinking about it in a congenial setting before starting experimental work, a technique that seems as useful today as ever. LITERATURE CITED 1 Bowes G Ogren WL Hageman R Biochem Biophys Res Commun 45 1971 716 722 Crossref Search ADS PubMed 2 Canvin DT Lloyd NDH Fock H Przybylla K CO2 Metabolism and Plant Productivity. Burris RH Black CC 1976 161 176 University Park Press Baltimore 3 Chilton MD Drummond M Merlo DJ Sciaky D Montoya AL Gordon MP Nester EW Cell 11 1977 263 271 Crossref Search ADS PubMed 4 Chollet R Ogren WL Bot Rev 41 1975 137 179 Crossref Search ADS 5 Cleland WW Andrews TJ Gutteridge S Hartman FC Lorimer GH Chem Rev 98 1998 549 561 Crossref Search ADS PubMed 6 Decker JP Plant Physiol 30 1955 82 84 Crossref Search ADS PubMed 7 Forrester ML Krotkov G Nelson CD Plant Physiol 41 1966 422 427 Crossref Search ADS PubMed 8 Kendall AC Keys AJ Turner JC Lea PJ Miflin BJ Planta 159 1983 505 511 Crossref Search ADS PubMed 9 Lea PJ Wallsgrove RM Miflin BJ Nature 275 1978 741 743 Crossref Search ADS 10 Leegood RC Lea PJ Adcock MD Hausler RE J Exp Bot 46 1995 1397 1414 Crossref Search ADS 11 Lorimer GH Trends Biochem Sci 8 1983 65 68 Crossref Search ADS 12 Lorimer GH Andrews TJ Nature 243 1973 359 360 Crossref Search ADS 13 Ogren WL Annu Rev Plant Physiol 35 1984 415 442 Crossref Search ADS 14 Ogren WL Bowes GH Nature New Biol 230 1971 159 160 Crossref Search ADS PubMed 15 Redei GP Annu Rev Genet 9 1975 111 127 Crossref Search ADS PubMed 16 Salvucci ME Portis AR Jr Ogren WL Plant Physiol 80 1986 655 659 Crossref Search ADS PubMed 17 Somerville CR Oxford Surveys of Plant Molecular and Cell Biology Miflin B 1 1984 102 133 Oxford University Press Oxford 18 Somerville CR Cell 100 2000 13 25 Crossref Search ADS PubMed 19 Somerville CR Ogren WL Nature 280 1979 833 836 Crossref Search ADS 20 Somerville CR Ogren WL Nature 286 1980 257 259 Crossref Search ADS 21 Somerville CR Portis AR Ogren WL Plant Physiol 70 1982 381 387 Crossref Search ADS PubMed 22 Somerville SC Ogren WL Proc Natl Acad Sci USA 80 1983 1290 1294 Crossref Search ADS PubMed 23 Tolbert NE Annu Rev Plant Physiol 22 1971 45 74 Crossref Search ADS 24 Zelitch I Annu Rev Plant Physiol 15 1964 121 142 Crossref Search ADS 25 Zelitch I Annu Rev Biochem 44 1975 123 145 Crossref Search ADS PubMed Author notes * E-mail [email protected]; fax 650–325–6857. Copyright © 2001 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Field, Christopher B.

doi: 10.1104/pp.125.1.25pmid: 11154288

Plant physiology is deeply entwined with climate change. On one hand, many plant processes are climate sensitive. Plants are potential victims of climate change, threatened by novel conditions that stress natural ecosystems and tax the creativity of agronomists. On the other, plants are also major regulators of climate. One aspect of this regulation involves the absorption and dissipation of solar energy at the earth's surface. A second involves the modulation of the water cycle through stomatal regulation of transpiration. In addition, plants influence climate through their role in the carbon cycle. Photosynthesis removes large amounts of CO2 from the atmosphere. Global gross primary production or photosynthesis on land fixes about 20 times more carbon than is released by fossil fuel combustion (TableI). Respiration by plants and heterotrophs, plus biomass combustion, add it back. When photosynthesis outpaces respiration plus combustion, the land biosphere is a sink for carbon, reducing the rate of CO2 accumulation in the atmosphere. When carbon losses outpace photosynthesis, the land is a carbon source. Table I. Summary of stocks and fluxes in the global carbon cycle CO2 sources averaged over the 1990s  Emissions from fossil fuel combustion and cement production 6.4 ± 0.5  Net emissions from tropical land use and land cover change 1.6 ± 1.0  Total anthropogenic emissions 8.0 ± 1.1 Carbon storage reservoirs averaged over the 1990s  Atmospheric increase 3.2 ± 0.2  Ocean uptake 2.0 ± 0.8  Northern Hemisphere forest regrowth 0.5 ± 0.5  Other terrestrial sinks 2.3 ± 2.0 Fluxes in the “background” carbon cycle of the 1980s  Land gross primary production 120  Land net primary production 56.4  Ocean net primary production 48.6 Stocks in the “background” carbon cycle of the 1980s  Atmospheric carbon in CO2 760  Land vegetation carbon 610  Soil carbon 1,580  Carbon in the ocean biosphere 3  Inorganic and organic carbon in ocean water 39,800 CO2 sources averaged over the 1990s  Emissions from fossil fuel combustion and cement production 6.4 ± 0.5  Net emissions from tropical land use and land cover change 1.6 ± 1.0  Total anthropogenic emissions 8.0 ± 1.1 Carbon storage reservoirs averaged over the 1990s  Atmospheric increase 3.2 ± 0.2  Ocean uptake 2.0 ± 0.8  Northern Hemisphere forest regrowth 0.5 ± 0.5  Other terrestrial sinks 2.3 ± 2.0 Fluxes in the “background” carbon cycle of the 1980s  Land gross primary production 120  Land net primary production 56.4  Ocean net primary production 48.6 Stocks in the “background” carbon cycle of the 1980s  Atmospheric carbon in CO2 760  Land vegetation carbon 610  Soil carbon 1,580  Carbon in the ocean biosphere 3  Inorganic and organic carbon in ocean water 39,800 All fluxes are Pg carbon per year. Stocks are Pg carbon. 1 Pg = 1015 g or 109 metric tons. The data are from references 26, 3, and 8. Open in new tab Table I. Summary of stocks and fluxes in the global carbon cycle CO2 sources averaged over the 1990s  Emissions from fossil fuel combustion and cement production 6.4 ± 0.5  Net emissions from tropical land use and land cover change 1.6 ± 1.0  Total anthropogenic emissions 8.0 ± 1.1 Carbon storage reservoirs averaged over the 1990s  Atmospheric increase 3.2 ± 0.2  Ocean uptake 2.0 ± 0.8  Northern Hemisphere forest regrowth 0.5 ± 0.5  Other terrestrial sinks 2.3 ± 2.0 Fluxes in the “background” carbon cycle of the 1980s  Land gross primary production 120  Land net primary production 56.4  Ocean net primary production 48.6 Stocks in the “background” carbon cycle of the 1980s  Atmospheric carbon in CO2 760  Land vegetation carbon 610  Soil carbon 1,580  Carbon in the ocean biosphere 3  Inorganic and organic carbon in ocean water 39,800 CO2 sources averaged over the 1990s  Emissions from fossil fuel combustion and cement production 6.4 ± 0.5  Net emissions from tropical land use and land cover change 1.6 ± 1.0  Total anthropogenic emissions 8.0 ± 1.1 Carbon storage reservoirs averaged over the 1990s  Atmospheric increase 3.2 ± 0.2  Ocean uptake 2.0 ± 0.8  Northern Hemisphere forest regrowth 0.5 ± 0.5  Other terrestrial sinks 2.3 ± 2.0 Fluxes in the “background” carbon cycle of the 1980s  Land gross primary production 120  Land net primary production 56.4  Ocean net primary production 48.6 Stocks in the “background” carbon cycle of the 1980s  Atmospheric carbon in CO2 760  Land vegetation carbon 610  Soil carbon 1,580  Carbon in the ocean biosphere 3  Inorganic and organic carbon in ocean water 39,800 All fluxes are Pg carbon per year. Stocks are Pg carbon. 1 Pg = 1015 g or 109 metric tons. The data are from references 26, 3, and 8. Open in new tab Over the last 25 years, understanding the role of plants and ecosystems in regulating atmospheric CO2 has been one of the central goals of global-change research. Although the understanding is not yet complete, the scientific framework is increasingly robust. It is unfortunate that the political framework for the use and abuse of the science is also well established. Arrhenius (1) first predicted that industrial activity could lead to climate warming through increased absorption of thermal radiation by elevated atmospheric CO2. Callendar's (5) estimates of changes in atmospheric CO2 from fossil fuel combustion, later confirmed by Keeling's pioneering work with long-term monitoring (21), established the first component of the Arrhenius scenario. The last quarter century of intensive research with climate models, temperature records, and satellite observations has gone a long way toward establishing the second. Early in the history of CO2 research it was clear that only a fraction of the CO2 emitted into the atmosphere from fossil fuel combustion was staying there. The other part was transferred to some kind of sink. Revelle and Seuss (25) and Bolin and Eriksson (4) calculated the expected transfer of CO2 into the world's oceans. Early syntheses of emissions, ocean uptake, and changes in atmospheric CO2 suggested that the budget was close to balance and that the carbon in the land biosphere was stable or slightly increasing during the industrial era (2). Another line of research indicated a critical problem with this approach. It was not accounting for CO2 emissions from land use change, calculated by Woodwell and colleagues (30) to be in the same range as the emissions from fossil fuel combustion. If a large flux to the atmosphere was essentially invisible to the understood parts of the carbon cycle, it must be balanced by an unknown or “missing” sink. Plant physiology provided a possible solution. In 1782 Senebier demonstrated that CO2 is necessary for photosynthesis. Increases in the rate of photosynthesis with increasing CO2 were documented around 1900 by Kreusler, Brown and Escombe, Treboux, and Pantanelli (23). With photosynthesis in C3 plants increasing by 40% to 70% under a CO2 doubling, it was reasonable to conjecture that the missing sink was somehow driven through CO2 fertilization. Ecosystems on land were the most likely candidate locations because ocean photosynthesis is not simply related to the CO2 concentration in the atmosphere. Though attractive, this explanation for the missing sink had at least two important problems. First, the deforestation flux calculated by Woodwell and colleagues was probably too large to be balanced by CO2 fertilization. Second, increased photosynthesis is not sufficient, by itself, to account for a large carbon sink. A large sink requires that the extra carbon fixed through photosynthesis must remain in the ecosystem for a substantial amount of time, on the order of decades. Houghton and others resolved the first problem with improved estimates of CO2 fluxes from land use change (11). Recent estimates of carbon emissions from land use and cover change are comparable, mostly in the range of 1 to 2 Pg year−1 (1 Pg = 1015g), but with a large uncertainty (Table I). The magnitude of the sink from CO2 fertilization is still not completely resolved. In the absence of a mechanistic formulation for the sink from CO2 fertilization, Bacastow and Keeling (2) estimated the CO2 fertilization effect as a residual. With estimates of the emissions from fossil fuel, plus uptake by the oceans, they assumed that the only missing term was the sink due to CO2 fertilization on land. Knowing the historical trajectory of emissions and ocean uptake, it was straightforward to calculate a CO2 sensitivity of the historical missing sink. Looking for a simple, reasonable form, Bacastow and Keeling suggested that the extra CO2uptake changes with the natural logarithm of the ratio of current to pre-industrial CO2. Their expression is simple, but not mechanistic. It is based on plant physiology only to the extent that it suggests accelerating plant growth with increasing atmospheric CO2. It has, however, been exceedingly important in efforts to understand options for managing the carbon cycle. The Intergovernmental Panel on Climate Change, the body asked by the world's governments to evaluate climate change and its impacts, stuck with this formulation for its major assessment reports in 1990 and 1995 (26). By this time, however, the scientific understanding of CO2 fertilization was becoming more multi-dimensional, with conflicting evidence from different approaches. It was also becoming more politicized. At