Plant Physiology

Minorsky, Peter V.

doi: 10.1104/pp.126.1.1pmid: N/A

Endo-1,4-β-Glucanase and Cellulose Synthesis Although cellulose is the major cell wall polysaccharide in plants, the enzymes that contribute to its production have generally proven to be recalcitrant to study by traditional enzymology. The study of cellulose-deficient, radial swelling (rsw) mutants of Arabidopsis is beginning to yield valuable insights into cellulose synthesis. For example, the gene RSW1, which appears to code for a glycosyltransferase, has recently been linked to cellulose production. In this issue, Lane et al. (pp. 278–288 ) examine the effects of the rsw2 mutation, which is non-allelic to RSW1. They report thatrsw2 is, in fact, allelic to KORRIGAN, a gene that encodes for a putative membrane-bound endo-1,4-β-glucanase whose dysfunction has previously been linked to abnormalities in cell expansion and division. The rsw2 mutant shows radial swelling in the root and hypocotyl; reduced axial growth; smaller leaves, stems, and flowers; and impaired anther dehiscence. These morphological aberrations are accompanied by gross anatomical abnormalities (Fig. 1). The effects of thersw1 and rsw2 mutations were found to be additive in part. It is interesting that cellulose synthesis in Arabidopsis reveals homologies to cellulose synthesis in Agrobacterium tumefaciens where two genes, celA and celC,also encode for a glycosyltransferase and an endo-1,4-β-glucanase, respectively. Fig. 1. Open in new tabDownload slide The reduced ability to synthesize cellulose causes gross anatomical abnormalities in the roots of the rsw2mutant of Arabidopsis (bottom) compared with wild type (top). (Note difference in magnification.) Fig. 1. Open in new tabDownload slide The reduced ability to synthesize cellulose causes gross anatomical abnormalities in the roots of the rsw2mutant of Arabidopsis (bottom) compared with wild type (top). (Note difference in magnification.) Magnetic Resonance Imaging (MRI) of Cavitating Xylem Vessels Transport of water through xylem vessels may become disrupted by the cavitation of water columns under high levels of tension or freezing temperatures. Such vessels would normally be lost to water transport unless mechanism sexist to reconnect the column. The mechanism by which cavitated vessels become repaired remains controversial, particularly in light of recent claims that cavitated vessels can be repaired even when water in neighboring conduits is under tension. In this issue, Holbrook et al. (pp. ) use high-resolution MRI to show that individual xylem vessels in grape (Vitis vinifera) vines do spontaneously refill following cavitation (Fig. 2). This non-destructive technique should prove most useful in studying the mechanisms underlying the repair of cavitated vessels. Fig. 2. Open in new tabDownload slide A high-resolution MRI image of a grapevine reveals individual cavitated vessels (arrows) that later recovered following watering. Fig. 2. Open in new tabDownload slide A high-resolution MRI image of a grapevine reveals individual cavitated vessels (arrows) that later recovered following watering. Unidentified Signal Regulates Apical Dominance in Pea The ramosus1 (rms1) mutant of pea (Pisum sativum) exhibits reduced apical dominance. Graftingrms1 scions to wild-type (WT) rootstocks restores the WT phenotype to the scion, suggesting that rms1 affects apical dominance by altering the levels of a branching inhibitor originating from the rootstock. Although cytokinins (CKs) originate in the root and have been implicated in apical dominance, they are poor candidates for this mysterious factor: CKs tend to promote branching when applied directly to buds, and rms1 mutants, in fact, have reduced levels of CKs in their xylem sap. Indole-3-acetic acid, another hormone long implicated in apical dominance, is also unlikely to be the mysterious branching inhibitor affected by the rms1 mutation given the basipetal polarity of its movement. In this issue, Foo et al. (pp. )report on the results of several grafting experiments that shed further light on this yet-to-be-identified branching inhibitor. First, they report that the grafting of a small (0.5–1.0 cm) WT interstock between an rms1 scion and rootstock almost completely inhibits lateral branching. Second, a WT and an rms1 shoot growing from the same mutant rootstock exhibit their normal differences in branching patterns, but if they are both grafted to a WT rootstock, the branching of both types of scions is inhibited. Third, the simultaneous grafting of rms1 scions to both WT and rms1rootstocks leads to an inhibition of branching in the mutant scion. Thus, all evidence points to the existence of a graft-transmissible, long distance inhibitor of branching in WT peas. Our Expanding Knowledge of β-Expansins Expansins are a family of plant proteins essential for acid-induced cell wall loosening. Sequence comparisons indicate that there are two classes of expansins, α and β, which despite sharing only about 20% amino acid identity, have in common, in addition to their cell wall-loosening abilities, a number of highly conserved motifs. To date most studies have focused on α-expansins, but in this issue two studies provide new insights into the molecular biology of β-expansin structure and function. Wu et al. (pp. ) report on their isolation and characterization of 13 of the more than 30 expansin DNAs in maize (Zea mays). Their data indicate that the expression patterns of α- and β-expansin genes run the gamut from general and overlapping to highly specific and localized. Unlike the case with Arabidopsis, the β-expansins of maize are more numerous and highly expressed than are α-expansins. Although β-expansins are apparently less common in dicots, their function in these plants is no less fascinating. A case in point is Cim1, a β-expansin from soybean (Glycine max) that increases 20- to 60-fold after treatment of CK-starved soybean suspension cells with CK. Previous studies have revealed that this accumulation stems from increased Cim1 stability.Downes et al. (pp. ) employ antibodies to Cim1 to reveal three processing intermediates that are involved in the maturation and degradation of this species of β-expansin. CK and auxin are reported to act synergistically to induce the accumulation of Cim1, and the onset of Cim1 expression is correlated with the growth of soybean cultures. Cim1 is rapidly and specifically degraded as soybean cultures reach stationary phase. Anion Channels and Al3+ Resistance The high levels of Al3+ that typify many types of acid soil are considered to be one of the major constraints to increasing crop yields worldwide. In many plants, the binding of toxic Al3+ by organic anions released from the roots comprises a major mechanism of Al3+ resistance. Reinforcing other recent findings published in Plant Physiology (Piñeros and Kochian, 2001; Zhang et al., 2001), two articles in the present issue offer mechanistic details of the anion channel that mediates this efflux of organic ions. Kollmeier et al. (pp. ) used the patch clamp technique to investigate Al3+-induced currents in the root tips of maize. Pre-incubation of intact roots with low concentrations of Al3+ induced a citrate- and malate-permeable anion channel in 80% of the protoplasts derived from the zone 1 to 2 mm from the root tip. When Al3+ was applied to the protoplasts in the whole-cell configuration, anion currents were elicited in 10 min. The anion channel blockers niflumic acid and 4,4′-dinitrostilbene-2,2′ disulfonic acid strongly inhibited both the Al3+-induced anion currents and the release of organic acids. The protein synthesis inhibitor cycloheximide had no effect on the elicitation of the anion current by Al3+, suggesting that channel activation is mediated posttranslationally. The data of Osawa and Matsumoto (pp. ) suggest that this posttranslational mechanism may involve protein phosphorylation, They report that K-252a, a broad-spectrum inhibitor of protein kinases, prevents the induction of malate release by Al3+ in an Al3+-resistant cultivar of wheat (Triticum aestivum). The transient activationof a 48-kD protein kinase was observed to precede the initiation of malate efflux, and its activation was abolished by K-252a. K-252a rendered this normally resistant cultivar sensitive to the toxic effects of Al3+. LITERATURE CITED 1 Piñeros M Kochian LV A patch-clamp study on the physiology of aluminum toxicity and alu-minum tolerance in maize: identification and characterization of Al3+-induced anion channels. Plant Physiol 125 2001 292 305 Google Scholar Crossref Search ADS PubMed WorldCat 2 Zhang WH Ryan PR Tyerman SD Malate-permeable channels and cation channels activated by aluminum in the apical cells of wheat roots. Plant Physiol 125 2001 1429 1441 Google Scholar Crossref Search ADS PubMed WorldCat 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)

Raikhel, Natasha V.

doi: 10.1104/pp.126.1.3pmid: N/A

Anniversaries provide opportunities for reflection, and so, after a year at the helm of Plant Physiology, it seems timely to look back at the changes and innovations that have been made to our journal during the past year, and to look forward to our aspirations for the coming year. Although in some ways it is too soon to know the effects of the past year's innovations on our journal, several indicators suggest that we are on the right track. Institutional subscriptions at 2000 year-end were up 7% from 1999 and there has been a gratifying growth in the number of research papers submitted. Among the major innovations of the past year was the publication of three special issues. The first of these (December 2000), a celebration of the completion of the Arabidopsis Genome Initiative, marked the first time that the number of people perusing Plant Physiology on the Internet surpassed the high standard set by our precocious younger sister, The Plant Cell. Even if this is in part due to the success of the publicity and promotion that surrounded this issue, it is still exciting to see that the first special issue was successful in introducing our journal to an even wider audience. We had even higher interest for our 75th Anniversary Issue, published in January 2001. The highlight of this special issue was the 42 invited commentaries summarizing conceptual breakthroughs during the last quarter century in various areas of plant biology. The conceptual breakthrough articles will be published as a separate book that will be made available at our 75th Anniversary Symposium on Plant Biology 2001 to be held in Providence, Rhode Island. Our third special issue, put together by two Associate Editors, Susan Wessler and Vicki Chandler, and published in March 2001, was devoted entirely to the grasses, specifically to their importance as a collective model genetic system alongside Arabidopsis. Responses to these special issues have been so positive that we plan to publish an Arabidopsis special issue and a Grass special issue annually. An Arabidopsis special issue will be published in June 2001 and a Grass special issue will be published in December 2002. In January, the Journal cover debuted a new look, but it is the new features inside the cover that are most important to Plant Physiology's readers. On the Inside, a column written monthly by our staff writer Peter Minorsky, capsulizes the essence of half a dozen or so articles that exemplify the scope and quality of content found in each issue. Peter also writes The Hot and Classic, a column that highlights selected major plant biology publications of the past and present. Soon, Peter plans to introduceNews from the Archives, a column that will highlight interesting papers that have fallen into near oblivion. With the advent of molecular biology and genetic engineering, plant biology has graduated from the cloistered walls of academia to the global spotlight. Other new front-of-the-journal subjects such as Breakthrough Technologies, Genome Analysis, and Editor's Choice contributions serve as creative outlets for scientists to address a variety of issues coming into prominence as a result of plant biology's new importance. The GMO-related Editor's Choice articles published over the last few months will be collected in a separate booklet to be made available at the 75th Anniversary Symposium in Rhode Island. Soon, with Willi Gruissem's help, I plan to start a new series of Editor's Choice articles devoted to topics of interest to young readers in our profession. We have asked many people—some still actively working and some well-known retired plant biologists—to share their differing career experiences and thoughts on what has kept them engaged and excited in science. We thought it very worthwhile for people new to our field to know about those people who helped shape plant biology. Thanks largely to the efforts of Lauren Ransome, Plant Physiologyhas been successful in advertising our current and forthcoming articles and keeping our readers informed about the many exciting articles. In response to impressions, real or imagined, that submissions toPlant Physiology took too long to publish, new internal procedures have been established to reduce turnaround time from submission to first decision from 6 to 4 weeks. To shorten production time for acceptance for publication, proofs of articles are now sent electronically to authors and in April we began publishing individual articles on-line ahead of print. In June 2001, we will be introducing on-line submission and review. We anticipate that this will make it easier to submit papers as well as expedite submitted manuscripts for evaluation by the Board and reviewers, thereby reducing overall turnaround time for submission to publication dramatically. Several on-line enhancements have been made to the on-line journal. Readers are now able to submit electronic letters to the editor with comments about individual articles directly on-line. You will see this option when you open full text of an article in the right-hand dialog box that appears with every article. The Journal is now a participant in CrossRef, an initiative designed to facilitate citation linking to journals outside the HighWire Press family. And finally, Plant Physiology is participating in PubMed Central (PMC), a Web-based archive of journal literature for all of the life sciences developed by the National Center for Biotechnology Information at the U.S. National Library of Medicine. With PMC, the National Center for Biotechnology Information is taking the lead in preserving and maintaining open access to the literature in electronic form, just as the U.S. National Library of Medicine has done for decades with the printed biomedical literature. PMC aims to fill the role of world-class library in the digital age. Plant Physiology fully supports this endeavor and is releasing back content to PMC with a 12-month delay in conjunction with the release of back issues on the HighWire site. We hope to shorten this delay as the reality of the impact on subscriptions becomes clear. Our main objective is to publish innovative science of the highest quality. In our new Instructions to Authors, we state that work reported in Plant Physiology should be vigorously executed, provide new information, and move the field to the next level. Our journal will maintain its broad scope, but we are determined that the work we publish feature cutting edge research. The editors feel that on average the papers published in Plant Physiology are a bit too lengthy for maximum impact. While Plant Physiology must remain committed to publishing complete data in a format that is large enough to see easily (at least in the print version), text length will be subjected to more stringent control, with particular emphasis on ensuring that “Discussions” explore but do not repeat “Results.” It has been a year since the new, international Board took over the journal, and their hard work, ideas, and feedback—both positive and negative—have been most useful. Together with the superb and highly professional efforts of our editorial staff (led by Melissa Junior, with the help of Lauren Ransome, Kim Davis, and Stephanie Butto) and our very talented science writer, Peter Minorsky, we are succeeding in making a good journal even better! Finally, I wish to sing praise for the unsung heroes and heroines of the Plant Physiology family—our reviewers. Their expertise, promptness, and thoughtful criticisms have always been a great source of pride and amazement to me. Think of all the time each of us has saved because these individuals have so generously used their vast experience and knowledge to select only the most rigorous of scientific papers for publication in Plant Physiology. Think also of the valuable advice they have provided for our submissions that fell short. All of us owe these unrecognized and unpaid guardians of quality an immense debt of gratitude. 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)

Buchanan, Bob B.

doi: 10.1104/pp.126.1.5pmid: 11351062

Bob B. Buchanan Member of the National Academy of Sciences Although much has been learned since the field was put on a scientific basis at the turn of the last century, our knowledge of food allergies is far from complete. It is still unclear, for example, why only certain individuals are affected and why, even among them, the problem is often restricted to childhood. It is also not clear why the allergies caused by various nuts and aquatic animals tend to persist and be lifelong. Milk, egg, soy, and wheat are the major food allergies in children, whereas peanut, tree nuts, shellfish, and fish are most prevalent in adults. The field is complicated by the fact that many more people believe they suffer from food allergies than is actually the case. Thus, although up to 20% of Americans have a perceived food allergy, the problem can be medically diagnosed in only about 2% of the population (Altman and Chiaramonte, 1996). The issue is further clouded by confusion with food intolerance and by evidence that allergies are increasing rapidly in developed countries for reasons that are only beginning to be understood. These factors collectively contribute to the lack of understanding that has long been a part of the food allergy field. Aside from limited attention drawn to the increased prevalence, food allergy has historically attracted little notice. However, with the advent of genetic engineering and its application to the production of food, the situation has changed dramatically. The development and commercialization of a variety of food crops with transgenes has thrust the allergy issue onto a public stage and given the field unprecedented exposure worldwide. Although not yet apparent, I believe the allergy and food technology fields will benefit from this attention in the long term, akin to the progress made in understanding the cellular immune system as a result of publicity brought by the acquired immune deficiency syndrome epidemic. WHY THE SUDDEN INTEREST? The increased public awareness of food allergy has arisen from a combination of three factors: reasoned concern, fear through ignorance, and political motivation. The first two factors are expected and limited in scope. The third, which was unanticipated and amplifies the second, stems from the goal of certain individuals and environmental organizations to delay the commercial development of genetic engineering, especially as applied to food. The allergy issue was selected because of its vulnerability: In addition to its enigmatic nature mentioned previously, opponents of genetic engineering recognized early on that it is difficult to determine with absolute certainty whether a protein introduced into a food by genetic engineering is a potential allergen. In retrospect, one wonders why the allergy issue was not raised earlier—for example, in the countless plant breeding programs since World War II—that significantly have not converted nonallergenic into allergenic foods. A new allergen has been introduced independently of plant breeding. The introduction of kiwi, a relatively obscure fruit, led to the development of a new allergy in the general population of the developed world. Interest in the allergy issue has been heightened by knowledge that a protein known to be an allergen in one species remains an allergen when transferred by genetic transformation to a second species. An example of such a protein, now widely known, is the Brazil nut allergen (2S protein) transferred to soybean. The allergenicity associated with the original 2S protein in Brazil nut was found to be retained after it was overexpressed in soybean (Nordlee et al., 1996). Although not surprising, this example is reassuring in documenting that the scientific community is capable of detecting and identifying a known allergen that has been transferred from one species to another by genetic engineering. As a result of the allergy tests, the transgenic soy product in question was not further developed as a commercial product. In this commentary, I shall identify the issues surrounding the allergy issue and discuss their scientific validity, rather than the production of hypoallergenic foods by genetic engineering—a research focus of a number of laboratories, including ours. I then turn to a discussion of the tools available to address the concerns and where we are in their resolution. It will be seen that a solution to this problem appears to lie on the near horizon. WHAT ARE THE ISSUES? Concern about the genetic modification of food appears to stem from three questions: Is the protein of interest an allergen? Has the protein of interest become an allergen as a result of the transformation and selection process? Has the transformation and selection process in some unknown way altered a normal cellular protein so that it has become an allergen? SCIENTIFIC BASIS FOR THE CONCERNS The first question, whether a particular protein is an allergen, is valid and should be answered. The second question, based on the conversion of the protein of interest into an allergen (for example, by glycosylation) also relates to a change that is biochemically feasible. One would think that indications of such a change would have surfaced with significantly abundant proteins in earlier plant breeding programs. Nonetheless, this point should be tested, at least until we have a greater understanding of the fate of transgenic proteins in plants. The last question, which raises the possibility that a given protein of the cell could become an allergen as a result of transformation and selection, is less tenable. However, this question, like the other two, will continue to be raised until additional experience has been gained and consumers have expressed confidence in genetically modified foods, especially those based on a protein to which the human population has not been previously exposed. CURRENT TOOLS FOR SOLVING THE PROBLEM The question of whether a transgene product is an allergen or whether its presence unintentionally renders a food product more allergenic than its nonengineered counterpart is addressed in several ways, including: (a) comparing the predicted amino acid sequence of the transgene product with that of known food allergens; (b) determining the abundance of the protein in food as significant food allergens typically represent one percent or more of the total protein; (c) examining the expressed protein for characteristics often associated with known food allergens, such as glycosylation, heat stability, and presence of disulfide bonds; and (d) monitoring the digestibility of the transgene product in simulated mammalian gastric and intestinal fluids. Although numerous nonallergens show one or more of the properties often associated with allergens, each analysis provides indirect evidence that is of some predictive value. Moreover, the tests to determine these properties were included in a decision tree that was proposed byMetcalfe et al. (1996). As far as I know, the protocol suggested in that tree has been closely followed in the industrial development of transgenic food products. However, as a result of recent problems in introducing new transgenic foods, it has become clear that an additional test is needed, namely an animal model for testing genetically modified products. An animal model is needed to provide a direct test of the allergenic properties for proteins showing potential evidence of allergenicity. Such tests cannot be done on humans directly, ethical considerations aside. Present populations have not been exposed to the engineered food in question and, as a result, would not show an adverse reaction, even if the food contained an allergen. In developing the decision tree, Metcalfe et al. (1996) pointed out the desirability of including an animal model, but did not do so because none “have been shown to predict the allergic potential of introduced proteins.” Animal models were also a major topic of discussion at a recent conference dedicated to allergy issues, “Assessment of the Potential Allergenicity of Genetically Engineered Foods” held December 5 and 6, 2000, at the National Center for Food Safety and Technology (Summit-Argo, IL). The advantages and disadvantages of each model were considered at the meeting: Brown Norway rat, guinea pig, dog, pig, and various mouse models. To be beneficial, it was considered that an animal model should: (a) show an allergic response to allergens in humans, but not to nonallergens; (b) show an allergen profile similar to that of humans—for example, the response to a strong allergen (peanut) > moderate allergen (milk) > a nonallergen (spinach leaf); (c) have a gastrointestinal system similar to humans; and (d) ideally, show an epitope response similar to humans. This latter feature was considered a desirable but not a mandatory feature in view of the wide range of epitopes that humans can recognize. The advantages, disadvantages, and current status of each model were discussed in Summit-Argo. It was agreed that, although decisive progress has been made, none of the current models meets these criteria because characterization and testing is still ongoing. Therefore, at this point it is not clear which of the models will prove to be of most value in detecting and assessing food allergens. I am personally prone to the dog because, perhaps as a reflection of having a gastrointestinal system similar to humans (Strombeck and Guilford, 1990), it is unique among animal models in having natural allergies as far as is known. The dog shows clinical symptoms typical of food allergy in humans, i.e. vomit and diarrhea (Ermel et al., 1997;del Val et al., 1999). Advances made using the dog will, therefore, benefit dogs as well as humans because of similarities in their allergic response. In recognition of these features, our laboratory started a project to determine the suitability of the dog as a predictor of allergens in humans in collaboration with Dr. Oscar L. Frick (University of California, San Francisco) and Drs. Laura Privalle and Greg del Val (Syngenta, Research Triangle Park, NC). Initiated 3 years ago, this study is now entering its final stage and is yielding encouraging results. The results, which will be published when the study is complete, suggest that the dog will be useful as an animal model. That point withstanding, the other models mentioned above warrant continued study, because, in the end, each of several could present a particular advantage in detecting and characterizing allergens in humans. One precautionary note seems in order. While proceeding with allergy testing, we must be careful not to overregulate and impose undue restrictions to stifle innovation. Rather, we should seek to formulate a balanced policy that insures food safety without hindering product development. CLOSING COMMENTS Great strides have been made in our understanding of food allergy since the problem was originally recognized by Hippocrates almost 2.5 millennia ago. Despite this rich history, large gaps remain in our knowledge and they are of such nature as to lend an element of mystery to the field. These features have led certain individuals and environmental groups to target food allergy in an effort to slow the commercial development of genetically modified crops and foods and, at the same time, utilize the issue as a fund-raising mechanism. Their efforts have been successful not only by having the intended effect, but also by negatively influencing science funding, especially in Europe. The net result has been that the participating organizations have experienced financial gain and genetically modified crops derived from research in developed countries are now being grown disproportionately in the developing world. For example, between 1999 and 2000, the area used for growing transgenic crops increased by 2% in industrial countries, whereas the area in developing counterparts, although still relatively small in total hectares, grew by 51% (James, 2000). The long-term economic effect of the shift in emphasis to developing countries could significantly impact research on transgenic crops in developed countries unless the situation changes. Such an impact on research would eventually adversely affect hunger and nutrition worldwide because, as recently pointed out in this series (e.g. Borlaug, 2000), continued progress in the genetic engineering of crops is critical to feeding future world populations. I believe, however, the problem to be transitory and that, once appropriate allergen testing capability is in place, health concerns will abate and the development of transgenic foods will continue apace. As seen above, the needs to bring about this change are not extensive. What seems to be most lacking at this stage is an animal model to identify transgenic plant proteins that either are, or have become, allergens in humans. Such a model is especially important for proteins to which humans have not been exposed. Had a reliable model been available, it is likely that StarLink corn could have avoided current problems (for example, see Barboza, 2000). Animal test data would have been available to allay consumer concern once the product was on the market. I am confident that, with progress now being made, one or more animal models will soon be available to serve as a reliable indicator of allergens in human and that a safe but reasonable testing policy will be formulated. Once such testing capability is in hand, the public will respond in a positive manner. In the long term, the food allergy and technology fields will likely benefit, rather than suffer, from this pause in their development. LITERATURE CITED 1 Altman DR Chiaramonte LT Public perception of food allergy. J Allerg Clin Immunol 97 1996 1247 1251 Google Scholar Crossref Search ADS WorldCat 2 Barboza D. December 4, 2000. Negligence suit is filed over altered corn. New York Times; Sect C:2 3 Borlaug NE Ending world hunger: the promise of biotechnology and the threat of antiscience zealotry. Plant Physiol 124 2000 487 490 Google Scholar Crossref Search ADS PubMed WorldCat 4 del Val G Yee BC Lozano RM Buchanan BB Ermel RW Lee YM Frick OL Thioredoxin treatment increases digestibility and lowers allergenicity of milk. J Allerg Clin Immunol 103 1999 690 697 Google Scholar Crossref Search ADS WorldCat 5 Ermel RW Kock M Griffey SM Reinhart GA Frick OL The atopic dog: a model for food allergy. Lab Anim Sci 47 1997 40 49 Google Scholar PubMed OpenURL Placeholder Text WorldCat 6 James C (2000) Global review of commercialized transgenic crops: 2000. The International Service for the Acquisitiion of Agri-biotech Applications, no. 21.http://www.isaaa.org/publications/briefs/Brief_17.htm 7 Metcalfe DD Astwood JD Townsend R Sampson HA Taylor SL Fuchs RL Assessment of the allergenic potential of foods derived from genetically engineered crop plants. Crit Rev Food Sci Nut Suppl 36 1996 S165 S186 Google Scholar Crossref Search ADS WorldCat 8 Nordlee JA Taylor SL Townsend JA Thomas LA Bush RK Identification of a Brazil-nut allergen in transgenic soybeans. N Engl J Med 334 1996 688 692 Google Scholar Crossref Search ADS PubMed WorldCat 9 Strombeck DR Guilford WG Small Animal Gastroenterology Ed 2 1990 346 355 Stonegate Publishing Co. Davis, CA 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)

Prakash, Channapatna S.