the single leaf level, the model of Farquhar and colleagues (7) provided a reliable framework for evaluating the response of C3 plants to elevated CO2. With a combination of robustness and simplicity, this model has become almost a standard component in analyses of the global carbon cycle. At higher levels of organization, however, results were mixed. Some growth chamber experiments indicated dramatic increases in plant growth under elevated CO2. These results rapidly became a rallying point for groups opposed to limits on CO2emissions. If elevated atmospheric CO2 could lead to large increases in plant growth, it might produce a dramatic “greening of the earth” and plant uptake so large that it would eventually completely balance emissions from fossil fuel combustion. At least that was the argument in a famous 1991 video (14). Other studies, including work by Oechel, Strain, and Bazzaz, suggested much different responses, sometimes with relatively rapid decay of the initial growth stimulation and only small CO2 responses in the long term (19). By around 1990 it was clear that the basic questions about the CO2 sensitivity of carbon storage could not be solved without moving to larger scales of space and time. The key issues concerned not the instantaneous response of photosynthesis to CO2, but changes in photosynthetic capacity, biomass allocation, nutrient availability, and longevity of the plant and soil pools receiving the extra photosynthate. To address these issues many new studies have moved to the scale of entire ecosystems. Some emphasize vegetation near natural CO2springs (24). Others utilize a technology called FACE, or Free Air CO2 Enrichment, in which an ecosystem is exposed to a computer-controlled cloud of elevated CO2(10). These ecosystem-scale experiments document a number of artifacts associated with earlier CO2 exposure techniques. For example, many examples of down-regulation of photosynthesis can be traced to limited rooting volume in pot experiments. On the other hand, the largest growth responses to elevated CO2occur in isolated plants, where an initial increase in growth produces a positive feedback through an increase in canopy size (20). Over many experiments, plant growth responses to approximately doubled CO2 range from small decreases to increases greater than 100%, with mean increases around 50% for C3 crops and 30% for woody plants (22). The potential for this extra growth to drive carbon storage is still incompletely known. In some experiments, increases in respiration parallel increases in photosynthesis, minimizing the potential for storage (13). In others, carbon accumulates in biomass or soils (19). But even this is not a true index of long-term sink potential. Initial storage is almost unavoidable, as photosynthesis spurts ahead of respiration. The initial carbon storage in an experiment with an instantaneous CO2 doubling is very difficult to relate to that ecosystem's potential for long-term storage (17). In fact, some of the negative feedbacks on storage, such as ecosystem-scale nutrient limitation, may develop only after several years of increased growth under elevated CO2(18). Of the global-scale models in wide use today, some postulate strong feedbacks from nutrient limitation, whereas others ignore the possibility completely (9). Long-term experiments are marching toward the evidence to reject one hypothesis or the other. Yet other long-term regulators of nutrient availability, from nitrogen fixation to exposure of soils with available phosphorus, are still very difficult to simulate with experiments or models. Progress on this front will require fundamental advances in understanding the factors that control whole-ecosystem nutrient budgets. These include retranslocation among plant tissues, the efficiency of foraging for nutrients, nutrient losses, controls nitrogen fixation, and the potential for limitation by nutrients other than nitrogen. Experimental studies are providing increasingly refined estimates of net primary production or plant growth responses to CO2 fertilization, and models are translating these into carbon sinks with increased sophistication. From the global end of the spatial scale, atmospheric methods are specifying the magnitude and location of the sinks. These methods work backwards from the spatial distribution of CO2 concentrations in the atmosphere to infer spatial patterns of sources and sinks. This is essentially equivalent to using the entire atmosphere as a bunch of gas exchange chambers, with observed or modeled winds constituting the flows between them. Though this method is sensitive to a number of kinds of errors, the rich spatial and temporal patterns in atmospheric CO2 suggest its potential (Fig.1). Several atmospheric studies over the last decade indicate the existence of a large sink on land, especially in the middle to boreal latitudes of the northern Hemisphere (27). Similar studies augmented with information about13C in CO2 and O2, useful as probes to separate land from ocean sinks, confirm that much of the sink is on land and suggest that it has increased in the last decade (3). Eddy flux measurements, which quantify ecosystem CO2 fluxes on a scale of 104 to 106m2, also confirm the existence of carbon sinks in an increasing number of temperate, boreal, and tropical forests (e.g. 29), though measurements in a few sites do not necessarily provide a regional perspective. Fig. 1. Open in new tabDownload slide Temporal and spatial patterns in the concentration of atmospheric CO2 showing the secular trend due to human emissions, the large seasonal fluctuations due to the terrestrial biosphere, and the spatial concentration differences that provide a basis for flux calculations with model inversions. Redrawn from reference 6, with updates from http://www.cmdl.noaa.gov/. Fig. 1. Open in new tabDownload slide Temporal and spatial patterns in the concentration of atmospheric CO2 showing the secular trend due to human emissions, the large seasonal fluctuations due to the terrestrial biosphere, and the spatial concentration differences that provide a basis for flux calculations with model inversions. Redrawn from reference 6, with updates from http://www.cmdl.noaa.gov/. Atmospheric CO2 is clearly rising. And there is definitely a CO2 sink on land, probably averaging 2 to 3 Pg C year−1 during the 1990s, and as large as 4 Pg C year−1 in some years (3). Is CO2 fertilization causing none, some, or all of the sink? This question can be approached from two perspectives. One is to simulate the CO2 fertilization directly and to compare the estimate with the land sink. Using this approach, Kohlmaier and colleagues (16) estimated CO2fertilization to be about the magnitude required to explain the terrestrial sink. On the other hand, several more recent studies have concluded that the net primary production responses needed to generate the historical sink are too large to be consistent with CO2 as the sole driver (28). A second approach for estimating the role of CO2fertilization is to estimate the likely sinks due to other mechanisms and ask what is left for CO2. Increasing evidence points to carbon sinks from a number of other mechanisms. In boreal and temperate latitudes, the regrowth of previously harvested forests appears to be important (15). Forest thickening due to fire suppression also appears to be a contributor. Agricultural practices that increase organic matter inputs to soil can also contribute to a sink (12). Because each of these processes is poorly known, it is not yet possible to employ them in precise estimates of the role of elevated CO2 in the terrestrial sink. It looks likely, however, that CO2 fertilization accounts for one-half of the sink or less. Even if CO2 fertilization is not the dominant driver of the terrestrial sink, it is still a substantial factor in the global carbon cycle. Future changes in CO2fertilization will significantly modulate the rate at which CO2 increases in the atmosphere. Understanding that modulation and how it will change in coming decades will be a major contribution to a sustainable future. LITERATURE CITED 1 Arrhenius S Lond Edinb Dublin Philos Mag J Sci 41 1896 237 276 Crossref Search ADS 2 Bacastow R Keeling CD Carbon and the Biosphere. U.S. Woodwell GM Pecan EV 1973 86 135 Department of Commerce Springfield, VA 3 Battle M Bender ML Tans PP White JWC Ellis J Conway T Francey RJ Science 287 2000 2467 2470 Crossref Search ADS PubMed 4 Bolin B Eriksson E The Atmosphere and the Sea in Motion, Rossby Memorial Volume. 1959 130 143 Rockefeller Institute Press New York 5 Callendar GS Tellus 10 1958 243 248 Crossref Search ADS 6 Conway TJ Tans PP Waterman LS Thoning KW Kitzis DR Masarie KA Zhang N J Geophys Res 99 1994 22831 22855 Crossref Search ADS 7 Farquhar GD von Caemmerer S Berry JA Planta 149 1980 78 90 Crossref Search ADS PubMed 8 Field CB Behrenfeld MJ Randerson JT Falkowski P Science 281 1998 237 240 Crossref Search ADS PubMed 9 Goudriaan J Shugart HH Bugmann H Cramer W Bondeau A Gardner RH Hunt LA Lauenroth WK Landsberg JJ Linder S Noble IR Parton WJ Pitelka LF Stafford Smith M Sutherst RW Valentin C Woodward FI The Terrestrial Biosphere and Global Change. Walker B Steffen W Canadell J Ingram J 1999 106 140 Cambridge University Press Cambridge, UK 10 Hendry GR Kimball BA Agric Forest Meteorol 70 1994 3 14 Crossref Search ADS 11 Houghton RA Tellus 51B 1999 298 313 12 Houghton RA Hackler JL Lawrence KT Science 285 1999 574 578 Crossref Search ADS PubMed 13 Hungate BA Holland EA Jackson RB Chapin FS III Mooney HA Field CB Nature 388 1997 576 579 Crossref Search ADS 14 Institute for Biospheric Research (1991) The Greening of Planet Earth. Western Fuels Association, video 15 Kauppi PE Mielikäinen K Kuusela K Science 256 1992 70 74 Crossref Search ADS PubMed 16 Kohlmaier GH Siré E Janecec A Keeling CD Piper SC Revelle R Tellus 41B 1989 487 510 Crossref Search ADS 17 Luo Y Reynolds JF Ecology 80 1999 1568 1583 Crossref Search ADS 18 McMurtrie RE Comins HN Global Change Biol 2 1996 49 59 Crossref Search ADS 19 Mooney HA Canadell J Chapin FS III Ehleringer J Körner C McMurtie R Parton WJ Pitelka L Schulze E-D The Terrestrial Biosphere and Global Change: Implications for Natural and Managed Ecosystems. Walker BH Steffen WL Canadell J Ingram JSI 1999 141 189 Cambridge University Press Cambridge, UK 20 Norby RJ Nature 381 1996 564 565 Crossref Search ADS 21 Pales JC Keeling CD J Geophys Res 70 1965 6053 6076 Crossref Search ADS 22 Poorter H Roumet C Campbell BD Carbon Dioxide, Populations, and Communities. Körner C Bazzaz FA 1996 375 412 Academic Press San Diego 23 Rabinowitch E Photosynthesis and Related Processes II, Part 1 1951 Interscience Publishers New York 24 Raschi A, Miglietta F, Tognetti R, van Gardigen PR, eds, Plant Responses to Elevated CO2: Evidence from Natural Springs. Cambridge University Press, Cambridge, UK 25 Revelle R Suess HE Tellus 9 1957 18 27 Crossref Search ADS 26 Schimel D Enting IG Heimann M Wigley TML Raynaud D Alves D Siegenthaler U Houghton JT, et al., eds, Climate Change 1994: Radiative Forcing of Climate Change and an Evaluation of the IPCC IS92 Emission Scenarios. 1995 33 71 Cambridge University Press Cambridge, UK 27 Tans PP Fung IY Takahashi T Science 247 1990 1431 1438 Crossref Search ADS PubMed 28 Thompson MV Randerson JT Malmström CM Field CB Global Biogeochem Cycles 10 1996 711 726 Crossref Search ADS 29 Wofsy SC Goulden ML Munger JW Fan S-M Bakwin PS Daube BC Bassow SL Bazzaz FA Science 260 1993 1314 1317 Crossref Search ADS PubMed 30 Woodwell GM Hobbie JE Houghton RA Melillo JM Moore B Science 222 1983 1081 1086 Crossref Search ADS PubMed Author notes * E-mail [email protected]; fax 650–325–3748. Copyright © 2001 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Ort, Donald R.