doi: 10.1104/pp.126.1.8pmid: 11351063

Channapatna S. Prakash “Whoever could make two ears of corn, or two blades of grass grow upon a spot of ground where only one grew before would deserve better of Mankind, and do more essential service to his country, than the whole race of politicians put together.” — The King of Brobdingnag, Gulliver's Travels by Jonathan Swift, 1727. “I believe that we have now reached a moral and ethical watershed beyond which we venture into realms that belong to God, and to God alone. Apart from certain medical applications, what actual right do we have to experiment, Frankenstein-like, with the very stuff of life? … ” — Prince Charles Windsor, heir to the British throne (Windsor, 1998). Throughout the history of humankind, there have been those who have embraced change and those who have clung to the old ways because they felt at least the risks were known. Few Edisons or Einsteins were properly recognized during their lifetime. And, since feeding ourselves was the primary occupation of mankind for most of our recorded and prerecorded history, changes in food production have been accepted slowly. The first person to try to scratch out a garden most assuredly heard derisive laughter as the mighty hunters headed off in pursuit of meat. So, we should not be surprised that eons of history are being replayed as we enter the era of biotechnology. As the fates of human society and crops have been inextricably intertwined since the dawn of civilization, an appreciation of our agricultural past may guide us in addressing societal concerns and also in ensuring minimal negative consequences from scientific pursuits. Farmers have embraced the new technology because it makes them more efficient, protects or increases yields and reduces their reliance on chemicals that, other things being equal, they would prefer not to use. Crops enhanced by biotechnology are being grown on nearly 110 million acres in 13 countries. Food ingredients produced from biotech crops are found in thousands of food products consumed worldwide. However, while no unequivocal evidence of harm to our health or the environment from these crops is known or expected, there is an intense debate questioning their value and safety. Societal anxiety over this so-called genetically modified (GM) food is understandable, and it is fueled by a variety of causes, including consumer unfamiliarity, lack of reliable information on the current safeguards in place, a steady stream of negative opinion in the news media, opposition by activist groups, growing mistrust of industry, and a general lack of awareness of how our food production system has evolved. The scientific community has neither adequately addressed public concerns about GM foods nor effectively communicated the value of this technology. Clearly, societal acceptance is pivotal to the continued development and application of biotechnology in food and agriculture. Two decades ago, many agricultural scientists rightfully saw the emerging recombinant DNA technology as a potent tool in enhancing crop productivity and food quality while promoting sustainable agriculture. Much of this early excitement and expectation was met with successive breakthroughs in scientific research on plant gene transfer methods, identification of valuable genes, and the eventual performance of transgenic crops. Plant breeders saw the technology as an additional means of crop improvement that could complement existing methods. For the first time, plant breeding was subjected to rigorous testing, and a regulatory framework was developed to oversee the commercialization of GM crops on a case-by-case basis. There has been widespread acceptance and support for biotechnology from the scientific community. Accumulated experience and knowledge of decades of crop improvement combined with expert judgment, science-based reasoning and empirical research has led to scientists' confidence that GM crops may pose no new or heightened risks that could not be identified or mitigated, and that any unforeseen hazard will be negligible, manageable, or preventable. Risks from GM crops should be monitored and measured, but concerns about these risks must also be balanced against the enormous benefits from this technology and weighed against alternative options. The strong trust that the American public has in its regulatory agencies (FDA, USDA, and EPA) has helped gain higher public acceptance of GM food in this country than in other nations. Mutant Food and Monarch Butterflies Despite the promised benefits, global negative reaction to GM crops ranges from mild unease to strong opposition. Typical questions asked about GM crops include: Is it ethical for scientists to modify living organisms around us? Is it morally right to tamper with our food supply? Is the genetic modification of crops inherently hazardous? Despite the built-in safeguards, can we unwittingly make our foods unsafe? What about the long-term consequences of consuming such foods? Do GM crops affect the environment or the wild ecosystem, reducing crop biodiversity, beneficial insects, or the revered monarch butterfly? Could these crops lead to the development of noxious “superweeds”? Are we introducing these crops into our environment without fully understanding the consequences of such action? What about genetic pollution? Can these genes be transferred to other organisms including humans and animals? In addition, there are also larger and even more important sociopolitical issues such as anxiety about the control of food and agricultural systems, including questions about the pervasive impact of globalization. How can scientists allay public concerns considering the complexities of these issues? Creating an awareness of agricultural history may provide a good beginning for our efforts to help alleviate consumer unease about GM foods. It may also educate scientists about the relevance of the societal context to our research. Most risk issues related to current GM crops are not unique when placed in the context of how agriculture was developed through crop domestication over many millennia and how we have bred modern crop varieties in the past century. As Frary and Tanksley (2000) put it, “The issue is not whether we should modify the genetics of crop plants. We embarked on that road thousands of years ago when plants were first domesticated. Instead of simply judging the vehicle through which we make genetic changes, we need to weigh the potential consequences that such modifications hold for the society and the environment.” Crop Evolution and Human Civilization Agriculture evolved independently in many places on this earth, but the earliest evidence of farming dates 10,000 years ago in present day Iraq (Heiser, 1990). For much of the 200,000 or so years prior to agriculture, humans lived as nomadic hunters, gatherers, and scavengers surviving solely on wild plants and animals. Subsequent domestication of these wild plants and animals from their natural habitats launched agriculture, thus radically transforming human societies. This occurred initially in the Fertile Crescent, the Andean region in South America, Mexico, and parts of Asia, but diffused throughout much of the globe. A change from the nomadic lifestyle to farming led us to become community dwellers, eventually spawning the development of languages, literature, science, and technology as people were freed from the continuous daily task of finding food. Some regions caught on much faster than others, by margins of thousands of years (Diamond, 1999). Plants have also evolved or, more accurately, they have been changed rapidly by human intervention (Harlan, 1992). Every crop plant grown today is related to a wild species occurring naturally in its center of origin, and progenitors of many of our crops are still found in the wild. Early humans must have tried eating thousands of feral plant species from a pool of a quarter of a million flowering plants before settling down on less than one thousand such species, which were subsequently tamed and adapted to farming. A little over 100 crop species are now grown intensively around the world, with only a handful of them supplying us with most of what we now eat. Through a process of gradual selection, our ancestors chose a very tiny section of the wild plant community and transformed it into cultivated crops. Some profound alterations in the plant phenotype occurred during such selection, and these include determinate growth habit; elimination of grain shattering; synchronous ripening; shorter maturity; reduction of bitterness and harmful toxins; reduced seed dispersal, sprouting and dormancy; greater productivity, including bigger seed or fruit size; and even an elimination of seeds, such as in banana. These changes reduced the survivability of crops in the wild, and thus a feature that transcends all of our crops is the reduction of weedy traits from wild plants. Present crops are thus totally dependent upon human care for their survival, and modern crop varieties would persist in the wild “no longer than a Chihuahua would last in the company of wolves” (Trewavas, 2000). Most crops that supply our food were thus obtained at the end of the Stone Age, often from a relatively narrow pool of extant wild genetic diversity. Additional diversity arose within such cultivated crops through new mutations and natural hybridization, and through judicious selection and perpetuation by farmers who maintained them as land races. Varied uses and preferences brought forth further diversification such as in corn (popcorn, sweet corn, dent corn, broom corn, and flour corn for tortilla and corn bread) or the derivatives of ancestral cabbage (kale, kohl rabi, brussels sprouts, cabbage, cauliflower, and broccoli). With the advent of transoceanic navigation and the “discovery” of the New World, crops were moved around the world rapidly, often achieving prominence in adopted homes far beyond their natural centers of origin or domestication. For instance, the United States is the leading producer of corn and soybean in the word, yet these crops are native to Mexico and China, respectively. The world's largest traded commodity, coffee, had a humble origin in Ethiopia, but now much of it is produced in Latin America and Asia. Florida oranges have their roots in India, while sugarcane arose in Papua New Guinea. Food crops that are now so integral to the culture or diet in the Old World, such as the potato in Europe, chili pepper in India, cassava in Africa, and sweet potato in Japan, were introduced from South America. For that matter, every crop in North America other than the blueberry, Jerusalem artichoke, sunflower, and squash are borrowed from elsewhere! A few sources of our food are also recent domesticates. Chinese gooseberry occurs wildly in China and is not edible. But careful breeding made it palatable, and it was re-christened “Kiwi fruit” in New Zealand after its introduction there early in the 20th century. The modern strawberry with big fruits is a product of the accidental crossing of two wild species from Virginia (United States) and Chile in France in the mid-18th century. Rapeseed, grown in India for centuries, was altered recently through classical breeding to eliminate the toxic erucic acid and smelly glucosinolates to result in canola—Canadian oil. Triticale, a completely new crop, was artificially sythesized a few decades ago by combining the genomes of wheat and rye (two distinct genera that do not interbreed in nature). It is now grown on over three million acres worldwide. Modern bread wheat itself is also a fairly recent crop in the evolutionary time scale, having arisen only about 4,000 years ago through hybridization of tetraploid (pasta or durum) wheat with inedible goat grass. From Mesopotamia to Mendel While humans have always molded the evolution of crop plants, such changes imposed by farmers occurred over several millennia, leading to rich crop diversity—especially in traits related to their planting or consumption. At the same time, global population grew very slowly until the mid-19th century. It took 1,800 years for the global population to climb from an estimated 300 million around the time when Christianity began, to reach its first billion. But it took only 12 years to add the last billion, rising from five billion people in 1987 to six billion two years ago. Fortunately, parallel scientific developments in agriculture ensured that food production kept pace with the population explosion of the past century (Conway, 1999). Beginning with Mendel's study of peas, knowledge of genetics helped usher in scientific crop development, resulting in high-yielding varieties. Food production increased in every part of the world in the past few decades, including in Africa. Per capita food consumption has also increased steadily everywhere except in parts of sub-Saharan Africa. In the United States and Canada, where such scientific developments and their applications were most intense, one average farmer now produces enough to feed nearly 150 people! In crops subject to intensive scientific attention—corn, wheat, and rice—the productivity levels increased severalfold. For example, U.S. corn growers averaged 26 bushels of corn per acre in 1928 and 134 bushels per acre in 1998 (National Corn Growers Association, 2001). Such a prodigious increase in agricultural production was underpinned by scientific crop improvement methods along with other developments, including the use of irrigation, improved soil fertility management, mechanization, and control of diseases and pests (Conway, 1999). To develop better crop varieties, scientists have used an array of tools. Artificial crossing, or hybridization, helped us assimilate desirable traits from several varieties into elite cultivars. When desired characteristics were unavailable in the cultivated plants, genes were liberally borrowed from wild relatives and introduced into crop plants. When a crop variety refused to mate with the wild species, various tricks were employed to force them to intermingle, such as the use of the carcinogenic chemical colchicine or by rescuing the hybrid embryos with tissue culture methods. Hybrid vigor was exploited in crops such as corn and cotton to boost productivity. When existing genetic variation within the cultivated germplasm was not adequate, breeders created new variants using ionizing irradiation (gamma ray, x-ray, neutron), mutagenic chemicals (ethyl methane sulfate, mustard gas), or through somaclonal variation (cell culture). Most people who are concerned about modern biotechnology have little or no knowledge of the processes that have been used to transform crops in the past. Nor are they likely aware that crops have been continually altered over time or that, without human care, they would cease to exist. Using a variety of tools over the past few decades, plant breeders have radically transformed our crop plants by altering their architecture (such as the development of dwarf wheat and rice), shortening growing seasons, developing greater resistance to diseases and pests (all crops), and developing bigger seeds and fruits (Figs.1 and 2). These crops are also more responsive to management and better adapted to diverse ecological conditions. Improved food quality also resulted through fewer toxins (canola), better digestibility (beans), increased nutrition (high-protein corn), better taste, longer shelf life (thus withstanding long transportation and storage), and enhanced freshness in many vegetables and fruits. A 1,000-fold increase in the marble-sized wild Lycopersicon resulted in the modern tomato that can now weigh as much as a kilogram (Frary and Tanksley, 2000). Fig. 1. Open in new tabDownload slide Cultivated tomato (left) and its wild relativeLycopersicon pimpinellifolium (right; approximate diameter of smaller tomato = 1 cm). (Photo kindly provided by Steve Tanksley.) Fig. 1. Open in new tabDownload slide Cultivated tomato (left) and its wild relativeLycopersicon pimpinellifolium (right; approximate diameter of smaller tomato = 1 cm). (Photo kindly provided by Steve Tanksley.) Fig. 2. Open in new tabDownload slide Modern corn hybrid (right), its wild relative teosinte (left), and their hybrid (cob in the center). (Photo kindly provided by John Doebley.) Fig. 2. Open in new tabDownload slide Modern corn hybrid (right), its wild relative teosinte (left), and their hybrid (cob in the center). (Photo kindly provided by John Doebley.) Modern farming has steadily increased the supply of relatively safe, affordable, and abundant food not only in the developed world, but also in most developing countries. An average American family now spends only 11% of its income on food and yet has access to better food choices with more variety and nutrition than ever before. Without scientific developments in agriculture, we would otherwise be farming on every square inch of arable land to produce the same amount of food! Using gene transfer techniques to develop GM crops thus can be seen as a logical extension of the continuum of devices we have used to amend our crop plants for millennia. When compared to the gross genetic alterations using wide-species hybridization or the use of mutagenic irradiation, direct introduction of one or a few genes into crops results in subtle and less disruptive changes that are relatively specific and predictable. The process is also clearly more expeditious, as the development of new cultivars by classical breeding typically takes from 10 to 15 years. The primary attraction of the gene transfer methods to the plant breeder, however, is the opportunity to tap into a wide gene pool to borrow traits, obviating the constraints of cross-compatible crop species. Addressing Public Concerns While direct gene transfer is still a relatively new approach, many concerns arising from its use may be addressed with the “benchmark” of conventionally bred varieties, as we have the accumulated experience and knowledge with the latter for more than a century. While it seems logical to express a concern such as “I don't know what I am eating with GM foods!” it must be remembered that we really never had that information before with classically bred crops. With GM crops, at least we know what new genetic material is being introduced, so we can test for predictable and even many unpredictable effects. Consider, for example, how conventional plant breeders would develop a disease-resistant tomato. They would introduce chromosome fragments from its wild relative to add a gene for disease resistance. In the process, hundreds