doi: 10.1104/pp.125.1.29pmid: 11154289

A great deal of importance has happened in research investigating photosynthetic response to environmental stress in the 25 years since the last anniversary issue of Plant Physiology. However, from my perspective, the importance of one set of discoveries stands out from the others for its far reaching influence on how we think about the photosynthetic response to a wide range on environmentally imposed limitations. As little as 15 years ago it was generally held that the success of plants in their environment was dictated by strategies that maximized the rate of photosynthesis. Further, maximum photosynthetic capacity was thought to be largely a static characteristic of individual leaves that was established during development. This view has now given way to the recognition that the regulation of photosynthesis in response to the environment is highly dynamic and dominated by a photoprotective process, the non-photosynthetic thermal dissipation of absorbed light (4, 10, 14), which was entirely unknown at the time of Plant Physiology's 50th Anniversary. This brief overview describes what is currently understood about this centrally important photoprotective process and highlights areas of current inquiry that may presage a detailed mechanistic understanding in the near future. MOST PLANTS ENCOUNTER EXCESS LIGHT CONDITIONS ON A DAILY BASIS Most days plants encounter light intensities that exceed their photosynthetic capacity. Exactly what constitutes excess light for a leaf depends on its instantaneous environmental conditions and can vary over an exceedingly wide range of irradiance levels. For example, irrigated field-grown sunflower is typical of C3 crop plants, exhibiting maximum photosynthetic capacity during mid-morning with photosynthesis declining throughout the afternoon as stomatal conductance declines in response to declining leaf water potentials (21). Thus even under conditions which may not generally be considered stressful, stomatal conductance can substantially restrict CO2 entry into leaves, rendering even moderate irradiances in the top of a crop canopy in excess of photosynthetic capacity. A DYNAMIC PROCESS ENABLING LEAVES TO REGULATE THERMAL DISSIPATION OF EXCESS ABSORBED LIGHT IS AT THE CENTER OF PLANT PHOTOPROTECTION When environmental conditions prevent the maintenance of a high capacity for photosynthetic and photorespiratory carbon metabolism to utilize absorbed light, the likelihood for the photosynthetic generation of biologically damaging molecules including reduced and excited species of oxygen, peroxides, radicals, and triplet state excited pigments increases dramatically (1). Although some plants can reduce the amount of incident light that is absorbed through strategic leaf and chloroplast movements, rapid reduction in light absorption appears to play only a minor role in the challenge of coping with excess light. The development of the techniques and biophysical interpretation of pulse modulated fluorescence in the mid-1980s by Bradbury and Baker (2) bolstered by important additions and refinements by many others (e.g. 7, 8, 20) provided the basis for a new understanding about the dynamic trade-off between photosynthetic efficiency and photoprotection (Fig.1). A wide range of studies on many different species revealed that frequently over one-half of the light absorbed by photosystem II (PSII) chlorophylls in healthy, fully functional leaves can be redirected by a process that operates within the antenna ensemble of PSII, which harmlessly discharges excess photon flux energy as heat (3, 4, 10, 14). This thermal dissipation process is measured and often called non-photochemical quenching, referring to the fact that the thermal dissipation of chlorophyll excited states competes with fluorescence emission as well as with photochemistry (i.e. photosynthesis). Fig. 1. Open in new tabDownload slide Model depicting the conversion of the thylakoid membrane at excess light from the high efficiency state (top) to the photoprotected state (bottom). The excess light condition is sensed by a very large ΔpH that initiates the non-photosynthetic thermal dissipation of absorbed light as described in the text. The major elements involved in the conversion between the high efficiency and photoprotected states are highlighted by the transition from blue to red. PSII, Photosystem II complex; PSI, photosystem I complex; b6f, cytochrome b6f complex; P680, reaction center chlorophyll of PSII; QA and QB, quinone acceptors of PSII; PQ and PQH2, plastoquinone and reduced plastoquinone; Cyt, cytochrome; FeS, Rieske iron sulfur protein; PC, plastocyanin; P700 and P700 +, reduced and oxidized forms of the reaction center chlorophyll of PSI; Ao, primary acceptor of PSI; FeS, bound iron sulfur acceptors of PSI; Fd, soluble ferredoxin; Chl*, excited chlorophyll molecule; Z, zeaxanthin; V, violaxanthin; CP22, minor PSII pigment protein (also called PsbS) required for regulated thermal energy dissipation and believed to instigate protonation-dependent reorganization in LHCII. Fig. 1. Open in new tabDownload slide Model depicting the conversion of the thylakoid membrane at excess light from the high efficiency state (top) to the photoprotected state (bottom). The excess light condition is sensed by a very large ΔpH that initiates the non-photosynthetic thermal dissipation of absorbed light as described in the text. The major elements involved in the conversion between the high efficiency and photoprotected states are highlighted by the transition from blue to red. PSII, Photosystem II complex; PSI, photosystem I complex; b6f, cytochrome b6f complex; P680, reaction center chlorophyll of PSII; QA and QB, quinone acceptors of PSII; PQ and PQH2, plastoquinone and reduced plastoquinone; Cyt, cytochrome; FeS, Rieske iron sulfur protein; PC, plastocyanin; P700 and P700 +, reduced and oxidized forms of the reaction center chlorophyll of PSI; Ao, primary acceptor of PSI; FeS, bound iron sulfur acceptors of PSI; Fd, soluble ferredoxin; Chl*, excited chlorophyll molecule; Z, zeaxanthin; V, violaxanthin; CP22, minor PSII pigment protein (also called PsbS) required for regulated thermal energy dissipation and believed to instigate protonation-dependent reorganization in LHCII. ΔpH AND THE INTERCONVERSION OF XANTHOPHYLLS PLAY A CRITICAL ROLE IN REGULATING THERMAL ENERGY DISSIPATION IN PSII Following the initial observations of Krause and Behrend (11) there is now a great deal of compelling evidence that excess light conditions are sensed or signaled by a large ΔpH (i.e. low-lumen pH), which forms when ATP utilization is restricted by CO2 availability or by stress-induced dysfunction in the enzymology of carbon reduction (4, 10, 14). It is not always recognized, even by everyone working in this area of research, that ΔpH formation is exceedingly non-linear with light intensity (19). A ΔpH sufficient to drive net ATP synthesis (approximately 2.5 units) and thus photosynthetic CO2 reduction is formed at 0.1% of full sunlight (15) and increases only on the order of 25% when the irradiance level is increased 1,000-fold. Thus only when the lumen pH is driven to very low values does photoprotective thermal energy dissipation within PSII become engaged. Building on the ground breaking work of Yamamoto and colleagues (22), and Demmig-Adams, Björkman, and their coworkers (5), there is now a large body of experimental data supporting the notion that the low lumen pH activates violaxanthin de-epoxidase (4), which in turn converts violaxanthin, a xanthophyll pigment bound to the PSII light harvesting complex (LHCII), to zeaxanthin (and antheraxanthin). Thus, as depicted in Figure 1, zeaxanthin accumulates at the expense of violaxanthin under excess light initiating thermal energy dissipation. Well-characterized mutants of Arabidopsis lacking functional violaxanthin de-epoxidase are unable to engage photoprotective energy dissipation in PSII, pointing to an obligate role for zeaxanthin in this process in higher plants (14). A second critical role of low lumen pH is the instigation of protonation-induced conformational change in one or more of the so-called minor LHC proteins of PSII. Although indirect evidence for several potential candidate LHCs has been reported, a recent breakthrough was made by Niyogi and colleagues showing that a deletion mutation in the gene encoding the minor PSII LHC PsbS (also called CP22) prevents thermal energy dissipation in PSII (12). Moreover, the mutation in PsbS also prevents an accompanying ΔpH- and zeaxanthin-dependent light scattering change that is thought to reflect a protonation-induced protein conformational change within PSII. The fact that this mutation in PsbS does not interfere with efficient light harvesting, water oxidation, or xanthophyll cycling supports a dedicated role of this chlorophyll- and xanthophyll-binding protein in photoprotective energy dissipation rather than photosynthetic light harvesting. THE BIOPHYSICAL MECHANISM OF ZEAXANTHIN/ΔpH-DEPENDENT ENERGY DISSIPATION WITHIN PSII IS UNRESOLVED As already mentioned, there is compelling evidence that the presence of zeaxanthin within the PSII LHC ensemble and the generation of a large ΔpH across the thylakoid membrane (i.e. very low lumen pH) are simultaneously required to engage photoprotective thermal energy dissipation. One attractive proposal for the underlying biophysical basis for the reversible conversion between the high efficiency and photoprotective states centers on a lowered calculated energy of the xanthophyll excited state accompanying the conversion of violaxanthin to zeaxanthin (23). Thus the formation of zeaxanthin was envisioned to introduce a new, energetically favorable pathway that dramatically promoted thermal dissipation of excited chlorophyll molecules in the LHCII ensemble. However, very recently two different experimental procedures were devised to directly measure the energy levels of the previously inaccessible S1 states of highly conjugated carotenoids (6, 18). These studies convincingly illustrated that the energy gap between the S1 states of violaxanthin and zeaxanthin is too small to account for their differential quenching capabilities. A second proposal for the quenching mechanism arose from evidence that ΔpH-dependent accumulation of zeaxanthin results in the reversible oligomerization of LHCII (9). Aggregation was suggested to cause changes in orientation among the pigments bound to LHCII proteins, allowing pigment interaction leading to concentration quenching of chlorophyll excited states (i.e. increase in the thermal dissipation of absorbed light energy). In this proposal the xanthophyll cycle has an indirect role in thermal dissipation by mediating a critical conformational change within the PSII antenna. Although the energy gap between the S1 states of violaxanthin and zeaxanthin is now known to be only about one-half as large as previously thought, it is nevertheless true that direct quenching could contribute and thus may partner with changes in LHCII aggregation during the thermal dissipation process. Most importantly, this is a highly active area of research currently being explored from several different directions that point to exciting and perhaps surprising discoveries on the horizon. WHAT HAPPENS IN PSI WHEN A LARGE PROPORTION OF THE LIGHT ENERGY ABSORBED BY PSII IS DISSIPATED AS HEAT? Rarely discussed in the primary literature or in reviews on photoprotection in plants is the participation of PSI in thermal dissipation of excess absorbed light energy. At low irradiance levels when photosynthetic membranes are in the high-efficiency state (Fig.1), leaves demonstrate an efficiency (i.e. quantum yield) for CO2 reduction that is close to the theoretical maximum (13). This exceptionally high efficiency is possible only because the amount of light absorbed by the antenna serving the two photosystems is closely balanced. Thus it is inescapable that at high irradiance levels when PSII photoprotective thermal dissipation is engaged, PSI will be absorbing many more photons than it is receiving electrons from PSII. Cyclic electron flow around PSI may utilize some of this excess, but the capacity of this pathway is modest in comparison to the excess photon load when zeaxanthin/ΔpH-dependent energy dissipation is fully engaged in PSII. Energy dissipation in PSI is much less studied than for PSII, but it is a reasonable notion that the photochemical yield in PSI is indirectly regulated by the photochemical yield in PSII. The central basis for this belief is that the oxidized