of unknown and unwanted genes would also be introduced, with the risk that some of them could encode toxins or allergens, armaments that wild plants deploy to survive. Yet we never routinely tested most conventionally bred varieties for food safety or environmental risk factors, and they were not subject to any regulatory oversight. We have always lived with food risks, but in the last few decades we have become increasingly more adept at asking questions. To address the concern about long-term health consequences of GM foods, it is instructive to recognize that we worried little about such impacts when massive amounts of new proteins (and unfamiliar chemicals) were introduced into our foods from wild species or when unknown changes were created through mutation breeding. When new foods from exotic crops are introduced, we often assimilate them easily into our diets. What's more we rarely, if ever, before asked the same questions that we now pose about GM crops. Many so-called functional foods, health foods, and nutraceuticals have been entering into the mainstream American diet lately, with little or no regulation or testing. We do not question the long-term health implications of these food supplements, even though they involve relatively large changes in our food intake. In contrast, the GM foods currently on the market have been tested extensively and judged to be substantially equivalent to their conventional counterparts, with just one or two additional proteins present in miniscule amounts (introduced into a background of thousands of proteins). And, those proteins are broken down either during processing or digestion, with little long-term consequence. In food products such as oil, starch, and sugar, such proteins are not even found. A nagging potential problem with a new protein in food is that it could be a potential allergen. As most food allergens are now well studied, we know that they are found in few defined sources (peanut and other grain legumes, shellfish, tree nuts, and a handful of other foods) and share many similar structural features. Moreover, they must be present in huge proportions in our food, and we must be sensitized to them over time for them to cause any adverse effects. Thus, it is highly unlikely for new allergens to be introduced into our food supply from GM plants. Historical Absence of Zero Risk There is no such thing as safe food, and there never has been! That is not to suggest that all of our foods are dangerous, only an acknowledgment that trace levels of such contaminants as toxins and carcinogens are present in everything we eat. But a primary rule of toxicology, articulated over 400 years ago by Paracelsus, refers to the importance of dosage: “Every substance is a poison, but it is the dosage that makes it poisonous” (Poole and Leslie, 1989). While not alarming, our daily food naturally contains thousands of chemicals, and many of them are shown to be carcinogenic or hazardous in lab animal studies with huge doses. We consume roughly 5,000 to 10,000 natural toxins daily, as plants have evolved to produce an array of chemicals to protect themselves against pests, diseases, and herbivores (Ames et al., 1990a). For instance, roasted coffee has over 1,000 chemicals, of which 27 have been tested and 19 of them found to be rodent carcinogens (Ames and Gold, 1997). The fat-soluble neurotoxins solanine and chaconine are present in potatoes and can be detected in the bloodstream of all potato eaters (Ames et al., 1990b). Naturally then, when crops are bred for resistance to pests by transferring genes through conventional methods, the resistance is often accompanied by an increase in such toxic compounds. Thus, it is not true that we never had problems with conventionally bred varieties. Any crop variety found to pose a real health risk was promptly removed from the market, but those varieties (in contrast to GM crops) were never routinely tested. One pest-resistant celery variety produced rashes in agricultural workers and subsequently was found to contain 6,200 ppb of carcinogenic psoralens compared to 800 ppb in the control celery (Ames et al., 1990). This celery was removed from cultivation and that was also the case with the potato variety Lenape, which contained very high levels of toxic solanine. We have always learned from trial and error with all innovations. Similarly, crop improvement practices evolved over time with continued refinement. It is common, though, for human nature to generate an exaggerated fear of new innovations while perceiving older or “natural” products as always more benign. Huber (1983) discusses this double standard in the larger context of risk regulation. We have always been lenient toward existing known and greater hazards, even as we create “gatekeepers” to minimize new risks. Thus, we fail to recognize and “exorcise” much larger older risks. While most food hazards arise from pathogens such as Escherichia coli 0:157, Listeria monocytogenes, andSalmonella enterica along with mycotoxins produced by fungi (and thus a function of food storage and handling), certain foods containing toxic compounds are known to produce adverse health consequences over time. Cassava, eaten by a large population in Africa, contains cyanogenic glucosides, which cause limb paralysis if consumed before extensive processing. Solanin in tomato and potato is known to cause spina bifida. Vetch pea, a common legume known for its hardiness—and thus popular in India among poor farmers—contains highly dangerous neurotoxins that cause untold misery. Phytohemagglutinin, found in undercooked kidney beans, is toxic. And peach seeds are extremely rich in cyanogenic glucosides. None of these were subject to any mandatory testing before they were introduced into the food chain, nor are they subject to any regulation now. But if the current regulatory standards imposed on GM crops were to be invoked for traditional crops, most of them would fail to meet their requirements. Humans have built-in natural defenses that protect us against normal exposure to toxins. But, according to Ames and Gold (1997), we have not evolved to achieve “toxic harmony” with everything we eat, because natural selection occurs much too slowly and because much of what is in our diet today was not eaten at all when we were hunter-gatherers. A balanced mixture of foods normally provides adequate nutrition. However, none of the crops grown today were selected with our nutritional requirements in mind. Instead they were chosen intuitively, by our ancestors, from among the edibles that could be found around them. Thus, the most important food crop in the developing world—rice—has no provitamin A and little iron in its endosperm. This has led to horrific problems, such as blindness among millions of children due to vitamin A deficiency, and iron-deficiency anemia in nearly a billion women dependent on a rice diet. Biotechnology research, far from causing any new food safety problems, has already demonstrated its potential in enhancing the nutritional quality of our food and is also being employed to reduce harmful toxic compounds that exist in our food. What about the Environment? All of us have to eat to live, and organized food production is the most ecologically demanding endeavor we have pursued. Agricultural expansion over the millennia has destroyed millions of acres of forestland around the world. Alien plant species have been introduced into non-native environments to provide food, feed, fiber, and timber, and as a result have disrupted local fauna and flora. Certain aspects of modern farming have had a negative impact on the biodiversity of crop plants and on air, soil, and water quality; nevertheless, it sustains and nurtures most of the world's six billion people with adequate nutrition and affordable food. How can we address the potential environmental concerns of GM crops in the context of our experience with traditional crop variety deployment? We have continuously introduced genes for disease and pest resistance through conventional breeding into all of our crops. Traits, such as stress tolerance and herbicide resistance, have also been introduced in some crops, and the growth habits of every crop have been altered. The risk of crop gene flow to weedy relatives has always existed, and such “gene flow” occurs where possible. Thus, it is comforting to recognize that no major “superweeds” have developed since the advent of modern plant breeding, although there have been a few instances of crops ever becoming weedy or of weeds becoming more invasive due to gene transfer from crops. Most noxious weeds, such as kudzu, water hyacinth, and parthenium, resulted from the introduction of semidomesticated wild plants into non-native environments without the checks and balances of their native pests. Yet, there are probably no dwarf plants among the wild Oryzaspp. and Triticum spp. populations in the Middle East or Asia, despite the fact that we now have been growing diminutive rice and wheat varieties for decades. The risk of gene transfer to wild plants is exacerbated when crops are planted in an area with compatible weedy relatives (as often seen in their centers of origin), when such species are promiscuous out-crossers (canola), or, most importantly, when the introduced genes enhance the reproductive fitness of the recipient weeds (although most genes introduced into crop plants, conventional or biotech, have little value in the wild). The risk of gene transfer to weeds is similar with both conventional and GM crops and is not contingent on how we introduced these genes into plants. We must be vigilant to ensure that weeds do not become noxious as a result of any new crop variety. The current case-by-case testing and monitoring approach with biotech crops is a good regimen for the future, while the past experience with conventional crops provides assurance that such risks will be minimal and manageable. Crop biodiversity is another issue of concern. The popularity of high-yielding varieties has already narrowed the genetic variation found in major crops. Biotechnology, if employed strategically, can reverse this through the recovery of older varieties that were discarded for lack of certain features (such as resistance to new disease strains), because modern gene transfer can restore such traits. Biotechnology research is also enabling the development of better methods for ex situ preservation of germplasm, such as cryopreservation, whereby valuable germplasm is being stored and thus saved from extinction. The introduction of corn with a single transferred Bt gene has led to some concern about its ecological impact. While this concern should not be dismissed, it should be balanced with our hindsight and experience with corn itself, an introduced alien species now grown on 75 million acres in the United States, where none existed about 1,000 years ago. A crop introduced into a new environment entails the wholesale introduction of thousands of new genes. When grown on massive amounts of land, it exerts considerable ecological impact on the native fauna and flora, including beneficial insects. In contrast, the introduction of one or two genes into this background of 50,000 genes present in corn will have relatively less effect on the environment. While the initial fear about the reported damage to monarch butterflies from Bt corn has not held up in additional studies, one also needs to consider the negative impact of alternate practices (such as pesticide sprays) and recognize the potential for positive impacts on beneficial insects by the GM crop due to the specificity of the insect target(s). For that matter, any concern about “gene pollution” pales in comparison to the massive “risk” of alien crop introduction, as 95% of the crop area in the United States now consists of such introduced crops. Concern about horizontal transfer of genes from GM crops to other organisms, such as bacteria, has also been expressed. But it appears highly unlikely that the risk is dependent upon the method of gene introduction. An inherent feature of biotechnology is that it lends itself easily to molecular detection of introduced genes, but a true measure of risk can only come in comparisons with classically bred crops where little or no such studies have been performed. Concerns such as random gene insertion, gene instability, and genomic disruption due to gene transfer have been expressed, but they are unlikely to be unique to GM crops or of any significance considering our current knowledge of genomic flux in plants. Worries about mixing genes from unrelated species ignore the history of plant breeding and the existing overwhelming sequence similarity of genes across kingdoms. Nevertheless, scientific research aimed at risk analysis, prediction, and prevention, combined with adequate monitoring and stewardship, must continue so that negative ecological impact from GM crops will be kept to a minimum. Most problems raised by science can be solved by additional science itself. For example, appropriate promoters may ensure that pollen will not express genes toxic to beneficial insects, while gene expression strategies, such as sterile pollen, could reduce the risk of gene flow. One must also recognize the potential positive impact of GM crops on the environment, such as decreasing agricultural expansion to preserve wild ecosystems; improving air, soil, and water quality by promoting reduced tillage, reducing chemical and fuel use; improving biodiversity through resuscitation of older varieties and promotion of beneficial insects; and cleaning up contaminated soil and air through phytoremediation. As we chart ahead with more exciting developments in biotechnology, such as genomics, and grapple with issues arising from consumer acceptance of innovations, historical knowledge on societal adoption of technological innovations may provide some valuable perspectives to scientists. Many innovations that would be good candidates for generating consumer apprehension and concern today were introduced in the past without concern because the public was less informed about innovation. The precautionary principle was never invoked to ensure the scientific certainty that crop varieties developed using nuclear irradiation or chemical mutagens were safe. And food labeling was never demanded for bread wheat improved with the addition of hundreds of unknown goat grass genes. Many other innovations that are now commonplace in our lives were met with skepticism and opposition when first introduced. Such fear of technology was especially more pronounced in food-related innovations (e.g. Pasteurization, canning, freezing, the microwave oven). However, once consumers recognize that new innovations can enhance their quality of life and once they understood that risks are either minimal or manageable, such technology eventually could enjoy public acceptance. This includes even those “disruptive” technologies that replace older ones (e.g. cars versus horse buggies, compact disc versus cassette tape). Nevertheless, there are historical instances of useful innovations that have not been readily accepted due to a variety of reasons, such as recalcitrance to adapt (e.g. Dvorak versus QWERTY keyboard), entrenched economic interests opposing change (e.g. the metric system in the United States; Beta versus VHS videotape), ideological opposition (e.g. plant breeding during Stalin-era Soviet Union by Lysenko), exaggerated notions of risk (e.g. food irradiation), ill-timed product introductions, and serious conflicts with societal values and beliefs. Humans and crops will always be mutually dependent on each other's survival, and the guided evolution of crops will continue but increasingly will be more knowledge-based and responsible. An appreciation of the history of agricultural development however may provide us with a useful roadmap for devising appropriate strategies to informing and rationalizing societal responses to crop improvement. Paraphrasing the American philosopher George Santayana, ignoring history may condemn us to repeat it, but an understanding of the past may as well lead us to an enlightened future. ACKNOWLEDGMENTS I am grateful to many contributors to my Internet discussion list Agbioview (www.agbioworld.org) for enriching my knowledge on these issues. I thank Gregory Conko, Tom DeGregory, Paul Gepts, Dan Holman, Richard Levine, Alan McHughen, and Neal Stewart for helpful comments on the manuscript. LITERATURE CITED 1 Ames BN Gold LS Chemical carcinogens: too many rodent carcinogens. Proc Natl Acad Sci USA 87 1990 7772 7776 Google Scholar Crossref Search ADS PubMed WorldCat 2 Ames BN Gold LS Pollution, pesticides and cancer misconceptions. What Risk? Bate R 1997 173 190 Butterrworth-Heinemann Boston 3 Ames BN Profet M Gold LS Dietary pesticides (99.99 percent all natural). Proc Natl Acad Sci USA 87 1990a 7777 7781 Google Scholar Crossref Search ADS WorldCat 4 Ames BN Profet M Gold LS Nature's chemicals and synthetic chemicals: comparative toxicology. Proc Natl Acad Sci USA 87 1990b 7782 7786 Google Scholar Crossref Search ADS WorldCat 5 Conway G The Doubly Green Revolution. 1999 Comstock Publishing Associates Ithaca, NY 6 Diamond J Guns, Germs and Steel: The Fates of Human Societies. W.W. 1999 Norton New York 7 Frary A, Tanksley S (2000) The origin of crop species-accelerated evolution with mankind at helm. SCOPE-GM Food Controversy Forum. http://scope.educ.washington.edu/gmfood/commentary/show.php?author=Frary(April 3, 2001) 8 Harlan J Crops and Man. 1992 American Society for Agronomy and Crop Science Society of America, Inc. Madison, WI 9 Heiser CB Jr Seed to Civilization: The Story of Food. 1990 Harvard University Press Cambridge, MA 10 Huber P Exorcists vs. gatekeepers in risk regulation. Regulation 7 1983 23 32 Google Scholar OpenURL Placeholder Text WorldCat 11 National Corn Growers Association (2001) Corn Production in the U.S.: Hinton Cal. http://www.ncga.com/03world/main/index.html (April 3, 2001) 12 Poole A Leslie GB A Practical Approach to Toxicological Investigations. 1989 Cambridge University Press Cambridge, UK 13 Santayana G The Lite of Reason. 1998 Prometheus Books Amherst, NY 14 Swift Jonathan Gulliver's Travels, Unabridged Edi-tion, 1999. 1727 Dover Publications, Albuquerque NM 15 Trewavas A (2000) GM is the best option we have. Agbio-view.http://agbioview.listbot.com/cgibin/subscriber?Act=view message&list id=agbioview&msg num=395&start num=410 16 Windsor Prince Charles Seeds of disaster. 1998 Daily Telegraph London , June 10, 1998 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)