primary donor of PSI, P700 +, is a strong quencher of excited states in the PSI antenna and can accumulate when PSI photochemistry outpaces PSII. Although the photophysical mechanism of this quenching of chlorophyll excited states remains a matter of debate, it does provide a reasonable means to balance PSI light energy utilization via zeaxanthin/ΔpH-dependent energy dissipation in PS II. Thus, when PSI absorbs more light quanta than it receives electrons from PSII, P700 becomes oxidized and stays oxidized until an electron comes along from PSII. In this way, as depicted in Figure 1, thermal energy dissipation in PSI by P700 + quenching tracks the ΔpH-dependent regulation of PSII thermal energy dissipation (17). LESSONS AND PROSPECTS Although photodamage has been documented in crops grown outside of their ancestral geographic range, the vast majority of plants in native habitats and even most crops under cultivation deal successfully with excess light avoiding photodamage even under daunting environmental challenges. Photoprotection is a complex process that includes an array of alternative electron acceptors to utilize excess absorbed light when CO2 is limiting, intricate pathways to detoxify photosynthetically produced reactive molecules, as well as a variety of repair processes to prevent the accumulation of photodamage. However, the regulated thermal dissipation of absorbed light is without question the keystone of photoprotection. There is a great deal of importance that is not yet understood about the mechanism and regulation of thermal dissipation, but the recent emergence of molecular genetic approaches portend rapid and exciting progress (14). Emerging directly from these recent discoveries on regulated thermal dissipation is a current view of the regulation of leaf photosynthesis as a balancing act in which photoprotection is traded for photosynthetic efficiency (16). It appears that evolution has refined the photosynthetic apparatus with an emphasis on high efficiency at limiting light with regulatory features to ensure that high intensities can be endured without the accumulation of photodamage. Although this view is admittedly an oversimplification, it is almost certainly true that when irradiances are high (e.g. mid-day at the top of the canopy) factors such as maintenance of water status take physiological precedence over maximizing photosynthesis. Although the trade-off between efficiency and photoprotection is clear, from an agricultural prospective it is less apparent how well the dynamic range of the trade-off is suited for agricultural environments and productivity goals. It seems possible, even likely, that forfeiture of photosynthetic efficiency may, under some circumstances, exceed that required to prevent photodamage thus reducing photosynthetic productivity more than necessary. Genetic variation in the ability of crop plant varieties to maintain photosynthetic efficiency at somewhat higher irradiances (i.e. higher ΔpH values) may prove to be an important factor in the search for improved photosynthetic productivity of crops. LITERATURE CITED 1 Asada K Advances in Photosynthesis: Photosynthesis and the Environment Baker NR 5 1996 123 150 Kluwer Academic Publishers Dordrecht, The Netherlands 2 Bradbury M Baker NR Biochim Biophys Acta 765 1984 275 281 Crossref Search ADS 3 Demmig B Winter K Krüger A Czygan F-C Plant Physiol 84 1987 218 224 Crossref Search ADS PubMed 4 Demmig-Adams B Adams WW Annu Rev Plant Physiol Plant Mol Biol 43 1992 599 626 Crossref Search ADS 5 Demmig-Adams B Adams WW Heber U Neimanis S Winter K Krüger A Czygan F-C Bilger W Björkman O Physiol Plant 92 1990 293 301 Crossref Search ADS 6 Frank HA Bautista JA Josue JS Young AJ Biochemistry 39 2000 2831 2837 Crossref Search ADS PubMed 7 Genty B Briantais J-M Baker NR Biochim Biophys Acta 990 1989 87 92 Crossref Search ADS 8 Horton P Hague A Biochim Biophys Acta 932 1988 107 115 Crossref Search ADS 9 Horton P Ruban AV Rees D Pascal AA Noctor GD Young AJ FEBS Lett 200 1991 298 302 10 Horton P Ruban AV Walters RG Annu Rev Plant Physiol Plant Mol Biol 47 1996 655 684 Crossref Search ADS PubMed 11 Krause GH Behrend U FEBS Lett 200 1986 298 302 Crossref Search ADS 12 Li X-P Björkman O Shih C Grossman AR Rosenquist M Jansson S Niyogi KK Nature 403 2000 391 395 Crossref Search ADS PubMed 13 Long SP Postl WF Bolhár-Nordenkampf HR Planta 189 1993 226 234 Crossref Search ADS 14 Niyogi KK Annu Rev Plant Physiol Plant Mol Biol 50 1999 333 359 Crossref Search ADS PubMed 15 Ort DR Oxborough K Annu Rev Plant Physiol Plant Mol Biol 43 1992 269 291 Crossref Search ADS 16 Osmond CB Photoinhibition of Photosynthesis from Molecular Mechanisms to the Field. Baker NR Bowyer JR 1994 1 24 Bios Scientific Publishers Oxford 17 Owens TG Advances in Photosynthesis: Photosynthesis and the Environment Baker NR 5 1996 1 23 Kluwer Academic Publishers Dordrecht, The Netherlands 18 Polı́vka T Herek JL Zigmantas Åkerlund H-E Proc Natl Acad Sci USA 96 1999 4914 4917 Crossref Search ADS PubMed 19 Portis AR McCarty RE J Biol Chem 249 1974 6250 6254 Crossref Search ADS PubMed 20 Schreiber U Schliwa U Bilger W Photosynth Res 10 1986 51 62 Crossref Search ADS PubMed 21 Wise WW Frederick JR Alm DM Kramer DM Hesketh JD Crofts AR Ort DR Plant Cell Environ 13 1990 923 931 Crossref Search ADS 22 Yamamoto HY Pure Appl Chem 51 1979 639 648 Crossref Search ADS 23 Young AJ Frank HA J Photochem Photobiol 36 1996 3 15 Crossref Search ADS Author notes * E-mail [email protected]; fax 217–244–0656. Copyright © 2001 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Hillier, Warwick; Babcock, Gerald T.

doi: 10.1104/pp.125.1.33pmid: 11154290

THE CONCEPT AND PHYSICAL REALITY OF REACTION CENTERS (RCs) The capture of solar radiation and the conversion of its free energy into chemical energy involves a sequence of reactions that occur within a physical structure called the photosynthetic RC. Following the initial capture of a photon by antenna pigments, the photon is transferred to the RC pigments, where it gives rise to a separation and stabilization of charge across the photosynthetic membrane. Figure 1 depicts this process and illustrates the time scales typically involved. One feature of the photochemistry is that all photosynthetic RCs undergo charge separation with a quantum yield approaching unity, which makes them marvelous molecular machines. Fig. 1. Open in new tabDownload slide Primary processes in the photosynthetic RC. Here, P represents the charge-separating (bacterio) chlorophyll pigments (the “primary electron donor”) and A represents the first “stable” acceptor. Energy transfer from the antenna pigments leads to photoexcitation of P on the fs-ps time scale (left). Charge separation produces oxidized P+ and A– on the ps-ns scale (center). The recombination of P+A– to produce PA, heat, and potentially damaging chemical species is efficiently prevented by further forward electron transfer that is now proton coupled. These more complex chemical processes ultimately produce stable photosynthetic products and occur, initially, on the ns and μs time scales (right). Fig. 1. Open in new tabDownload slide Primary processes in the photosynthetic RC. Here, P represents the charge-separating (bacterio) chlorophyll pigments (the “primary electron donor”) and A represents the first “stable” acceptor. Energy transfer from the antenna pigments leads to photoexcitation of P on the fs-ps time scale (left). Charge separation produces oxidized P+ and A– on the ps-ns scale (center). The recombination of P+A– to produce PA, heat, and potentially damaging chemical species is efficiently prevented by further forward electron transfer that is now proton coupled. These more complex chemical processes ultimately produce stable photosynthetic products and occur, initially, on the ns and μs time scales (right). The first notions of the operation of a photosynthetic RC originated with the photosynthetic unit experiments of Emerson and Arnold (9), which demonstrated that approximately 2,500 chlorophyll molecules were involved in the release of just one molecule of O2. Thus, a photosynthetic unit contains numerous pigments but the photochemically active chromophores are present in much lower concentration. This pioneering concept led to the distinction of two types of pigments: the light-harvesting, but photochemically inactive, antenna chromophores; and the photochemically active RC pigments. The antenna pigments physiologically increase the absorption cross section of the RC dramatically. Moreover, they ensure that the potentially reactive intermediates containing unpaired electron spins (e.g. semiquinones) generated by single photon photochemistry are efficiently converted by a second photochemical event to products (e.g. hydroquinones) that contain only paired spins. For efficient energy transfer between the antenna and the RC, the RC absorbs at longer wavelengths, effectively forming a trap for excitation energy. Despite these conceptual advances, more than 35 years passed before the first physical isolation of a pigment protein RC complex was reported (17). Since that time, many other RCs have been isolated and characterized biophysically and biochemically. STRUCTURAL AND OPERATIONAL INSIGHTS Insight into the molecular organization of the RC has been derived, initially, from spectroscopic studies and, subsequently, from the development and analysis of high-resolution crystal structures of several photosynthetic organisms. The first RC structurally resolved (3 Å) was of the purple bacterial RC from Rhodopseudomonas viridis (7), for which the 1988 Nobel Prize was awarded. This was soon followed by the elucidation of several other purple bacterial structures. We are now witnessing the appearance of detailed RC structures from oxygenic systems, most notably the 4Å structure of photosystem I (PSI; 13). Good progress is also being made toward achieving two- and three-dimensional structures of photosystem II (PSII) crystals. It is surprising that the structures of all of the different RCs show a dimeric core with a pseudo-C2 axis of symmetry. This feature is illustrated in Figure 2 in the example of a purple bacterial RC. The holoprotein is shown on the left. The charge-separating RC pigments contained within the structure (Fig. 2, right) are aligned along the C2 symmetry axis with the two photochemically active (bacterio) chlorophyll pigments positioned in close proximity. Exciton coupling between these two pigments provides a red shift in the optical spectrum that contributes substantially to forming the low-energy trap discussed above. The conversion of photons to chemical potential involves photoexcitation and initial charge separation to produce an oxidized (bacterio) chlorophyll and reduction of one of the other chlorin pigments in the RC. From this chlorin, the electron migrates to reduce a quinone in less than a nanosecond (Fig. 2). It is interesting that the strength of the dimer exciton coupling has changed substantially during the course of oxygenic RC evolution from photosynthetic bacteria. The bacteria usually have strong couplings, approximately 2,000 cm−1, whereas the plant and algal RCs have a much weaker coupling, typically approximately 300 cm−1 (8). The weaker coupling in the oxygenic RCs increases the thermodynamic efficiency of photon capture so that a significant improvement in useful free energy capture from the photon is realized. Subsequent proton-coupled electron transfer steps (Fig. 1) stabilize the charge separation effectively and ensure the near-unity quantum efficiency of photosynthesis. Fig. 2. Open in new tabDownload slide Structure of the purple bacterial RC (Rhodobacter sphaeroides from MH. Stowell et al; Protein Data Band file no. 1AIJ). The heterodimeric RC (left) is comprised of a C2-symmetrical heterodimer of the L and M polypeptides shown in orange and blue respectively. A third subunit, H, is also shown in green. The pseudo-C2symmetric cofactor arrangement and the active pathway of electron transfer are indicated on the right. Charge separation from the RC Chl dimer (P) to the BChl monomer to B-Phe occurs in approximately 3 ps down the active L branch. This is followed by charge stabilization with electron transfer to the quinones. The phytyl and isoprenoid chain have been removed for visualization. Fig. 2. Open in new tabDownload slide Structure of the purple bacterial RC (Rhodobacter sphaeroides from MH. Stowell et al; Protein Data Band file no. 1AIJ). The heterodimeric RC (left) is comprised of a C2-symmetrical heterodimer of the L and M polypeptides shown in orange and blue respectively. A third subunit, H, is also shown in green. The pseudo-C2symmetric cofactor arrangement and the active pathway of electron transfer are indicated on the right. Charge separation from the RC Chl dimer (P) to the BChl monomer to B-Phe occurs in approximately 3 ps down the active L branch. This is followed by charge stabilization with electron transfer to the quinones. The phytyl and isoprenoid chain have been removed for visualization. A remarkable aspect of the RC structures is the occurrence of two almost identical electron acceptor pathways arranged along the C2 axis relative to the primary charge-separating dimer (bacterio) chlorophyll (Fig. 2). This finding posed a key question: Does electron transfer involve both branches? In the purple bacterial RC, only one branch is active although the inactive branch can be forced into operation with modification of amino acid side chains on the active branch (1). The strong asymmetry imposed on primary charge separation photo-chemistry in the purple bacterial RC results from two homologous polypeptides that function as a heterodimer. A heterodimer is also involved in the core of the RCs of PSI and PSII. However, some RCs, such as heliobacteria (2) and green sulfur bacteria (6, 18), contain two identical homodimeric polypeptides, and electron transfer is potentially bifurcated. Genetic sequence information has greatly improved the understanding of the origin of the RC proteins. From the sequence analysis, it became clear that the purple bacteria RC is remarkably similar to that of PSII, and PSI was also discovered to have similarity with that of the green sulfur bacteria (6, 10). Further elaboration with 16S-rRNA phylogenic trees (5) and broader homology comparison (14) revealed a close interrelationship among many RCs. Recent structural comparisons between PSI and PSII, for example, show a distinct structural homology, which suggests that even these two RCs likely share a common ancestor (13). TYPES OF RCs The general details of RC structure and function described above persist among photosynthetic organisms, but differences in detail have become apparent. Today, we recognize six different classes of photosynthetic RCs. The principal variations lie in the RC pigments (chlorophyll versus bacteriochloropyll), the size and nature of the antenna pigment array, the associated longest wavelength maximum and strength of the pigment exciton coupling, and the thermodynamic coupling of the primary donor chlorophyll dimer (P) to its acceptor system (i.e. its midpoint reduction potential). Figure3 presents a summary of the various RCs, cofactors, and electron transport chains. The six classes of RC divide into two forms: the type I and type II RCs (10, 15). The type I RCs comprise PSI, the gram-positive heliobacteria, and the green sulfur bacteria, all of which share iron-sulfur clusters as electron acceptors. The type II RCs from PSII, purple bacteria and the green filamentous bacteria, share quinone acceptors that serve as two-electron reductants. Two of these RCs, from heliobacteria and the green filamentous bacteria, have only been recognized quite recently and there may be others that await discovery—the field continues to progress rapidly. Fig. 3. Open in new tabDownload slide Electron-transfer pathways for the two different types and six classes of RCs shown according to the midpoint potentials of key redox components. In Type I RCs (left), iron-sulfur clusters are used as the electron acceptors. Type I is subdivided into three classes: PSI, green sulfur bacteria, and heliobacteria (see text). In Type II RCs, quinones are used as the first “stable” electron acceptors (left). Type II is also subdivided into three classes: PSII, purple bacteria, and green filamentous bacteria. Intermediates in the scheme have the following designations: the RC primary donor, P; transient initial (bacterio) pheophytin acceptor, (B) Ph; “stable” quinone acceptors, QA and QB; transient initial chlorophyll (A0) and quinone (A1) acceptors; “stable” iron-sulfur cluster acceptors, FX, FA, and FB; and final NADP acceptor (NADP). The electron donors are a Tyr residue, TyrZ, and a cluster of 4 manganese ions for PSII, a plastocyanin molecule (PC) for PSI, and cytochrome c for the bacterial RCs. The intermediate electron transfer complexes, cytochromebc1 and b6f, are boxed. Fig. 3. Open in new tabDownload slide Electron-transfer pathways for the two different types and six classes of RCs shown according to the midpoint potentials of key redox components. In Type I RCs (left), iron-sulfur clusters are used as the electron acceptors. Type I is subdivided into three classes: PSI, green sulfur bacteria, and heliobacteria (see text). In Type II RCs, quinones are used as the first “stable” electron acceptors (left). Type II is also subdivided into three classes: PSII, purple bacteria, and green filamentous bacteria. Intermediates in the scheme have the following designations: the RC primary donor, P; transient initial (bacterio) pheophytin acceptor, (B) Ph; “stable” quinone acceptors, QA and QB; transient initial chlorophyll (A0) and quinone (A1) acceptors; “stable” iron-sulfur cluster acceptors, FX, FA, and FB; and final NADP acceptor (NADP). The electron donors are a Tyr residue, TyrZ, and a cluster of 4 manganese ions for PSII, a plastocyanin molecule (PC) for PSI, and cytochrome c for the bacterial RCs. The intermediate electron transfer complexes, cytochromebc1 and b6f, are boxed. Further differentiation in photosynthetic organisms is found in the structure and arrangement of the antenna pigments associated with each RC. The RC from heliobacteria features a very simple organization with a core containing approximately 40 chlorophyll g and no additional auxiliary peripheral antenna proteins (2). Building on this organizational theme are RCs from PSI and green sulfur bacteria, which contain large numbers (∼100) of pigments attached directly to the polypeptides that bind the RC components (13), as well as an extensive external antenna array with which the RCs communicate in a controlled way. At the other extreme are the RCs from purple bacteria and PSII, which contain only six to eight pigments arranged along the C2 symmetry axis and are fundamental to the charge separation process. These RCs rely on a substantial antenna system as conduits of excitation energy. This antenna system is bound to polypeptides distinct from the RC polypeptides. OXYGENIC PHOTOSYNTHESIS The incorporation of two RCs in series during the evolution of plant and algal photosynthesis represents a brilliant strategy for using an inexhaustible supply of water in the unlikely role of reductant without sacrificing the ability to use photons in the red (λ > 600 nm) region of the spectrum. For a single RC oxidizing water and reducing NADP, photons of about 500 nm or shorter would be required to span the entire redox-potential range between the two products (O2 and NADPH) with sufficient irreversibility to ensure a high quantum yield. Using two photoreactions in series, this energetic requirement is relaxed, and photons in the longer wavelength region (680 nm [PSII] and 700 nm [PSI]) become useful. The overall quantum requirement for water oxidation increases from four photons to eight: PSII:2H2O→4hv  O2+4 H·;PSI:2NADP→4hv  2NADPH but lower-energy photons, up to 700 nm, are able to drive the process. Coordination of two photosystems, however, requires significantly greater sophistication to balance the incoming excitation energy to the RCs associated with the two photosystems. To meet this requirement, PSI and PSII demonstrate significant differences in pigment composition and placement of the antenna proteins as a function of the light quality. In plants, this situation is highlighted by the lateral heterogeneity between the two RCs, which results in the physical separation of the PSII RC to the grana-appressed region and the PSI RC to the nonappressed region of the thylakoid (3). This division of RCs forms the basis of the biochemical isolation procedure that has been the cornerstone of much of the biochemical and biophysical work with PSII (4). The charge-stabilizing reactions that occur following primary charge separation in the RC are coming into sharper focus. A key realization has been that these reactions are often proton coupled; that is, the motion of the electron must be coupled in some fashion to the motion of a proton (Fig. 1). This marks the conversion from pure photon and electron chemistry to chemistry that involves nuclei as well. Okamura and his coworkers have produced seminal results on electron/proton coupling on the reducing side in the bacterial RC (16). Results that incorporate these and other thoughts concerning proton/electron coupling in PSII are emerging as the underlying mechanism that drives water oxidation to produce O2. To oxidize water, potentials upwards of 1 V must be generated in PSII; moreover, the observations by Joliot and Kok (12) that O2 evolution follows a four-flash oscillatory pattern necessitates that the oxidizing equivalents produced by P680+ must be stored. To accomplish this, P680+ (midpoint reduction potential at pH 7, approximately 1.2 V) is intimately coupled to a redox-active Tyr (TyrZ) and an inorganic Mn4Ca1ClXcluster where the water oxidation reaction is catalyzed. Current insights into the cluster structure have been largely driven by x-ray absorption spectroscopy, which predicts that the catalytic manganese complex is organized as pair of manganese-oxo dimers (20). The water oxidation reaction is more difficult to access. Recent H2 16O/H2 18O exchange measurements (11) show rapid exchange of substrate water (ms time regime), supporting the notion that substrate water is bound terminally to manganese. A number of other measurements concerning the water oxidation reaction have led to a metalloradical model for PSII water oxidation. In this proposal, TryZ is directly involved in hydrogen atom abstraction from the substrate water terminally bound at the manganese cluster (19). This proposal, as with most hypotheses, has attracted both supporters and detractors! It is certain that in the next few years the structure of PSII and the oxygen-evolving complex will be resolved at high resolution, as will be the structures of many other photosynthetic RCs. Already the last 25 years have seen tremendous advances in the understanding of photosynthetic RCs. Long-held concepts have been challenged, reinvestigated, and changed as the results of new structural, dynamic, biochemical, and molecular biological insights. These have been stimulating and exciting developments; undoubtedly they will continue. LITERATURE CITED 1 Allen JP Willams JC FEBS Lett 438 1998 5 9 Crossref Search ADS PubMed 2 Amesz J J Photochem Photobiol 30 1995 89 96 Crossref Search ADS 3 Anderson JM Andersson B Trends Biol Sci 7 1982 288 292 Crossref Search ADS 4 Berthold DA Babcock GT Yocum CF FEBS Lett 134 1981 231 234 Crossref Search ADS 5 Blankenship RE Photosynth Res 33 1992 91 111 Crossref Search ADS 6 Buttner M Xie DL Nelson H Pinther W Hauska G Nelson N Proc Natl Acad Sci USA 89 1992 8135 8139 Crossref Search ADS PubMed 7 Deisenhofer J Epp O Miki K Huber R Michel H Nature 318 1985 618 624 Crossref Search ADS PubMed 8 Diner BA Babcock GT Oxygenic Photosynthesis: The Light Reactions. 1996 213 247 Kluwer Dordrecht, The Netherlands 9 Emerson R Arnold W J Gen Physiol 16 1932 191 205 Crossref Search ADS PubMed 10 Golbeck JH Proc Natl Acad Sci USA 90 1993 1642 1646 Crossref Search ADS PubMed 11 Hillier W Messinger J Wydrzynski T Biochemistry 37 1998 16908 16914 Crossref Search ADS PubMed 12 Joliot P Kok B Bioenergetics of Photosynthesis. 1975 387 412 Academic Press New York 13 Krauβ N Schubert WD Klukas O Fromme P Witt HT Saenger W Nat Struct Biol 3 1996 965 973 PubMed 14 Mulkidjanian AY Junge W Photosynth Res 51 1997 27 42 Crossref Search ADS 15 Nitschke W Rutherford AW Trends Biol Sci 16 1991 241 245 Crossref Search ADS 16 Okamura MY Paddock ML Graige MS Feher G Biochim Biophys Acta 1458 2000 148 163 Crossref Search ADS PubMed 17 Reed DW Clayton RK Biochem Biophys Res Commun 30 1968 471 475 Crossref Search ADS PubMed 18 Sakurai H Kusumoto N Inoue K Photochem Photobiol 64 1996 5 13 Crossref Search ADS 19 Tommos C Babcock GT Acc Chem Res 31 1998 18 25 Crossref Search ADS 20 Yachandra VK Sauer K Klein MP Chem Rev 96 1996 2927 2950 Crossref Search ADS PubMed Author notes * Corresponding author; e-mail [email protected]; fax 517–353–1793. Copyright © 2001 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Lorimer, George H.