Machuka, Jesse

doi: 10.1104/pp.126.1.16pmid: 11351064

Jesse Machuka Few would disagree that the many claims and counterclaims concerning what biotechnology can or cannot do to solve Africa's food insecurity problem have mainly been made by non-Africans. It is no wonder that Florence Wambugu's (1999) excellent article titled “Why Africa needs agricultural biotech” is now widely cited by those who support the view that developing countries, particularly in SubSaharan Africa (SSA), stand to gain the most from modern biotechnology applications. The article explained in a nutshell some of the potential benefits Africa stands to gain by embracing biotechnology. Although opinions differ regarding the role biotechnology can play in African development, all (hopefully!) must agree about the urgency to eradicate the perpetual cycle of hunger, malnutrition, and death in a world of plenty. It is an acknowledged fact that Africa is endowed with tremendous natural (including genetic) and human wealth that has yet to be harnessed to the benefit of its people. Sadly, some of this reservoir of resources have been disintegrating and the trend is bound to accelerate unless urgent measures are taken to stop and reverse this drift. Since farming is the most important source of income and sustenance for about three quarters of the population of SSA, there is no doubt that agricultural biotechnology (agbiotech) can make very substantial contributions toward increasing food production by rural resource-poor farmers, while preserving declining resources such as forests, soil, water, and arable land (Bunders and Broerse, 1991). However, application of modern biotechnology tools is not likely to significantly reduce the contributions that conventional disciplines such as soil science, breeding, plant health management, agronomy, agricultural economics, and social sciences make to enhance crop production. In villages, constraints to crop production include pests, diseases, weeds, environmental degradation, soil nutrient depletion, low fertilizer inputs, inadequate food processing amenities, poor roads to markets, and general lack of information to make science-based decisions that underlie farming methodologies and systems. For some of these constraints, biotechnology is the most promising recourse to alleviate them. For example, an insect known as Marucapodborer is the major constraint restricting increased grain legume production in Africa, often causing up to 100% crop failure during severe attacks on important crops such as cowpea (Fig. 1). Many decades of conventional breeding efforts have failed to control this pest. However, recent research in U.S. universities and at the International Institute of Tropical Agriculture based in Ibadan, Nigeria, shows that this pest can be controlled by applying biotechnology tools. This is just one of the myriad problems facing food production systems in Africa for which biotechnology can provide at least some solutions. Although biotechnology has potential downsides, the major “concerns” in Africa are not so much about justifying its role in agricultural production—the “why” question. It is conceivable that the millions of dollars being wasted each year by antibiotech activists elsewhere could go a long way to help build badly needed capacity for agbiotech research in Africa! The key issues revolve around questions of where, when, how, and who will do biotechnology for Africa's benefit? If we are thinking of ultimate answers, then there is probably only one answer: biotechnology for Africa should mostly be done in Africa and mostly by Africans themselves, now. And yes, this is being realistic, and it can be done, if there is consensus and goodwill. Fig. 1. Open in new tabDownload slide Podborer larvae infest legume pods. Inset, Podborer larva on cowpea callus in bioassay to test efficacy of cowpea pest resistance characters. Fig. 1. Open in new tabDownload slide Podborer larvae infest legume pods. Inset, Podborer larva on cowpea callus in bioassay to test efficacy of cowpea pest resistance characters. Despite many years of agricultural and other “development” aid and promises by different agencies related to increased food security and poverty eradication, those of us who live in Africa do not have confidence that things are getting any better. Because of this history, some are either pessimistic or skeptical, but the majority remain cautious and optimistic, that modern biotechnology opens new opportunities to address constraints that have led to declining harvests in farmers' fields in the midst of an expanding population. Richard Manning (2000) makes a good point when he suggests that one way to feed the increasing world population is to help “third world scientists to feed their own people, while ensuring sensitivity to culture and environment that we missed in the first green revolution” (http://www.mcknight.org/crop-frontier.htm). For SSA, the pertinent question is, how does the international community of public and private institutions and donors, governments, scientists, and other actors help African scientists (and farmers!) to feed their own people? It is crucial that scientific information reaches farmers in the rural areas who have space to practice farming and that other actors such as agricultural scientists and extensionists interact with farmers to attain acceptance and use of new technologies for sustainable food production and development. In this regard, we must have it in mind that life science technologies that offer hope to farmers, such as agbiotech, belong to the farmer. We must also ensure that the technology not only reaches farmers but that they understand it and are empowered to use it. Furthermore, our starting point is not the “ignorant peasant” but the practices, techniques, experience, and knowledge of the African farmer built over the centuries (Duprez and DeLeener, 1988). A good example of how biotechnology can reach rural farmers involves a special program by the Biotechnology Development Co-operation of the Netherlands Government, the Kenyan Ministry of Research, Science and Technology, and the small-scale farming system stakeholders. The program structure is designed to ensure that biotechnology reaches the small farmer (end-user) through a bottom-up approach steered by the Kenya Agricultural Biotechnology Platform. The composition of farmers includes male and female farmers, oxen owners, different age groups from different subvillages, etc. Projects under the Kenya Agricultural Biotechnology Platform funding bring together collaborators who include scientists from research institutions such as universities, national agricultural research centers, and farmers. A Farming Systems Research Program ensures that farmers participate in the research as partners with scientists, extensionists, and other actors and enables scientists also to utilize indigenous knowledge in research and development. This prevents “cut and paste” approaches that may be foreign market-driven and which tend to provide short-term, quick-fix solutions to unique problems faced by small-scale farmers in Africa who have developed their own unique crops, cropping, and farming systems that cannot be changed without their full and careful involvement. Since 1992, Farmers Research Groups and Farmers Extension Groups, established along the lines of Farming Systems Research Programs, have been in existence in the Lake Zone of Tanzania for purposes of farmer participatory research. This experience shows that such participatory methods increase farmers' inputs in the decision-making process as well as in the dissemination of research products through their involvement in field trials, farmers' and “on-station” field days, PRA surveys, and farmer-to-farmer diffusion of information through Village Extension Workers rather than institutional extension (Fig. 2). Since Farmers Research Groups represent different geographic zones and hence different agro-ecological and farming systems, linkage mechanisms that bring together their experiences need to be established to allow horizontal and vertical dissemination of technologies as well as collaboration in the SSA region. Obviously, this is not the only way that research results from the laboratory reach farmers' fields, but it illustrates the fact that applied agbiotech research can similarly be targeted and tied to meet specific needs of rural farmers, both in the short- and long-term, in the face of scant resources. With African farmers and scientists working together to set the research agenda, there is hope that the research will focus on uniquely African (“orphan”) crops such as millet and sorghum that are very important in marginal, famine-prone regions such as the Sahel and Horn of Africa. Root and tuber crops such as yam, sweet potato, and cassava may also begin to receive the attention they deserve. Fig. 2. Open in new tabDownload slide The bottom up approach: Farmers and scientists discuss “crazy top” disease (inset) in maize caused by the downy mildew pathogen Peronosclerospora sorghi in Ogbomoso, southwestern Nigeria. Fig. 2. Open in new tabDownload slide The bottom up approach: Farmers and scientists discuss “crazy top” disease (inset) in maize caused by the downy mildew pathogen Peronosclerospora sorghi in Ogbomoso, southwestern Nigeria. Although Africa lags far behind other regions when it comes to public information and awareness of biotechnology issues, excellent work is being done by organizations such as the Nairobi-based African Biotechnology Stakeholders Forum and South African-based AfricaBIO to educate the general public in biotechnology. Opportunities abound for scientists in Africa to get involved in these efforts that are urgently needed if Africans are going to decide for themselves what biotechnology can do for them rather than let others decide for them, especially anti-genetically modified organism activists! There is also urgency to educate policy makers in African governments and the private sector concerning the need to support and invest in biotechnology Research and Development (R&D). At the same time, the international donor community needs to begin to trust Africans and allow them to manage their research agenda for themselves. They can take the cue from very successful initiatives undertaken by the Rockefeller Foundation in Africa. There are enough African scientists around to make a difference on farmers fields if resources are properly channeled for agricultural R&D. African scientists and science managers in government and other institutions as well as farmers, on the other hand, need to be efficient and faithful in the way they manage research programs and funds if they are going to be trusted with money by national and international donors. The current success in tissue culture-aided production and multiplication of disease-free planting materials for cassava, yam, banana, plantain, citrus, and flowers in countries such as Kenya and Ghana is attracting private sector companies who are seeing the potential to invest in successful new biotechnologies. On November 8–11, 2000, the Strategic Alliance for Biotechnology Research in African Development (SABRAD) held a workshop in Accra, Ghana, that brought together more than 150 participants from southern, East, Central, and West Africa as well as partners from the U.S. 1890 Land Grant Universities, U.S. Department of Agriculture, Food and Agricultural Organization of the United Nations, United Nations Environment Program, International Agricultural Research Centers, other non-governmental organizations, private companies, and journalists. International Agricultural Research Centers were represented by the Mexican-based International Maize and Wheat Improvement Centre and International Institute of Tropical Agriculture. The theme of this first SABRAD Workshop was “Enabling Biotechnology for African Agriculture.” Increasing education and awareness and formulation of regulatory (policy) frameworks that would allow access to modern biotechnology for R&D were identified as key priorities for enabling biotechnology for African development that targets resource-poor rural farmers. The one thing that was unique at the Accra meeting was that Africans themselves were at the center of discussions to work out plans for enabling biotechnology to take root in their respective countries. The action plans agreed upon will be implemented through networking between regions. The ultimate socio-economic impact is food self-sufficiency and improved living conditions of resource-poor farmers who were identified as the target recipients for products generated from biotechnology applications. We live in a world that has become an increasingly interdependent “global village” due to advances in information and transportation technology. In this global village, millions have plenty of food to throw away, while millions of others die daily because they have nothing to eat. It is not always true that those with surplus food do not care about those who die in near and far away places! In Africa itself, there are many that have plenty of food, acquired either genuinely or by stealing public wealth, and who still watch their hungry neighbors die helplessly. Although Africans are thankful for development and relief aid, they are uncomfortable about their condition of continuous dependence on handouts that come in many forms, including food and expatriate foreign aid, with no permanent solutions apparently in sight. The SABRAD initiative is one step in the right direction that deserves support from all those who want to help African scientists and farmers to feed their own people. LITERATURE CITED 1 Bunders FG Broerse EW Appropriate Biotechnology in Small-Scale Agriculture: How to Reorient Research and Development. 1991 CAB International Wallington, Oxon, UK 2 Duprez H DeLeener P Agriculture in African Rural Communities. Macmillan and Technical Centre for Agricultural and Rural Co-operation. 1988 CTA London 3 Manning R Food's Frontier: The Next Green Revolution. 2000 North Point Press New York 4 Wambugu F Why Africa needs agricultural biotech. Nature 400 1999 15 16 Google Scholar Crossref Search ADS WorldCat 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)

Siedow, James N.

doi: 10.1104/pp.126.1.20pmid: 11351065

James N. Siedow Recent issues of Plant Physiology have contained a marvelous series of essays dealing with issues and controversies that surround the introduction and use of crops developed through the application of recombinant DNA technologies and genetically modified organisms (GMOs). These articles have provided considerable insight and thoughtful analysis of some of the major issues related to this timely topic. Among the points raised throughout these essays is the important role that GMOs will play as one of the components needed to enhance future agricultural productivity. Continued improvements in crop quality and productivity are crucial if we are to be in a position to feed the world of 10 billion people that will come into existence sometime after the middle of the current century. In the first essay in this series, Chris Somerville admonished plant biologists to make their voices heard in the ongoing GMO debate. However, plant biologists who make their voices heard on this issue, and that should include every member of the American Society of Plant Biologists, need to be knowledgeable on many aspects relating to GMOs, a number of which go beyond the science involved. One difficulty with many plant biologists in this regard is that we know a lot about the biology but often much less about the agricultural, sociopolitical, and economic issues that bear on the discussions surrounding GMOs. This is particularly true when talking about GMOs in terms of world agriculture. I will admit to having been relatively ignorant of agriculture worldwide myself until several years ago when I first read the book written by M.J. Chrispeels and D.E. Sadava,Plants, Genes and Agriculture, which remains an excellent primer on the topic. Recognizing this general deficiency, I would like to recommend three books to anyone interested in the larger topic of feeding the world's population and in particular to those of you who are publicly engaged in the GMO debate. The first book, Feeding the Ten Billion: Plants and Population Growth, is written by Lloyd T. Evans. Evans is a crop physiologist from Australia and takes the interesting tack of following the progressive development of agriculture through time, going from a population of five million about 10,000 years ago, to the six billion reached a couple of years ago. Evans notes at the outset that the book is not meant to be an all-inclusive history of agriculture, and it is not. However, much agricultural history is woven throughout the fabric of the text in a very readable fashion. Evans also does a good job of illustrating how advances in our understanding of plant biology have been incorporated into agricultural practices. It is interesting that although plant physiology began to be applied to agriculture in a knowledgeable manner in the first half of the 19th century, until the advent of the Green Revolution after 1960, the major contributor to increases in the world food supply was the extension of arable land. Increased production since then has been obtained through rising yields, a feature that is beginning to show some signs of slowing down. The subject of arable land provides an illustration of why Evan's book is worth reading. I have often seen it stated that most, or even all, of the arable land on the earth is already under cultivation, suggesting there is no more land available for that purpose. Worldwide, this is not true, but the actual situation is complex. There is a lot of potentially arable land that is currently not under cultivation but much of it is undisturbed forest and wetland, whereas other land is arable but marginal. Arable land is being lost all the time to urbanization and replaced with previously uncultivated land, keeping the total roughly constant. The book is filled with topics like this that will help the reader better understand the complexities of the issues related to producing enough food to keep up with population growth. Most plant biologists should come away from reading this book with a better sense of world agriculture in terms of where we are today, how we got there, and the constraints that will drive its development over the next 50 years. In a more philosophical vein, Evans begins the book by juxtaposing two views of the relationship between food production and population growth. The one view of Thomas Malthus has the supply of food being the driving variable and population growth dependent upon it and the other view is of Ester Boserup, who sees it the other way around, with population growth being the driver of agricultural development. Evans makes no attempt to resolve this issue, but keeps it front and center throughout the book and leaves it to the reader to ascertain which view might be closer to the truth. I would note that the correct answer, if one truly exists, would have a large bearing on the eventual acceptance of genetically modified crops, particularly in developing countries. The second book I recommend reading is Feeding the World: A Challenge for the Twenty-First Century by Vaclav Smil. Although the title is similar to that of Evans' book, the approach is quite different. Smil brings more of an ecological perspective to the topic and treats the subject from the standpoint of where we are now and where we need to go in the future. Smil has long addressed issues of sustainability. The often-quoted limit of four billion people that can be sustained if nitrogen were only applied following the principles of organic farming can be traced to him, although others have made similar calculations. Smil's book makes for good reading because he regularly searches for practical approaches (or as he calls it, “truth”) to achieving a sustainable agriculture that can support 10 billion people. It is interesting that he does this in part by appropriating the most legitimate points of both those who see only catastrophe on our present course and those who effectively see no limits to the number of people that the earth can sustain long term. As he does this, Smil also points out fallacies associated with many of the numbers that both of these camps regularly cite. As noted, Smil's goal is how to achieve long-term agricultural sustainability. To do that, he works his way up the food chain, from crop productivity through postharvest losses and onto food production, consumption, and human nutrition. In the process, he continually presents a message that there is considerable slack in the current system and that the prospects for more efficient use of existing resources at all levels are very real. Smil's background in ecology and his understanding of food chains shows up well in his discussion of nutrition and how an omnivorous world can be sustainable, but only if done in an intelligent way, which means more chicken and much less beef. It is equally important that the efficiencies Smil envisions are achieved with existing technologies and knowledge bases, although some of his approaches to optimizing plant physiological parameters are based on more ideal control of plant functioning than is presently attainable. The use of GMOs, pro or con, garners little mention. Far from being a drawback, this omission makes the book all the more important to read. It serves to remind us not only that GMOs are just one part of the solution to feeding a 10 billion-person world but also identifies what other components of the solution are likely to be. Smil's movement from Evans' primary focus on agriculture onto issues of ecological sustainability and nutrition represents a good segue into the third book, The Doubly Green Revolution: Food for All in the 21st Century by Gordon Conway. Conway is currently President of the Rockefeller Foundation and was the recipient of the American Society of Plant Physiologists' (ASPP) Leadership in Science Public Service Award last year. Although Conway is cited as being an agricultural ecologist, there is clearly a lot of economist in him. This makes for tough sledding in some parts of the book. On the other hand, this also leads to a wealth of interesting and useful data presented throughout the book. Conway has spent much of his career working with the international agricultural research centers, and he provides a more detailed picture of the world agricultural scene than either of the other two books. He also understands poverty and the many socioeconomic factors that contribute to the existence of significant numbers of underfed people in a world of sufficient food supplies. Opponents of GMOs often use this fact and point to poverty as the problem, not a lack of food. Conway makes it clear that, however true the latter is, alleviating poverty is not a practical or workable solution and does not address the future need to feed 60% to 70% more people than exist at present. Conway sympathizes with Smil's goal of achieving a more sustainable form of agriculture than that he sees associated with the first green revolution; hence, the notion of the next one being “doubly green.” He approaches this goal with several themes that appear regularly throughout the book. One is that he is much more supportive than Smil of the need to include new technologies in the mix needed to feed a world of 10 billion people. In that regard, GMOs (plants and animals) are addressed specifically, with Conway seeing the potential gains from the application of GMOs as far outweighing their perceived risks at this point. This is especially true when he talks of pest and disease management, where Conway envisions GMOs as being an important way out of the cycle of large-scale application of pesticides associated with the first green revolution. Conway also sees the need for far more broad-ranging partnerships than currently exist. He cites several examples where industry has either partnered with, or given technologies to, public agricultural research centers in developing countries. Conway is particularly upbeat about the possibility of companies acting as stewards of their technology in a way that benefits developing countries and protects their intellectual property rights in developed countries. However, this is not the only kind of partnership Conway envisions and another recurring theme is the need to empower and include local farmers in the new partnerships. He feels there is much to be learned on the ground from people who have spent decades or even centuries growing crops and surviving on a particular plot of land. In the end, Conway is calling for a comprehensive agricultural revolution, one that includes the technological, the ecological, and the sociological. He recognizes this will not be an easy task to accomplish but sees the cost of a failure to act as being extremely high. This book is the most difficult of the three to read, but I believe the reward is worth the effort to those who persevere. In summary, all three books are built around the same general theme: feeding the world in the middle of this century. Although there is much overlap in what they have to say, each tends to emphasize a different area when looking to the future. Evans looks more to the capabilities inherent in the biology of plants, Smil stresses a more environmentally based approach and the need to optimize our use of resources to achieve agricultural sustainability, and Conway brings the socioeconomic and cultural dimensions of the world food supply more to the fore. In total, these three books make for informative and important reading for any plant biologist. Before ending this essay, I would like to add a couple of my own thoughts related to the GMO debate and why the information provided in these three books is important for any plant biologist participating in that debate to know. First, in spite of their different outlooks, all three would agree that feeding a world of 10 billion inhabitants cannot be accomplished without making significant changes, particularly in the developing world, that run throughout the food chain, from agricultural quality and productivity to socioeconomics. However, the battle over the application of GMO technology to help feed the earth's growing population currently rests in the hands of the developed countries, whereas most of the people that will need to be fed are located in developing nations. The irony of this situation rests on the fact that thanks to modern agricultural practices, the population of the developed world has access to the most abundant, healthiest, and cheapest supply of food in the history of the human race. Simply stated, people in the developed world are spoiled when it comes to food, and they are in a position to be picky about what they chose to, or choose not to, eat. Opponents of GMOs do not need to prove whether any claim about the possible dangers of GMOs is true or not. Just raising the specter of a possible risk associated with GMOs in many people's minds is enough to make them say they do not want to eat any food containing GMOs. This decision is easily made because it comes with no apparent consequence for the cost, availability, or quality of the food they subsequently eat. That luxury is not afforded to someone in a country where food is nowhere near as cheap and available, as all three books make abundantly clear. Second, as a long-time member and recent Chair of ASPP's Public Affairs Committee, I believe the Society can justifiably be proud of the extent to which members of the Public Affairs Committee and the Society as a whole have been willing to participate in the public debate on GMOs. In doing so, we have attempted to behave as honest brokers, ensuring that the scientific issues underlying the GMO debate are presented in as fair and objective a manner as possible. This is not always an easy thing to do when it comes to GMOs, given how polarizing the issue is. It has become difficult to take a position that remotely feigns in the direction of one side of the GMO issue without immediately being seen as some sort of mindless lackey by people on the other side. The best way I know of to counter the latter charge is to develop support for one's arguments (pro or con) based on a thorough understanding of the subject. Knowledge truly is power in this case, and one can never be too knowledgeable on this most controversial, current, and important of topics. Just as Chris Somerville opened this series of essays with a call for plant biologists to make their voices heard, I would like to end the series with a second important recommendation: “Go read a book (or three).” LITERATURE CITED 1 Chrispeels MJ Sadava DE Plant, Genes and Agriculture. 1994 Jones and Bartlett London 2 Conway C The Doubly Green Revolution: Food for All in the Twenty-First Century. 1997 Cornell University Press Ithaca, NY 3 Evans LT Feeding the Ten Billion. 1998 Cambridge University Press Cambridge, UK 4 Smil V Feeding the World: A Challenge for the 21st Century. 2000 MIT Press Cambridge, MA 5 Somerville C The genetically modified organism conflict. Plant Physiol 123 2000 1201 1202 Google Scholar Crossref Search ADS PubMed WorldCat 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)