doi: 10.1104/pp.125.1.38pmid: 11154291

“Oh wad some Power the giftie gie us To see oursels as ithers see us! It wad frae monie a blunder free us.”   Robert Burns, 1786 The history of the chaperonins involves two diverse, seemingly unrelated observations dating back to the 1970s—the genetics of bacterio-phage morphogenesis and the synthesis of Rubisco during the biogenesis of chloroplasts. They coalesce in the late 1980s with the demonstration by the groups of Georgopoulos and Ellis that GroEL and the Rubisco-binding protein are homologous to one another (9) and with our demonstration in 1989 of what it was the chaperonin proteins really do (7). GENETICS OF BACTERIOPHAGE MORPHOGENESIS In the early 1970s Costa Georgopoulos isolated temperature-sensitive (ts), mutant strains ofEscherichia coli that were unable to support the growth of phage λ and a number of other bacteriophages (6 and references therein). The E. coli gene responsible for this phenotype was eventually given the name groE. By dint of some further genetics Costa, together with Barbara Hohn, identified thegroE gene product, a protein with a subunitM r of approximately 65 that we now know as GroEL (6). This enabled the groups of Roger Hendrix and Barbara Hohn to purify and record the first, now familiar, electron micrographs of GroEL. It was a 14-mer consisting of two heptameric rings stacked back to back. It also hydrolyzed ATP. Quite what GroEL did in the cell and how it was involved in phage morphogenesis was not known at this stage. Nevertheless, Hohn et al. (10) wrote “In groE mutants, bacteriophage T4 capsid protein aggregates in lumps at the cell membrane instead of forming normal capsids; bacteriophage λ head protein aggregates to form polyheads, spirals and inactive prehead-like particles and bacteriophage T5 tail assembly is affected. These phenomena could be explained by assuming an assembly aiding function present in normal cells but missing or malfunctional in the mutant.” In retrospect you can see that they were describing a malfunction in protein folding that leads to aggregation instead of the native state. But recall also that these observations were made immediately after Anfinsen's seminal work (1) that provided the dominating intellectual framework in the 1970s and 1980s in all matters related to protein folding. By then the prevailing dogma was that protein folding was a spontaneous event and suggesting that the folding of some proteins was assisted by other proteins was tantamount to heresy. With his bank of groE mutants Costa next designed a screen to look for suppressor mutations. This led to the discovery of GroES (6) and to evidence that the two groE gene products, GroEL and GroES, interacted with one another in vivo. Next Costa's group purified GroES, showed that in vitro it formed a 1:1 complex with GroEL in the presence of ATP, that GroES inhibits the ATPase activity of GroEL, and that it had a ring-like oligomeric structure. Still the function of GroEL and GroES remained elusive. Costa also reported two further genetic observations relating to GroEL and GroES the significance of which was not then immediately obvious but which, with the benefit of hindsight, would later become more significant. The first was that a temperature-sensitive mutation could be suppressed by over expressing GroEL and GroES (4). It is now apparent that many (not all) ts mutations are the result of a malfunction in protein folding at the restrictive temperature. Later, when we knew what GroEL and GroES really did, my colleagues at DuPont demonstrated that one could suppressts-mutations in a wide variety of structurally unrelated proteins simply by over-expressing both GroEL and GroES in the cells harboring the ts-mutant genes (15). The second important observation relates to the indispensable nature of the chaperonins. By the late 1980s it had become apparent that GroEL and GroES were an important part of the heat shock response. Using a genetic approach Costa's group demonstrated that both GroEL and GroES were indispensable proteins for bacterial growth at all temperatures between 20°C and 40°C (5). Again, the full significance of this result does not really become apparent until one knows the function of GroEL and GroES within the cell, assisting other proteins to fold. There are few events more central, more fundamental to the cell. BIOSYNTHESIS OF RUBISCO In the late 1970s, John Ellis and his colleagues found that one could study protein synthesis in chloroplasts by feeding radiolabeled amino acids to intact, isolated chloroplasts. The major product of such a synthesis is the large subunit of Rubisco. After incubating intact, isolated chloroplasts with [35S]Met for varying lengths of time, Barraclough and Ellis (2) quenched the reaction by osmotically lysing the chloroplasts and removed the green membranous material. They first subjected the soluble material to non-denaturing PAGE. After electrophoresis they either stained the gel with Coomassie or subjected it to autoradiography. They observed that the radioactivity was initially associated with a slower migrating band than the holoenyzme of Rubisco. Only later did radioactivity appear together with the holo-enzyme, implying a precursor-product relationship. When the slower migrating radioactive band was excised and subjected to SDS-PAGE, the radioactive band comigrated with the large subunit of Rubisco, whereas the Coomassie stain was associated with a protein with a subunit size of about 60 kD. They concluded that the synthesis of the holo-enzyme of Rubisco involved the transient formation of a complex with another large, oligomeric protein, the Rubisco subunit binding protein (RsuBP). Because at that time there was no intellectual framework into which the observation could be fitted, so pervasive was the Anfinsen view (1), this observation had little impact beyond those interested in chloroplast biogenesis or Rubisco. Ellis's group subsequently purified RsuBP and noted that it was an oligomer of approximately 750 kD with a subunit mass of approximately 60 kD and that it bound ATP (11). About then Harry Roy's group did a series of pulse-chase experiments following the fate of the Rubisco large subunits (13). They showed that the “hot” large subunits first sedimented as a 29S complex; this was the binary RsuBP•large subunit complex seen in the Ellis's non-denaturing gels. Next, in the chase the “hot” large subunits sedimented as a broad 7S species. With the benefit of hindsight and the crystal structure, I believe that these 7S species were a mixture of L2 dimers and (L2)2 tetramers en route to forming the (L2)4 octomeric core. They also showed that ATP was needed to chase the material out of the 29S complex into the 7S species. Finally, the 7S species was chased into the holoenzyme, which sediments at 18S. In retrospect these papers were very insightful, although at the time they were unappreciated given the prevailing dogma. In 1987 at a NATO meeting John Ellis first used the term “molecular chaperone” in referring to RsuBP. Later (3) he speculated on the existence of a class of proteins (molecular chaperones) “whose function is to ensure that the folding of certain other polypeptide chains and their assembly into oligomeric structures occur correctly.” He went on to suggest that molecular chaperones “do not form part of the final structure nor do they necessarily possess steric information specifying assembly.” Shortly after that Tony Gatenby and I decided to take a serious look at the RsuBP. By “serious” I meant mechanistic studies with purified materials. So I purified some RsuBP while Tony developed an E. coli-based in vitro transcription-translation system for making radiolabeled large subunits. However, our first experiment yielded a surprise. While the autoradiogram of the non-denaturing-PAGE gel clearly showed the presence of RsuBP-large subunit binary complex, the control, to which no RsuBP had been added, also showed the very same complex! I rudely suggested to Tony that he must have added RsuBP to both samples. Tony was not amused! But we did not wait long for an explanation, for within a short time John Ellis informed us that RsuBP was homologous to GroEL, which obviously (in hindsight) was present in the E. coli transcription-translation system. Our response was “GroEL? What's that?” CONNECTING PHAGE MORPHOGENESIS AND RUBISCO SYNTHESIS A protein in plants similar to GroEL was first observed by Tsuprun and Pushkin in 1981 (12). They reported electron micrographs and other properties of a protein from pea leaves indistinguishable from GroEL. However, in the absence of a function for this protein the significance of these results was not obvious or appreciated. A further 6 years were to pass before the connection between phage morphogenesis and Rubisco synthesis was established (9). This came about by the cloning and sequencing of RsuBP and of the GroE operon. RsuBP and GroEL were clearly homologous proteins. The gene for GroES was located immediately upstream of GroEL. The authors begin the discussion as follows: “We have described a ubiquitous, conserved, abundant protein that is associated with the post-translational assembly of at least two structurally distinct oligomeric protein complexes. We conclude that the role of this protein is to assist other polypeptides to maintain or assume conformations which permit their correct assembly into oligomeric structures.” The function of RsuBP in assisting the assembly of Rubisco in the chloroplast is immediately obvious. Less obvious was the function of GroEL and GroES in E. coli. It is clearly not there to assist the folding of Rubisco, a protein not normally found in E. coli; nor can the sole function of GroEL and GroES be the assembly of new phage particles. It