Minorsky, Peter V.

doi: 10.1104/pp.126.1.23pmid: N/A

The guard cells that flank the stomatal pores of leaves and stems must integrate and respond appropriately to a multitude of instantaneously varying stimuli. In addition to a background circadian rhythmicity, the principle physiological determinants of stomatal aperture are the respective levels of blue light, CO2, and the stress-induced hormone abscisic acid (ABA). The 1990s witnessed a blossoming in our understanding of how guard cells work, largely because of the advent of patch-clamp recording techniques, new ways to measure cytoplasmic free Ca2+([Ca2+]cyt), and the adoption of Arabidopsis as a model organism. This month's The Hot and the Classic presents a brief synopsis of the most cited guard cell research contribution for each year of the 1990s. 1990: Ca2+ Increase Precedes ABA-Induced Closure Although there had been much indirect evidence that Ca2+ fluxes might be involved in regulating the responses of guard cells to ABA, McAinsh et al. (1990) were able to demonstrate this conclusively by application of fura-2, a fluorescent Ca2+ indicator to Commelina communisguard cells. Physiological concentrations of ABA caused a 2- to 10-fold increase in [Ca2+]cyt. 1991: Multiple Stretch-Activated Channels Mechanosensitive ion channels in the plasma membrane of fava bean (Vicia faba) guard cell protoplasts were studied by the patch clamp technique (Cosgrove and Hedrich, 1991). Stretch-activated (SA) channels in outside-out patches were analyzed for channel conductance, kinetics, and ion selectivity. Three distinct SA channels were found that were permeable to Cl−, K+, and Ca2+. These SA channels may mediate ion transport across the plasma membrane directly, as well as influence the activity of non-SA channels via effects on membrane voltage and [Ca2+]cyt. 1992: Foreign Expression of Plant K+Channel Gene KAT1 had previously been cloned from Arabidopsis by complementation of Saccharomyces cerevisiae mutants deficient in K+ uptake. Schachtman et al. (1992)report that a single mRNA transcript from the ArabidopsisKAT1 cDNA confers the functional expression of a hyperpolarization-activated K+ channel inXenopus laevis oocytes. The channel encoded byKAT1 is highly selective for K+ over other monovalent cations and is blocked by tetraethylammonium and Ba2+. These characteristics demonstrate thatKAT1 encodes an inward-rectifying K+channel. 1993: Control of Outward K+ Channel by Cytoplasmic pH The activation by ABA of outward-rectifying K+ channels and its dependence on cytoplasmic pH were examined in stomatal guard cells of V. faba (Blatt and Armstrong, 1993). ABA caused a cytoplasmic alkalinization and a parallel rise in the outward-rectifying K+channel current. Acid loads, imposed with external butyrate, abolished the ABA-evoked K+ current. These results establish a causal link between cytoplasmic alkalinization and the activation of the outward K+ current by ABA and thus affirm a role for H+ in signaling and transport control in plants. 1994: Vacuolar K+ Channel More than 90% of the K+ released from guard cells during stomatal closure originates from the guard cell vacuole.Ward & Schroeder (1994) report upon a novel type of K+ channel in the vacuolar membrane of V. faba guard cells that is activated by physiological increases in [Ca2+]cyt. The Ca2+, voltage, and pH dependences, high selectivity for K+, and high density of the K+ channels in the vacuolar membrane suggest a central role for these channels in vacuolar K+ release. The authors also presented a model of a possible mechanism of Ca2+-induced Ca2+ release involving the vacuolar K+ channel and a previously described slow vacuolar channel. 1995: Protein Phosphatase Regulation of K+Channels Disruption of ABA sensitivity in wilty abi1-1 mutants of Arabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de-)phosphorylation contributes to stomatal control by ABA. Armstrong et al. (1995)stably introduced the abi1-1 mutant allele intoNicotiana benthamiana, and monitored its influence on ion channel activity in guard cells under voltage clamp. Expression of theabi1-1 gene was associated with 2- to 6-fold reductions in an outward K+ current and the desensitization of both inward and outward K+ currents to ABA. In guard cells from the abi1-1 transformants, the protein kinase antagonists H7 or staurosporine restored the normal responses of both types of K+ channels and stomatal aperture to ABA. These results implicate ABI1 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell K+ channels to ABA-evoked signal cascades. 1996: Ca2+ and CO2-Induced Closure Webb et al. (1996) used fura-2 fluorescence to measure increases in guard cell [Ca2+]cyt in stomatal guard cells of C. communisin response to increased CO2. Removal of extracellular Ca2+ both prevented the CO2-induced increase in [Ca2+]cyt and inhibited the associated reduction in stomatal aperture. These data suggest that an influx of Ca2+ is required for stomatal response to CO2. 1997: Activation of an Anion Channel by ABA ABA strongly activates slow anion channels in wild-type Arabidopsis guard cells (Pei et al., 1997). Protein phosphatase inhibitors suppressed ABA-induced anion channel activation and stomatal closing. ABA activation of slow anion channels and ABA-induced stomatal closing were abolished in wilty abi1 andabi2 mutant guard cells. These impairments in ABA signaling were partially rescued by kinase inhibitors in abi1 but not in abi2 guard cells. These data provide evidence that theabi2 locus disrupts early ABA signaling, thatabi1 and abi2 affect ABA signaling at different steps in the cascade, and that protein kinases act as negative regulators of ABA signaling in Arabidopsis. 1998: Farnesyltransferase and ABA-Induced Closure Protein farnesylation, a posttranslational modification process, mediates the COOH-terminal lipidation of specific cellular proteins such as Ras and G-proteins. Pei et al. (1998) report that deletion of the Arabidopsis farnesyltransferase geneERA1 or application of farnesyltransferase inhibitors resulted in ABA hypersensitivity of guard cell anion-channel activation and of stomatal closing (Pei et al., 1998). Double-mutant analyses ofera1 with the ABA-insensitive mutants abi1 andabi2 showed that era1 suppresses the ABA-insensitive phenotypes. Moreover, era1 plants exhibited a reduction in transpirational water loss during drought treatment. 1999: Phospholipase C and Ca Oscillations ABA induces oscillations in C. communis guard cell [Ca2+]cyt(Staxen et al., 1999). The pattern of the oscillations depended on the ABA concentration and is correlated with the final stomatal aperture. U-71322, an inhibitor of phosphoinositide-specific phospholipase, inhibited both ABA-induced oscillations in [Ca2+]cyt and stomatal closure. An inactive analog of U-71322 was without effect. These findings suggest a role for phosphoinositide-specific phospholipase in the generation of ABA-induced oscillations in [Ca2+]cyt and suggest the involvement of oscillations in [Ca2+]cyt in the maintenance of stomatal aperture by ABA. LITERATURE CITED 1 Armstrong F Leung J Grabov A Brearley J Giraudat J Blatt MR Sensitivity to abscisic-acid of guard-cell K+ channels is suppressed by abi1-1, a mutant Arabidopsis gene encoding a putative protein phosphatase. Proc Natl Acad Sci USA 92 1995 9520 9524 Google Scholar Crossref Search ADS PubMed WorldCat 2 Blatt MR Armstrong F K+ channels of stomatal guard-cells: abscisic-acid-evoked control of the outward rectifier mediated by cytoplasmic pH. Planta 191 1993 330 341 Google Scholar Crossref Search ADS WorldCat 3 Cosgrove DJ Hedrich R Stretch-activated chloride, potassium, and calcium channels coexisting in plasma-membranes of guard-cells of Vicia faba L. Planta 186 1991 143 153 Google Scholar Crossref Search ADS PubMed WorldCat 4 McAinsh MR Brownlee C Hetherington AM Abscisic acid-induced elevation of guard-cell cytosolic Ca2+ precedes stomatal closure. Nature 343 1990 186 188 Google Scholar Crossref Search ADS WorldCat 5 Pei ZM Ghassemian M Kwak CM McCourt P Schroeder JI Role of farnesyltransferase in ABA regulation of guard cell anion channels and plant water loss. Science 282 1998 287 290 Google Scholar Crossref Search ADS PubMed WorldCat 6 Pei ZM Kuchitsu K Ward JM Schwarz M Schroeder JI Differential abscisic acid regulation of guard cell slow anion channels in Arabidopsis wild-type and abi1 and abi2 mutants. Plant Cell 9 1997 409 423 Google Scholar PubMed OpenURL Placeholder Text WorldCat 7 Schachtman DP Schroeder JI Lucas WJ Anderson JA Gaber RF Expression of an inward-rectifying potassium channel by the Arabidopsis KAT1 cDNA. Science 258 1992 1654 1658 Google Scholar Crossref Search ADS PubMed WorldCat 8 Staxen I Pical C Montgomery LT Gray JE Hetherington AM McAinsh MR Abscisic acid induces oscillations in guard-cell cytosolic free calcium that involve phosphoinositide-specific phospholipase C. Proc Natl Acad Sci USA 96 1999 1779 1784 Google Scholar Crossref Search ADS PubMed WorldCat 9 Ward JM Schroeder JI Calcium-activated K+ channels and calcium-induced calcium-release by slow vacuolar ion channels in guard-cell vacuoles implicated in the control of stomatal closure. Plant Cell 6 1994 669 683 Google Scholar Crossref Search ADS PubMed WorldCat 10 Webb AAR McAinsh MR Mansfield TA Hetherington AM Carbon dioxide induces increases in guard cell cytosolic free calcium. Plant J 9 1996 297 304 Google Scholar Crossref Search ADS WorldCat 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)

Minorsky, Peter V.

doi: 10.1104/pp.126.1.25pmid: N/A

Why do so many scientific research articles languish in obscurity? There is no single answer. No doubt many have earned their ignominy by their flawed scholarship, poor exposition, or arcane subject matter, but this is not always the case. A search of the old literature often uncovers many seemingly worthy contributions that have inexplicably fallen into oblivion. Probably the most common reason for the neglect of these papers is their publication in unsuitable journals or in journals of limited circulation. The examplar par excellence of this phenomenon, of course, was Gregor Mendel's inscrutable choice of the little-known Verhandlungen des naturforschenden Vereines im Brunn as a vehicle for his scientific communications. Mendel's journal choice certainly accounted in part for the 35-year lag in the recognition of his fundamental contributions to the science of heredity. Mendel's case also exemplifies a second reason why some papers may be overlooked: they are simply too ahead of their time to be fully appreciated. News from the Archives, which will be an occasional feature of Plant Physiology, doesn't promise to expose any lost paradigms as big as Mendel's. The goals of this column are much more modest: first, to bring to light some lost observations from the dusty recesses of the archives; and second, to redress historical oversights. Suggestions from the readers of Plant Physiology concerning specific articles that have been treated unfairly by history or which deserve renewed consideration in light of recent discoveries are most welcome. This month's column explores the possible implications of some overlooked reports concerning the effects of two Na+ channel drugs, veratrine and tetrodotoxin (TTX), on plant cell mitosis. Veratrine, a Na+ Channel Agonist, Inhibits Mitosis in Plants Veratrine is a mixture of alkaloids produced by the sabadilla plant (Schoenocaulon officinale), a member of the Liliales variously placed in the Liliaceae or the Melanthiaceae (Fig.1). The two major alkaloid components of veratrine are veratradine and cevadine, both of which act as agonists of plasma membrane Na+ channels in animal cells. Na+ ions play a much more integral role in the basic transport processes of animal cells than they do in plant cells. In animal cells, the pumping activity of Na+-ATPases are the primary source of the electrogenic component of the membrane potential, whereas the same function is served by H+-ATPases in plant cells. This dichotomy in the use of Na+ and H+ is also seen in the cotransport systems of plant and animal cells, where Na+ is most often cotransported in animal cells and H+ is most often cotransported in plant cells. Perhaps most telling of the relative unimportance of Na+ to plant cell function is the fact that Na+ isn't even an essential mineral nutrient of most plants. In retrospect, therefore,Witkus and Berger's (1944) discovery that 0.1% solution of veratrine sulfate inhibits both spindle and cell plate formation in the meristematic cells of onion (Allium cepa) root tips is quite surprising. Of the veracity of Witkus and Berger's (1944) observations, there can be little doubt since Sharma and Sarkar (1956) and Kubiak (1971a, 1971b) independently arrived at the same conclusion. Fig. 1. Open in new tabDownload slide The sabadilla plant S. officinale is a source of the alkaloid mixture veratrine that is widely used as an agonist of Na+ channels in animal cells. (© Missouri Botanical Garden) Fig. 1. Open in new tabDownload slide The sabadilla plant S. officinale is a source of the alkaloid mixture veratrine that is widely used as an agonist of Na+ channels in animal cells. (© Missouri Botanical Garden) TTX, a Na+ Channel Antagonist, Also Inhibits Mitosis in Plants TTX is a toxin extracted from the puffer fishArothron nigropunctatus (Fig.2). Khora et al. (1997) reported that TTX inhibited mitosis at concentrations greater than 30 μm as evident by the fall of mitotic index. TTX at far lower concentrations (0.1–5.0 μm) significantly enhanced the frequencies of sister chromatid exchange (SCE), indicating a possible interference of the toxin in DNA replication and repair. Other authors, however, have previously found no effect of TTX on an inward Na+ current inZea mays (Roberts and Tester, 1997) or Triticum aestivum (Davenport and Tester, 2000) roots, or on the secretory network of Closterium acerosum (Domozych, 1999), or on the resting or action potential of Nitella mucronuta(Koppenhöfer, 1972). It is conceivable that the putative veratrine- and TTX-sensitive channel in plants may only be important during certain stages of plant cell mitosis. Fig. 2. Open in new tabDownload slide The puffer fish is a rich source of tetrodotoxin, a toxin most often used as a blocker of Na+chanels in animal cells. (© Jeffrey N. Jeffords) Fig. 2. Open in new tabDownload slide The puffer fish is a rich source of tetrodotoxin, a toxin most often used as a blocker of Na+chanels in animal cells. (© Jeffrey N. Jeffords) Veratrine and TTX Also Affect Ca2+ Currents in Heart Cells Ion channels are often named according to the dominant ion that they pass under physiological conditions, but this often leads to the impression that such channels are completely and absolutely specific for that ion. This is not always true, and the Na+ channel of animal cells is a case in point. Several studies have concluded that some Na+channels in heart cells are also permeable to Ca2+ under certain conditions (Lemaire et al., 1995; Aggarwal et al., 1997; Cole et al., 1997. Santana et al., 1998). Indeed, genomic evidence suggests that Ca2+ and Na+ channels have similar structures, reflecting a common ancestry (Spafford et al., 1999). Both are large monomeric proteins that include four homologous repeats and share extensive sequence homology in their transmembrane segments and S5-S6 linkers (Sato et al., 2001). Especially intriguing is the recent discovery that the activation of either the β-adrenergic receptor or protein kinase A transforms the Na+ channel in rat heart cells into one that is promiscuous with respect to ion selectivity, permitting Ca2+ ions to permeate as readily as Na+ (Santana et al., 1998). Ca2+ Currents: The Site of Action for Veratrine and TTX in Plant Cells? What is striking about the effect of veratrine on A. cepa cell mitosis is how similar it is to the effects of treatments such as caffeine (Samuels and Staehelin, 1996; Valster and Hepler, 1997) and intracellular Ca2+ chelators (Jurgens et al., 1994). These latter two agents are believed to act by disrupting intracellular Ca2+ gradients. In keeping with the new appreciation that some types of veratrine-sensitive Na+ channels may also serve as Ca2+ channels, it is tempting to speculate that veratine may serve as a Ca2+ channel agonist inA. cepa meristematic root cells. If the effect of TTX on Allium sister chromatid exchange during mitosis is related to its blocking effect on a Ca2+ current, then it follows that any factor that lowers [Ca2+]cytshould also increase the frequency of SCE. Ortı́z and Cortés (1990) reported that SCE frequency in the meristematic cells of A. cepa roots increased in a dose-dependent fashion when treated with EDTA. They proposed that deprivation of divalent cations (Ca2+ or Mg2+) probably play an important role in DNA replication and repair processes. Thus, all pharmacological evidence is consistent with the hypothesis that mitotic plant cells may employ a veratrine- and TTX-sensitive Ca2+ channel. LITERATURE CITED 1 Aggarwal R Shorofsky SR Goldman L Balke CW Tetrodotoxin-blockable calcium currents in rat ventricular myocytes: a third type of cardiac cell sodium current. J Physiol 505 1997 353 369 Google Scholar Crossref Search ADS PubMed WorldCat 2 Cole WC Chartier D Martin F Leblanc N Ca2+ permeation through Na+ channels in guinea pig ventricular myocytes. Am J Physiol 42 1997 H128 H137 Google Scholar OpenURL Placeholder Text WorldCat 3 Davenport RJ Tester M A weakly voltage-dependent, nonselective cation channel mediates toxic sodium influx in wheat. Plant Physiol 122 2000 823 834 Google Scholar Crossref Search ADS PubMed WorldCat 4 Domozych DS Perturbation of the secretory network in Closterium acerosum by Na+-selective ionophores. Protoplasma 206 1999 41 56 Google Scholar Crossref Search ADS WorldCat 5 Jurgens M Hepler LH Rivers BA Hepler PK BAPTA-calcium buffers modulate cell plate formation in stamen hairs of Tradescantia: evidence for calcium gradients. Protoplasma 183 1994 86 99 Google Scholar Crossref Search ADS WorldCat 6 Khora SS Panda KK Panda BB Genotoxicity of tetrodotoxin from puffer fish tested in root meristem cells of Allium cepa L. Mutagenesis 12 1997 265 269 Google Scholar Crossref Search ADS PubMed WorldCat 7 Koppenhöfer E Die Wirkung von Kupfer, TTX, Cocain und TEA auf das Ruhe- und Aktionspotential von Nitella. Pflüg Arch 336 1972 299 309 Google Scholar Crossref Search ADS WorldCat 8 Kubiak R The action of veratrine on mitoses in Allium cepa. Acta Biol Cracov Ser Bot 14 1971a 37 41 Google Scholar OpenURL Placeholder Text WorldCat 9 Kubiak R The action of veratrine on mitoses in Allium cepa. Genet Pol 12 1971b 289 291 Google Scholar OpenURL Placeholder Text WorldCat 10 Lemaire S Piot C Seguin J Nargeot J Richard S Tetrodotoxin-sensitive Ca2+ and Ba2+ currents in human atrial cells. Recept Chann 3 1995 71 81 Google Scholar OpenURL Placeholder Text WorldCat 11 Ortı́z T Cortés F Differences in the effectiveness of EDTA to induce SCEs and chromosomal aberrations in CHO and Allium cepa chromosomes. Cytobios 61 1990 187 193 Google Scholar PubMed OpenURL Placeholder Text WorldCat 12 Roberts SK Tester M A patch clamp study of Na+ transport in maize roots. J Exp Bot 48 1997 431 440 Google Scholar Crossref Search ADS PubMed WorldCat 13 Samuels AL Staehelin LA Caffeine inhibits cell plate formation by disrupting membrane reorganization just after the vesicle fusion step. Protoplasma 195 1996 144 155 Google Scholar Crossref Search ADS WorldCat 14 Santana LF Gomez AM Lederer WJ Ca2+ flux through promiscuous cardiac Na+ channels: slip-mode conductance. Science 279 1998 1027 1033 Google Scholar Crossref Search ADS PubMed WorldCat 15 Sato C Ueno Y Asai K Takahashi K Sato M Engel A Fujiyoshi The voltage-sensitive sodium channel is a bell-shaped molecule with several cavities. Nature 409 2001 1047 1051 Google Scholar Crossref Search ADS PubMed WorldCat 16 Sharma AK Sarkar SK Veratrine: its use in biochemistry. Caryologia 8 1956 240 249 Google Scholar Crossref Search ADS WorldCat 17 Spafford JD Spencer AN Gallin WJ Genomic organization of a voltage-gated Na+ channel in a hydrozoan jellyfish: insights into the evolution of voltage-gated Na+ channel genes. Recept Chann 6 1999 493 506 Google Scholar OpenURL Placeholder Text WorldCat 18 Valster AH Hepler PK Caffeine inhibition of cytokinesis: effect on the phragmoplast cytoskeleton in living Tradescantia stamen hair cells. Protoplasma 196 1997 155 166 Google Scholar Crossref Search ADS WorldCat 19 Witkus ER Berger CA Veratrine, a new polyploidy inducing agent. J Hered 35 1944 131 133 Google Scholar Crossref Search ADS WorldCat 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)

Holbrook, N. Michele; Ahrens, Eric T.; Burns, Michael J.; Zwieniecki, Maciej A.