has to be doing something of benefit toE. coli. So we (Pierre Goloubinoff, Tony Gatenby, and I) reasoned that if the synthesis of Rubisco in chloroplasts involves RsuBP, the chloroplast version of GroEL, then the synthesis of recombinant Rubisco in E. coli would also involve GroEL and perhaps also GroES. We had two plasmids, one encoding the cyanobacterial Rubisco operon for both large and small subunits and the other encoding the dimeric L2 Rubisco. While expression of these proteins in E. coli did yield some soluble, biologically active Rubisco, most of the Rubisco was an insoluble, inactive inclusion body. Pierre engineered a plasmid encoding the GroE operon so that one could over-express GroEL and GroES and Rubisco in the same E. coli cell. The total amount of Rubisco protein expressed by E. coli was approximately the same whether or not one also over-expressedGroE. What was dramatically different, however, was the fate of Rubisco. In the cells that over-expressed GroE nearly all of the Rubisco was soluble and biologically active. For this to occur required both GroEL and GroES to be over-expressed. This was true for both L2 and S4 · (L2)4 · S4forms of Rubisco. We thus concluded that the formation of active Rubisco in E. coli involved GroEL and GroES post-translationally at some point between the formation of the nascent polypeptide and the formation of the L2 dimer (8). At this point I felt we had a great opportunity to figure out exactly what GroEL and GroES did. We knew the identity of one of its in vivo substrates, the large subunit of Rubisco. We knew how to assay Rubisco. We had also narrowed the involvement of GroE to two possibilities: either the folding of the monomer and/or the association of the folded monomers to give the biologically active dimer. Some have described our next step as “very bold.” We purified GroEL and GroES to homogeneity and set out to do some neo-Anfinsen experiments using chemically denatured L2 Rubisco as the substrate. The results of the very first such experiment sent Pierre over the moon. “It works, it works! We've got to write a paper forNature!” I was less impressed. It was true that no active Rubisco was formed at all if one omitted any of the components (GroEL, GroES, Mg2+, or ATP). But compared with the control with native Rubisco, the recovery was miserable, approximately 1% as I recall. However, I spotted a potential flaw in the experimental protocol. Thinking that GroEL/ES would act as an enzyme, Pierre had used substrate (unfolded Rubisco) in molar excess. In short there was too much Rubisco in the pot. So Pierre was sent back to the bench with instructions to use equimolar amounts of unfolded Rubisco and GroEL 14-mers. Wow! Now we recovered >80%. It was almost too good to be true and so the very next day I repeated (successfully) the experiment with my own hands. Now I was over the moon! Now we could do the kind of experiments that enzymologists like to do with purified components instead of with messy, ill-defined mitochondrial or chloroplast extracts. It did not take us very long to work out some of the essential features, and we reported this in a paper toNature (7). The Nature paper contains many mechanistic insights. We begin by defining three species of Rubisco on the basis of circular dichroism: native Rubisco N, unfolded (in urea or GdnHCl) Rubisco U, and acid denatured Rubisco A. Rubisco A had some secondary structure, and we suggested that it might correspond to a molten globule, a term that was then fashionable but which I no longer consider very useful. We were lucky on two counts. First, we arbitrarily chose conditions for refolding that were non-permissive; i.e. no reconstitution of enzymatic activity occurred in the absence of the complete system (GroEL, GroES, and MgATP). Second, there was a fourth component needed that we luckily included, a monovalent cation K+ or NH4 +. Our preparations of GroEL and GroES were stored as ammonium sulfate precipitates, and enough NH4 + was carried over into the reaction mix to satisfy that particular requirement. We showed that ATP hydrolysis was required by quenching the reaction with hexokinase plus Glc (to convert the ATP to ADP). The yield of refolded Rubisco depended on whether one started with Rubisco U or Rubisco A, but the rate constant was the same regardless. We concluded that “reconstitution involves a common intermediate, the formation of which preceeds the same chaperonin-dependent rate-determining step. We propose that on removal of the denaturant by dilution, a rapid chaperonin-independent folding of Rubisco U to a state rich in secondary structure and resembling Rubisco A (the Rubisco I state), is followed by reaction with GroEL, GroES and Mg-ATP.” We noted that one required a slight molar excess of GroEL oligomers over Rubisco monomers. We attributed this to the instability of the Rubisco-I state and established that, absent a molar equivalent of GroEL oligomers, Rubisco-I underwent irreversible aggregation. We wrote “when either Rubisco U or Rubisco A was diluted into solutions containing the chaperonins, two competitive and mutually exclusive processes occurred. The unfolded or partly folded protomers either formed biologically unproductive aggregates or they formed a binary complex with GroEL, which prevented aggregation and directed the protomers along a biologically productive pathway.” Later, we amplified this in the discussion “It is clear that the concept of a stable substrate ‘patiently’ awaiting interaction with substoichiometric quantities of a catalyst does not apply here. Instead, there is an urgent need to stabilize this fickle intermediate by formation of a binary complex with GroEL.” We next demonstrated the existence of this binary complex by electrophoretic and immunological methods commenting that “the need to sequester unstable folding intermediates evidently exists in vivo as well as in vitro. The formation of a binary complex between the Rubisco large subunit and chloroplast GroEL was demonstrated long ago.” Here we refer to the Barraclough-Ellis experiment of a decade before (2). We next showed that the discharge of this binary GroEL-Rubisco-I complex depended on GroES and MgATP. We concluded that “the chaperonin-dependent reconstitution of Rubisco involves a strictly ordered set of reactions—first, the formation of a stable binary complex of GroEL•Rubisco-I, which at least in vitro can be demonstrated as an MgATP- and GroES-independent event, followed by the MgATP- and GroES-dependent discharge of folded and stable, but catalytically inactive monomers, which subsequently assemble into active dimers.” In our concluding paragraph we also noted that “the E. coli chaperonins clearly did not evolve to facilitate the folding or assembly, or both, of Rubisco, a protein not normally found inE. coli. It follows that the interaction between GroEL and the partly folded Rubisco cannot be specific for Rubisco alone. Instead, the specificity of the interaction must lie in some so far unidentified structural element of the partly folded protein. Because the native protein shows no detectable interaction with GroEL, this structural element must be missing from, or inaccessible in, the native protein.” It was not very long before purified, chemically denatured proteins were being thrown at every molecular chaperone and heat shock protein known to man. Gone were the messy experiments with organelles and other variants of chicken soup. In the ensuing 10 years much progress has been made unraveling the mechanistic and structural details of the chaperonin nano-machine (for reviews, see 16 and 14). Paradoxically, however, the problem that Tony Gatenby and I set out to investigate 12 years ago, how to refold hexadecameric, higher plant Rubisco S4 · (L2)4 · S4from its denatured subunits, remains unsolved. To date, despite several forlorn attempts both with or without chaperonins, not so much as a whiff of Rubisco activity has been reconstituted. This only goes to show that the grand old protein of plant biology still has a few secrets to divulge. LITERATURE CITED 1 Anfinsen CB Science 181 1973 223 230 Crossref Search ADS PubMed 2 Barraclough R Ellis RJ Biochim Biophys Acta 608 1980 19 31 Crossref Search ADS PubMed 3 Ellis RJ Nature 328 1987 378 379 Crossref Search ADS PubMed 4 Fayet O Louarn JM Georgopoulos C Mol Gen Genet 202 1986 435 445 PubMed 5 Fayet O Ziegelhoffer T Georgopoulos C J Bacteriol 171 1989 1379 1385 Crossref Search ADS PubMed 6 Friedman DI Olson ER Georgopoulos C Tilly K Herskowitz I Banuett F Microbiol Rev 48 1984 299 325 Crossref Search ADS PubMed 7 Goloubinoff P Christeller JT Gatenby AA Lorimer GH Nature 342 1989 884 889 Crossref Search ADS PubMed 8 Goloubinoff P Gatenby AA Lorimer GH Nature 337 1989 44 47 Crossref Search ADS PubMed 9 Hemmingsen SM Woolford C van der Vies SM Tilly K Dennis D Georgopoulos CP Hendrix RW Ellis RJ Nature 333 1988 330 334 Crossref Search ADS PubMed 10 Hohn T Hohn B Engel A Wurtz M Smith PR J Mol Biol 129 1979 359 373 Crossref Search ADS PubMed 11 Musgrove JE Johnson RA Ellis RJ Eur J Biochem 163 1987 529 534 Crossref Search ADS PubMed 12 Pushkin AV Tsuprun VL Solov'eva NA Shubin VV Evstigneeva ZG Kretovich WL Biochim Biophys Acta 704 1982 379 384 Crossref Search ADS 13 Roy H Bloom M Milos P Monroe M J Cell Biol 94 1982 20 27 Crossref Search ADS PubMed 14 Thirumalai D, Lorimer GH (2001) Ann Rev Biophys Struct Biol (in press) 15 Van Dyk TK Gatenby AA LaRossa RA Nature 342 1989 451 453 Crossref Search ADS PubMed 16 Xu Z Sigler PB J Struct Biol 124 1998 129 141 Crossref Search ADS PubMed Author notes * E-mail [email protected]; fax 301–352–5539. Copyright © 2001 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Farquhar, Graham D.; von Caemmerer, Susanne; Berry, Joseph A.

doi: 10.1104/pp.125.1.42pmid: 11154292

A BRIEF HISTORY Our model of photosynthesis (8) published some 20 years ago in Planta has had an impact and seen application that far exceeded our expectations. Perhaps it is useful to reflect on what this model did and why we published it. It is important to note that our model is not a complete model of photosynthesis. It makes no attempt to treat all of the steps in this important process; rather, it was a synthesis, a simplified view of the already (in 1980) overwhelming knowledge of the contributing mechanisms. In the years preceding our model a great body of work had accumulated describing the responses of CO2 exchange by leaves to a wide range of environmental conditions (temperature, CO2 concentration, light intensity, humidity, and oxygen concentration). These responses were quite reproducible, but difficult to explain. Pieces began to fall into place that informed our ignorance. Perhaps the pivotal event was the finding by George Bowes and Bill Ogren that O2 was a competitive inhibitor of CO2 fixation by Rubisco and an alternative substrate leading to a side reaction that fueled photorespiration. Others added findings that integrated photorespiration into photosynthetic carbon metabolism. This synthesis provided a plausible explanation of the manifold interactions between O2 and CO2 on the photosynthesis of leaves. In our model we linked equations describing Rubisco kinetics with others on the stoichiometry of the photosynthetic carbon reduction cycle and the photorespiratory carbon oxidation cycle, particularly on their energetic (electron transport and ATP synthesis) requirements. Building on the pioneering modeling of Hall, Tenhunen, Peisker, Laisk, and others, we then drew together biochemical and organelle level observations of the temperature dependencies of these phenomena, and combined them with an empirical equation for the dependence of “potential” electron transport rate on absorbed irradiance. Our model attempted to match generalized observations of the photosynthetic gas exchange of leaves with predictions from this mathematical summary of photosynthesis. We published our paper (8), “A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species,” in 1980. Susanne von Caemmerer was, at that stage, a PhD student with Graham Farquhar at the Research School of Biological Sciences, and Joe Berry had earlier been a visitor to Barry Osmond's laboratory there. Many of the principles had been discussed during the earlier visit, including what Joe Berry called the “teeter-totter” (and Graham Farquhar called it a “see-saw”) between two flux limitations. That is, that photosynthesis cannot go faster than a carboxylase activity limited rate, and also cannot go faster than an electron transport limited rate, but should move easily from one to another without the overall rate being much smaller than either limitation–few “frictional” losses. A working model of C3 photosynthesis including these principles was developed before Joe Berry left, and this was integrated into the first publication, a model of the C4 mechanism (2). WHY MODEL? Graham Farquhar was interested in modeling photosynthesis to answer the question raised in his collaboration with Ian Cowan on optimal behavior of stomata: What would the rate of CO2 assimilation be if stomatal conductance were slightly perturbed? Joe Berry was interested in how the CO2-concentrating system of C4 plants influenced CO2fixation and photorespiration by Rubisco in the bundle sheath cells. In both cases we needed a mechanism for representing the properties of C3 plants in the context of a larger analytical framework. Susanne von Caemmerer, who came to this environment with a degree in pure mathematics, used the model as a tool for making quantitative links between leaf biochemistry and gas exchange kinetics (18). Of course an underlying feeling was that one doesn't really understand something until one can describe it mathematically. The model has been subsequently used for pedagogic purposes, and also as a useful framework for fitting to data, and then extrapolating. All this has provided an interplay between model and measurement that has stimulated development of both fields. The interplay was also relevant to the direct issue of publishing our paper. It was initially rejected: it contained no data, and it was against Planta policy to publish papers that were solely models. Ian Cowan crafted a letter arguing that the modeled response curves were familiar to all the experimentalists who worked on gas exchange. We are grateful to the Planta editors for accepting the argument. Later Graham Farquhar was asked to referee all modeling papers sent to Planta. BRINGING BIOCHEMISTRY AND GAS EXCHANGE TOGETHER The original model development was aided by breakthroughs in understanding Rubisco oxygenase and oxygen inhibition of CO2 fixation (12) and Rubisco kinetics and their temperature dependence. It was also aided by developments in Rubisco activation, but only in the sense of providing sufficient activity to relate to rates in vivo. Understanding the pathway and stoichiometry of the photorespiratory cycle was also important. Improvements in gas exchange technology permitted measurement of photosynthesis, stomatal conductance, and intercellular partial pressure of CO2. Susanne von Caemmerer discusses many of these issues in her recent text (17). Later, the pool sizes of intermediates and enzyme activities were measured in relation to gas exchange. The parametrization of our model requires estimates of Rubisco kinetic constants and recently, plants with antisense to the small subunit of Rubisco have proved an ideal system for measuring the constants in vivo, as these plants are more completely Rubisco-limited than the wild type. Some contemporary gas exchange measurements include those of oxygen and its isotopes (17). CONTROL OF PHOTOSYNTHESIS Control of RuBP Regeneration In our original model, RuBP regeneration was controlled by electron transport, but could also be limited further by other components (lumped together) of the photosynthetic carbon reduction cycle. At higher [CO2], Tom Sharkey has drawn attention to a third limitation that may come into play, that of triose phosphate utilization. The details of all these interactions are yet to be fully modeled. An interesting, new, and complex approach is being taken by Agu Laisk who has been at the forefront of so much modeling of photosynthesis. He and colleagues are developing a detailed model (13) with dozens of enzymes and electron carriers so that regulation can be examined, as well as control of overall leaf photosynthesis. It could serve as a working encyclopedia of what is known about enzymes, pathways, and membrane transport. It is perhaps surprising that electron transport in whole-leaf models is still treated largely empirically. Fluorescence studies have been valuable in making progress. Engelbert Weis and Joe Berry (19) used our model to relate the rate of CO2 exchange to that of electron transport. This permitted a quantitative analysis of the relation between fluorescence quenching properties and electron transport. It separated the role of photochemical quenching and showed that there is a residual role that could be related to non-photochemical quenching. Bernard Genty and colleagues (11) developed a model that has a theoretically satisfying basis and simplicity, built on this finding. They showed that the quantum yield of photosystem II is related to the ratio of steady-state fluorescence to saturated fluorescence (ΦPS2 = 1 − Φ/Φm). The challenge remains that we still don't know the physical mechanism of non-photochemical quenching. Rubisco Activation and Its Control Activation/carbamylation and inhibition of Rubisco are an active area of research, experimentally and theoretically (17). Our original model did not include these processes, and with the understanding of that time their inclusion would have kept rates of photosynthesis below those observed in real life. Susanne's thesis work (18) showed that to achieve those rates with subsaturating [CO2], Rubisco would have to be fully active. Few researchers were allowing for subsaturation and the idea that Rubisco was a storage protein was then wide spread. The subsequent work on tight binding of RuBP and of inhibitors, and on carbamylation, has been an interesting case where model formulation has informed the comparison of in vitro and in vivo data. In the steady state, Rubisco activation is now generally thought to be regulated and to place no greater limitation on photosynthesis than is already there because of limited capacity for regeneration of RuBP (typically electron transport). However, a low level of activation may limit, for example, when light intensity suddenly increases. Transients Several groups have addressed the issue of transient changes in light intensity and how such changes affect photosynthesis (14, 17). The transients involve the times taken for stomata to respond, for Rubisco activation to increase, and for the pool of phosphorylated intermediates of the PCR cycle to autocatalytically build to the appropriate level. REGULATION VERSUS LIMITATION: The Supply of CO2 to Rubisco Limitations within the Leaf Many earlier models of photosynthesis assumed that the kinetics of CO2 assimilation were determined by resistances to diffusion within the chloroplast. The biochemical models challenged those views. Now the challenge is to modify the biochemical models with appropriate representations relating fluxes to concentrations. We need three-dimensional parameters analogous to the one-dimensional concept of resistance. Dave Parkhurst has made a start with more detailed descriptions of diffusion within the intercellular air spaces of leaves. There appears to be a significant drawdown in [CO2] from the substomatal cavities to the sites of Rubisco, much of it presumably across the cell walls and membranes, and an unknown contribution from the air spaces (17). Limitations by Stomata Chin Wong (20) showed that under many conditions where photosynthetic capacity was caused to change, the ratio of intercellular and ambient CO2 concentration (C i/C a) often remained constant, reducing with increasing leaf-to-air humidity difference. Tim Ball and Joe Berry (1) generalized this finding and effectively produced a succinct relationship between stomatal conductance and rate of photosynthesis. When combined with a biochemical model of photosynthesis, it has formed the basis for many studies modeling whole-leaf and canopy carbon assimilation. Ian Cowan described stomatal functioning in terms of optimization of carbon gain with respect to water loss, with the free parameter being tied to the statistics of rainfall (3). There is at present insufficient information for a mechanistic model of stomatal functioning. “Patchiness” Heterogeneity in stomatal supply has been recognized as a problem when it comes to assigning a concentration of CO2at the substomatal level. If the heterogeneity of stomatal opening comes about as the result of some imposed stress, one might be fooled into interpreting the data as a loss of photosynthetic capacity (16). The current quantitative mapping of photosynthesis to address this problem derives from analysis of chlorophyll fluorescence images (4). HOMOGENEITY OF LIGHT INTENSITY AND PHOTOSYNTHETIC CAPACITY We originally thought of our model as applying at the chloroplast level and were somewhat surprised that it seemed to work for a leaf. It assumed a homogeneous light environment, CO2concentration, and concentration of Rubisco. It emerged later that the same model should hold if light intensity and photosynthetic capacity co-vary in space (7). Although that can happen within a leaf or canopy when light is averaged over a day (9), it does not hold in detail with changes in light intensity on a time scale far shorter than that of photosynthetic adaptation—particularly a problem with light flecks deep in a canopy. Thus big-leaf models with the total Rubisco per unit ground area treated as a single leaf overestimate canopy photosynthesis. Models that differentiate sun and shade leaves largely overcome these problems (5) for broad-leaved species and grasses like wheat. Nevertheless, there is a need to introduce penumbral effects, especially for coniferous species. The analogous problem exists considering the internal volume of a leaf as well. SCALING TO THE GLOBAL LEVEL Models of photosynthesis and stomatal conductance are becoming embedded in larger models of the global carbon cycle and of land surface feedbacks on climate. The physiological properties affect atmospheric temperature and the hydrological cycle (10). Models now strive to simulate the mass and energy balance of the land surface with changing meteorological conditions over time—especially in climate models. Use of our model of photosynthesis has resulted in development of theory to couple satellite remote sensing of spectral reflectance to estimate the absorbed photosynthetically active radiation and the efficiency of its use by chloroplasts at a global scale with spatial resolution of about 1 km2. The coupling of these with stomatal models has improved the simulation of heat and water exchange between vegetated surfaces of continents and the atmosphere (15). The significance for quantitative understanding of the bioenergetics of our planet is just beginning to have an impact. MOVING TO LONGER TIME SCALES There is a need for longer-term modeling of photosynthesis. There is little known about and thus little predictive modeling of how Rubisco activity and electron transport capacity change with environmental conditions. In practice most applications follow the observed changes in leaf properties, either from direct observations or from measurements of leaf nitrogen levels, as the latter often give reasonable measures of capacity once the nitrogen tied up in cell walls is accounted for (6). Future developments are inextricably linked with modeling of growth and development and will necessarily involve considerations of cell division and expansion and of hormonal and other controls of gene expression. LITERATURE CITED 1 Ball JT Woodrow IE Berry JA Progress in Photosynthesis Research Biggins J IV 1987 221 224 Martinus Nijhoff Dordrecht, The Netherlands 2 Berry JA Farquhar GD Proceedings of the 4th International Congress on Photosynthesis, Reading, England 1977. Hall D. Coombs J. Goodwin T. 1978 119 131 The Biochemical Society London 3 Cowan IR T.J. Givnish, ed, On the Economy of Plant Form and Function. 1986 171 213 Cambridge University Press Cambridge, UK 4 Daley PF Raschke K Ball JT Berry JA Plant Physiol 90 1989 1233 1238 Crossref Search ADS PubMed 5 de Pury DGG Farquhar GD Plant Cell Environ 20 1997 537 557 Crossref Search ADS 6 Evans JR Oecologia 78 1989 9 19 Crossref Search ADS PubMed 7 Farquhar GD Phil Trans R Soc Ser B 323 1989 357 368 8 Farquhar GD von Caemmerer S Berry JA Planta 149 1980 78 90 Crossref Search ADS PubMed 9 Field CB Oecologia 56 1983 341 347 Crossref Search ADS PubMed 10 Field CB Plant Physiol 125 2000 25 28 Crossref Search ADS 11 Genty B Briantais J-M Baker N Biochim Biophys Acta 990 1989 87 92 Crossref Search ADS 12 Laing WA Ogren W Hageman R Plant Physiol 54 1974 678 685 Crossref Search ADS PubMed 13 Laisk A Oja V Dynamics of Leaf Photosynthesis: Rapid Response Measurements and Their Interpretations. 1998 Commonwealth Scientific and Industrial Research Organization Publications Collingwood, Victoria, Australia 14 Pearcy RW Gross IJ He D Plant Cell Environ 20 1997 411 424 Crossref Search ADS 15 Sellers PJ Dickinson RE Randall DA Betts AK Hall FG Berry JA Collatz GJ Denning AS Mooney HA Nobre CA Sato N Field CB Henderson-Sellers A Science 275 1997 502 509 Crossref Search ADS PubMed 16 Terashima I Wong SC Osmond CB Farquhar GD Plant Cell Physiol 29 1988 385 394 17 von Caemmerer S Biochemical Models of Photosynthesis. 2000 Commonwealth Scientific and Industrial Research Organization Publications Collingwood, Victoria, Australia 18 von Caemmerer S Farquhar GD Planta 153 1981 376 387 Crossref Search ADS PubMed 19 Weiss E Berry J Biochim Biophys Acta 894 1987 198 208 Crossref Search ADS 20 Wong SC Cowan IR Farquhar GD Nature 282 1979 424 426 Crossref Search ADS Author notes * Corresponding author; e-mail [email protected]; fax 61–2–6249–4919. Copyright © 2001 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)