doi: 10.1104/pp.126.1.27pmid: 11351066

Abstract Magnetic resonance imaging (MRI) was used to noninvasively monitor the status of individual xylem vessels in the stem of an intact, transpiring grape (Vitis vinifera) plant over a period of approximately 40 h. Proton density-weighted MRI was used to visualize the distribution of mobile water in the stem and individual xylem vessels were scored as either water or gas filled (i.e. embolized). The number of water-filled vessels decreased during the first 24 h of the experiment, indicating that approximately 10 vessels had cavitated during this time. Leaf water potentials decreased from −1.25 to −2.1 MPa during the same period. Watering increased leaf water potentials to −0.25 MPa and prevented any further cavitation. Refilling of xylem vessels occurred as soon as the lights were switched off, with the majority of vessels becoming refilled with water during the first 2 to 3 h in darkness. These measurements demonstrate that MRI can be used to monitor the functional status of individual xylem vessels, providing the first method to study the process of cavitation and embolism repair in intact plants. Transport of water through xylem vessels may become disrupted by breakage of water columns under high levels of tension or freezing temperatures (Tyree and Sperry, 1989). Because gas-filled vessels cannot transmit tensions, embolized vessels are permanently lost from the water transport system unless a mechanism exists to reconnect the water column. The idea that embolized vessels might be restored to their functional state is not new, but has generally been thought to be limited to situations in which the entire vascular system could be pressurized due to active solute transport by the roots (Cochard et al., 1994; Fisher et al., 1997). Recent studies, however, suggest that cavitated vessels may be repaired even when the water in neighboring conduits is under tension (Salleo et al., 1996;McCully et al., 1998; Zwieniecki and Holbrook, 1998; Pate and Canny, 1999; Tyree et al., 1999; Melcher et al., 2001). Embolism removal is thought to require positive pressures to force the gas into solution, making it difficult to understand how this process could take place against a background of negative water potentials (Holbrook and Zwieniecki, 1999). Although there has been some progress on how this local compartmentalization might occur (Zwieniecki and Holbrook, 2000), a mechanism that reconciles xylem tension and embolism repair has not, in our opinion, been fully articulated. A major factor limiting our understanding of embolism repair is the lack of an in vivo method for examining changes in the functional status of individual vessels. All of the methods currently used to study refilling, such as temporal changes in hydraulic conductivity (Zwieniecki and Holbrook, 1998), percent loss conductivity (Salleo et al., 1996), or proportion of gas-filled conduits (McCully et al., 1998;Pate and Canny, 1999), require destructive sampling. In this paper, we use high-resolution magnetic resonance imaging (MRI) to follow the status of individual xylem vessels in the stem of an intact grape (Vitis vinifera) plant. MRI is well suited for studies of embolism repair because it can image inside optically opaque subjects, it is noninvasive, it is compatible with longitudinal investigations, and it does not require the use of any exogenous chemical tracers (MacFall and Van As, 1996; Chudek and Hunter, 1997). However, previous studies using MRI to study water transport in plant stems do not have the spatial resolution to distinguish individual xylem vessels (Johnson et al., 1987; Köckenberger et al., 1997). In addition, MRI studies of water transport have tended to use plants small enough to fit entirely within the magnet (Kuchenbrod et al., 1996;Köckenberger et al., 1997). Because vines have large diameter xylem vessels and relatively flexible stems, they are well suited for MRI studies of water transport. Here we present the first direct observations of xylem cavitation and embolism repair in an intact plant. RESULTS As expected, regions of high water density in theM r images corresponded with xylem vessels (Fig. 1). At the start of the experiment, leaf water potential was −1.25 MPa and theM r image contained several regions with a low density of vessels, indicating that some cavitation had already occurred (Fig. 2). Over the next 24 h, approximately 10 additional vessels cavitated (Fig. 2). During this period, leaf water potentials fell to −2.1 MPa. There was no obvious spatial pattern to which vessels cavitated; on one side of the stem the cavitated vessels were somewhat clumped, whereas on the other side they were evenly dispersed. Fig. 1. Open in new tabDownload slide Cross section of grape stem using light microscope (A) or MRI (B). Scale bar in A = 2 mm. Note that the bark can be seen in the MRI image, but only the wood is present in the anatomical cross section (A). Fig. 1. Open in new tabDownload slide Cross section of grape stem using light microscope (A) or MRI (B). Scale bar in A = 2 mm. Note that the bark can be seen in the MRI image, but only the wood is present in the anatomical cross section (A). Fig. 2. Open in new tabDownload slide Time course of total number of vessels visible in the MRI images and leaf water potential as a function of time. A through C show representative MRI images, with white arrows marking vessels that were initially water filled (A), then gas-filled and hence not visible (B), and finally refilled with water (C). A QuickTime movie of all 39 images can be viewed at www.plantphysiol.org. Fig. 2. Open in new tabDownload slide Time course of total number of vessels visible in the MRI images and leaf water potential as a function of time. A through C show representative MRI images, with white arrows marking vessels that were initially water filled (A), then gas-filled and hence not visible (B), and finally refilled with water (C). A QuickTime movie of all 39 images can be viewed at www.plantphysiol.org. After the plant was rewatered, leaf water potentials increased rapidly to −0.25 MPa. During the period in which the lights remained on there was no change in the number of visible vessels in the MRI image. Once the lights were turned off, the number of water-filled vessels increased markedly (Fig. 2). After the first hour in the dark, the number of water-filled vessels was approximately equal to the start of the experiment. The number of water-filled vessels continued to increase over the next 12 h, although the rate of increase slowed with time. During this period there was no evidence of root exudation from a short side branch at the base that had been freshly cut. At the end of the experiment the plant was severed at the base and there were no visible signs of root exudation. DISCUSSION The images presented demonstrate that MRI can be used noninvasively to monitor the functional state of individual xylem vessels, thus opening new possibilities for studying embolism repair in intact plants. By combining MRI with more detailed physiological measurements such as sapflow and in situ thermocouple psychrometry, we will be able to delineate the conditions under which repair occurs. Because MRI can be used to image flow velocities (Bourgeois and Decorps, 1991; Köckenberger et al., 1997; Kuchenbrod et al., 1998) as well as water density, further studies could determine whether refilled vessels are able to subsequently transport water during periods of active transpiration. In addition, monitoring through more than one drying cycle could be used to determine if previously cavitated vessels are more prone to cavitation in the future. In the grapevine examined in this study, cavitation occurred while the plant was actively transpiring and the leaves were turgid. Cavitation appeared to occur at random within the stem, although some areas lost more vessels than did others. There was no evidence of embolism repair, as determined by the reappearance of water in xylem vessels, although the leaves were illuminated. This was true even after the plant was watered and leaf water potentials substantially increased. Repair was first observed in the measurement immediately after the lights were turned off, and continued to occur, although at a decreased rate, over the next 12 h. This suggests that in grapevines embolism repair may require both an increase in water potential and a cessation of flow through the xylem. Other species, however, are reported to repair cavitated vessels during periods of active transpiration (McCully et al., 1998). MRI studies of these species will allow us to pinpoint the conditions under which such repair occurs. Grapes are well known for their capacity to generate root pressure (Hale, 1727; Sperry et al., 1987). Prior to leaf expansion, grapes use root pressure to refill xylem vessels that had become air filled during the winter (Sperry et al., 1987). We did not observe any signs of root exudation during this study despite careful visual examination. However, in the absence of additional measurements, we recognize that the possibility of root pressure being responsible for the observed repair cannot be eliminated. The major technical breakthrough of this study is the application of high-resolution MRI to investigate the dynamic changes in xylem transport capacity at the level of individual vessels. Previous use of MRI to study water transport in plants has lacked the spatial resolution needed to determine the functional status of individual xylem vessels (Johnson et al., 1987; Kuchenbrod et al., 1996;Köckenberger et al., 1997). In addition, the small size of the plants used in previous studies makes it unlikely that substantial tensions were generated within the xylem. The major limitation to MRI studies of xylem transport arises from the need to have exclusive use of the expensive microscopy instrumentation for extended periods of time and the physical constraints on suitable plant material associated with having to position the region of interest within the MRI magnet. In the case of the instrument used in this study, this meant that one-half of the plant (either all of the leaves or all of the roots and soil) had to be threaded through a 4-cm-diameter constriction in the center of the magnet bore. The ability to observe xylem processes in vivo, however, greatly outweighs these limitations and provides a new approach for understanding factors influencing the maintenance of water transport capacity in the xylem. MATERIALS AND METHODS Observations were made on a grape (Vitis viniferaL. var. Concord) plant growing in an 8-L pot. The plant had been previously pruned such that at the time of measurements it had an unbranched shoot approximately 4 m in length. Measurements were made using a vertical wide-bore (89-mm) 500-MHz, 11.7-tesla magnetic resonance microscopy system (Bruker Instruments Inc., Billerica, MA) located at the Biological Imaging Center (California Institute of Technology). A laboratory-built 1-cm-diameter radio frequency (RF) surface coil and resonant tank circuit was mounted directly onto the stem and was used for both RFexcitation and reception. A single turn surface coil was utilized because of its high quality factor and its ease in placement along the stem. The loss in reception homogeneity due to this geometry was not a serious drawback because the coil diameter exceeded that of the stem by approximately 40%, and a single, small, tip angle RFexcitation pulse (approximately 3°) was utilized in the gradient echo sequence. After mounting the coil, the shoot was carefully inserted into the magnet bore. The portion of the plant protruding out the top of the magnet was coiled beneath a light assembly that provided approximately 300 μmol photons photosynthetically active radiation m−2s−1 to the leaves. Images were acquired using a two-dimensional Fourier transform gradient echo protocol with a small tip angle excitation pulse (Callaghan, 1991). The repetition time and echo time were equal to 50 and 4.5 ms, respectively. Two transverse image slices were acquired simultaneously at 55-min intervals over a period of 45 h. Each slice was 1.5 mm thick and separated by 1.75 mm. The in-plane image resolution was 20 × 20 μm. Signal averaging was required to obtain a satisfactory signal-to-noise ratio of order of 10 within vessels, and this limited the temporal resolution per acquisition to approximately 20 min. Because the MRI method used here visualizes the distribution of mobile water (Callaghan, 1991), vessels containing embolisms are easily distinguished from filled vessels. The plant was continuously illuminated during the first 31 h of the experiment. At 24 h into the experiment the plant was rewatered (approximately 3 L of water added to the pot). After 9 more h the lights were turned off and the imaging continued for an additional 12 h. Leaf water potentials were measured using a pressure chamber. To avoid substantial reductions in leaf area and thus changes in plant water balance during the MRI measurements, the water potentials of only six leaves were measured. After the MRI session was completed, the stem was sectioned in the plane where the MRI images were taken. The time-dependent populations of filled and embolized vessels were quantified from time lapse data through a fixed image plane. The number of water-filled vessels in each MRI slice was counted by comparing each image to the last frame (frame 39) of that slice's sequence. The final image was printed on a transparency and all distinct (i.e. water-filled) vessels were counted and circled. The other images (1–38) were then printed on paper and overlain, one at a time, on a back-lit reference image (frame 39). The number of missing vessels was counted for each of the frames and subtracted from the total number of vessels in frame 39. ACKNOWLEDGMENTS We wish to thank the greenhouse staff at the Huntington Gardens (Pasadena, CA) for generously providing greenhouse space. LITERATURE CITED 1 Bourgeois D Decorps M Quantitative imaging of slow coherent motion by stimulated echoes with suppression of stationary water signal. J Magn Reson 94 1991 20 33 Google Scholar OpenURL Placeholder Text WorldCat 2 Callaghan PT Principles of Nuclear Resonance Microscopy. 1991 Oxford University Press New York 3 Chudek JA Hunter G Magnetic resonance imaging of plants. Prog Nuclear Magn Reson Spectrom 31 1997 43 62 Google Scholar Crossref Search ADS WorldCat 4 Cochard H Ewers FW Tyree MT Water relations of tropical vine-like bamboo (Rhipidocladum recemiflorum): root pressures, vulnerability to cavitation and seasonal changes in embolism. J Exp Bot 45 1994 1085 1089 Google Scholar Crossref Search ADS WorldCat 5 Fisher J Angeles G Ewers FW Lopezportillo J Survey of root pressure in tropical vines and woody species. Int J Plant Sci 158 1997 44 50 Google Scholar Crossref Search ADS WorldCat 6 Hales S Vegetable Staticks. W. & J. Innys and T. 1727 Woodward London 7 Holbrook NM Zwieniecki MA Xylem refilling under tension: do we need a miracle? Plant Physiol 120 1999 7 10 Google Scholar Crossref Search ADS PubMed WorldCat 8 Johnson GA Brown J Kramer PJ Magnetic resonance microscopy of changes in water content in stems of transpiring plants. Proc Nat Acad Sci USA 84 1987 2752 2755 Google Scholar Crossref Search ADS PubMed WorldCat 9 Köckenberger W Pope JM Xia Y Jeffrey KR Komor E Callaghan PT A non-invasive measurements of phloem and xylem water flow in castor bean seedlings by nuclear magnetic resonance microimaging. Planta 201 1997 53 63 Google Scholar Crossref Search ADS WorldCat 10 Kuchenbrod E Kahler E Thurmer F Deichmann R Zimmermann U Haase A Functional magnetic resonance imaging in intact plants: quantitative observation of flow in plant vessels. Magn Reson Imaging 16 1998 331 338 Google Scholar Crossref Search ADS PubMed WorldCat 11 Kuchenbrod E Landeck M Thürmer F Haase A Zimmerman U Measurement of water flow in the xylem vessels of intact maize plants using flow-sensitive NMR imaging. Bot Acta 109 1996 184 186 Google Scholar Crossref Search ADS WorldCat 12 MacFall JS Van As H Magnetic resonance imaging of plants. Curr Top Plant Physiol 16 1996 33 76 Google Scholar OpenURL Placeholder Text WorldCat 13 McCully ME Huang CX Ling LE Daily embolism and refilling of xylem vessels in the roots of field-grown maize. New Phytol 138 1998 327 342 Google Scholar Crossref Search ADS PubMed WorldCat 14 Melcher PJ Goldstein G Meinzer FC Jones TJ Holbrook NM Huang C Water relations of coastal and estuarine Rhizophora mangle: the dynamics of embolism formation and repair. Oecologia 126 2001 182 192 Google Scholar Crossref Search ADS PubMed WorldCat 15 Pate JS Canny MJ Quantification of vessel embolisms by direct observation: a comparison of two methods. New Phytol 141 1999 33 43 Google Scholar Crossref Search ADS WorldCat 16 Salleo S Lo Gullo MA De Paoli D Zippo M Xylem recovery from cavitation-induced embolism in young plants of Laurus nobilis: a possible mechanism. New Phytol 132 1996 47 56 Google Scholar Crossref Search ADS PubMed WorldCat 17 Sperry J Holbrook NM Zimmermann MH Tyree MT Spring filling of xylem vessels in wild grapevine. Plant Physiol 83 1987 414 417 Google Scholar Crossref Search ADS PubMed WorldCat 18 Tyree MT Salleo S Nardini A Lo Gullo MA Mosca R Refilling of embolized vessels in young stems of laurel: do we need a new paradigm? Plant Physiol 120 1999 11 21 Google Scholar Crossref Search ADS PubMed WorldCat 19 Tyree MT Sperry JS Vulnerability of xylem to cavitation and embolism. Ann Rev Plant Physiol Plant Mol Biol 40 1989 19 38 Google Scholar Crossref Search ADS WorldCat 20 Zwieniecki MA Holbrook NM Short term changes in xylem water conductivity in white ash, red maple and sitka spruce. Plant Cell Environ 21 1998 1173 1180 Google Scholar Crossref Search ADS WorldCat 21 Zwieniecki MA Holbrook NM Bordered pit structure and vessel wall surface properties: implications for embolism repair. Plant Physiol 123 2000 1015 1020 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the Andrew W. Mellon Foundation, by the National Science Foundation (grant no. IBN 0078155), and by the U.S. Department of Agriculture (grant no. NRICGP 9800878). Core support for the imaging system was provided in part by the Human Brain Project (grant no. DA08944), with contributions from the National Institute on Drug Abuse and the National Institute of Mental Health (grant no. MH61223), and the National Center for Research Resources (grant no. RR13625). 2 Present address: Revise, Inc., 79 Second Avenue, Burlington, MA 01803. [w] The online version of this article contains Web-only data. The supplemental material is available at www.plantphysiol.org. * Corresponding author; e-mail [email protected]; fax 617–496–5854. 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)

Galston, Arthur W.

doi: 10.1104/pp.126.1.32pmid: 11351067

Recent discoveries on photoreceptors for both red and blue light compel a reexamination of certain older work, including some of my own. New data from molecular biology have removed the uncertainty in the interpretation of absorption and action spectra to define pigment systems active in photobiological phenomena. In 1968, while on sabbatical at the Department of Biophysics at King's College in London, I made use of their Universal Microspectrophotometer (UMSP-1, Zeiss, Göttingen, Germany) in an attempt to define the intracellular locale of phytochrome (Galston, 1968). I selected apical regions of etiolated oat (Avena sativa) coleoptiles and pea (Pisum sativum) epicotyls, known to have abundant phytochrome, exposing the plant material briefly to ambient white light during subsequent manipulations. I prepared either hand-cut sections, three to four cells thick, from fresh tissue, or thinner (15–20-μm) sections from frozen material with a cryostat-microtome. After being mounted in either glycerol, water, or paraffin oil, the sections were scanned with a 0.5- to 1.0-μm diameter spot to obtain an automatically recorded absorption spectrum of each of the various parts of the cell. The system obviously worked well because the absorption spectra of slightly green chloroplasts showed characteristic chlorophyll and carotenoid peaks, and repeated scans coincided closely (see Fig. 1 in Galston, 1968). For observations on phytochrome, I exposed the sections alternately to 5 min of either actinic 650-nm (red [R]) or 750-nm (far-red [FR]) light, and took a new absorption spectrum at the same location after each actinic exposure. With a 0.5-μm-diameter scanning spot, I noted distinct localized spectral shifts following the actinic R and FR treatments. These shifts appeared only in isolated areas of the nucleus, especially near the nuclear membrane, but not elsewhere in the cell. Most of the observed spectral changes occurred at or in the vicinity of the expected wavelengths for phytochrome transformation, but some did not. Feeling somewhat unsure of the significance of some of the data because of deviations from phytochrome's known peaks, I mailed my results to Sterling Hendricks (U.S. Department of Agriculture, Beltsville, MD; now deceased), the discoverer of phytochrome, and solicited his opinion. To my pleasant surprise, his response was so strongly positive that he volunteered to (and did) submit my manuscript to the Proceedings of the National Academy of Science. At his suggestion, the paper includes the raw spectrophotometric data from the recording. Hendricks even mentioned that an “unusual” R-/FR-induced reversible shift in the 580 to 620 nm region that I had noted for a glycerol-mounted section (see Fig. 4 in Galston, 1968) confirmed his previous observations with glycerol-mounted dried films of phytochrome in gelatin. Bolstered by this support from Hendricks, I concluded that my findings were evidence for the occurrence of phytochrome in the nucleus. I hypothesized that my data strengthened “the view that the pigment interacts in some way with genetic material, although the localization at or near the nuclear membrane may indicate control of passage of materials between nucleus and cytoplasm.” To my knowledge, there has never been a refutation or even a repetition of these experimental findings. Nevertheless, as Linda Sage has observed in her account of the history of phytochrome research (Sage, 1992), “skepticism greeted this interpretation, because there appeared to be too little phytochrome to generate such an absorbance change.” For example, Spruit (1972), discussing the feasibility of phytochrome microspectrophotometry, concluded that “… . it appears doubtful whether local phytochrome concentrations inside the cell can be found sufficient to obtain spectrophotometric readings, comparable in sensitivity with such readings made on bulk samples.” To add to the skepticism, immunolocalization evidence obtained by Pratt et al. (Coleman and Pratt, 1974; Pratt and Coleman, 1974) revealed phytochrome only in the cytoplasm, not in plastids, mitochondria, or nuclei. With regard to the spectrophotometric argument, I pointed out orally at meetings that the situation would be altered if, in fact, the majority of the phytochrome were aggregated as particulate material in the nucleus, forming an optically dense body that would give a stronger absorption signal. At the time, this suggestion was discounted because there was no evidence for the presence of phytochrome in the nucleus. However, only a little later, Quail et al. (1973) reported that cytoplasmic phytochrome, localized in the centrifugal supernatant fraction after prolonged dark or FR, became pelletable after exposure to R. This suggested that the location and status of phytochrome in the cell might depend on prior illumination and perhaps other conditions. Within the last several months, this situation has become considerably clearer. Quail et al. (Martinez-Garcia et al., 2000) have shown that cytoplasmic phytochromes A and B bind to a nuclear transcription factor after R irradiation, thus moving to the nucleus after photoactivation. This binding of phytochromes to promoters turns on the expression of light-activated genes. Others (Kircher et al., 1999; Yamaguchi et al., 1999) have presented similar evidence for light-driven movement of phytochrome from cytoplasm to nucleus, leading Nagatani (2000) to summarize the situation as follows in a Science Perspective: “phytochromes perceive a light stimulus, move into the nucleus, interact with PIF3 which is bound to the G-box motif of a light activated gene, and switch the gene on.” Of course, it is not clear that the signals I detected resulted from such nuclear phytochrome complexes, but at least any theoretical objections to accepting that they might have represented such signals have now vanished. It all depends on the details of the experiment. We now know that phytochrome in dark-grown seedlings resides largely in the cytoplasm and moves to the nucleus only after irradiation with red light transforms it to the FR absorbing form of phytochrome (Pfr). This movement takes some time, and in the case of the predominant phytochrome A, is accompanied by a loss of photoreversibility. Thus, for my results to be meaningfully connected to phytochrome, the conditions of prior irradiation of the tissue and the time interval involved before measurement of photoreversibility were critical. If I got these conditions right, it could only have been due to chance, because of course at the time I was ignorant of these relevant parameters. I can only say that preparation of tissue sample took place under the subdued light of the laboratory incandescent bulb, and required 10 to 15 min before the sample could be placed into the beam of the spectrophotometer. As reported above, the sample was then exposed for 5 min to actinic red light of unknown fluence rate before the first absorption spectrum was taken, then to 5 min of actinic far-red of unknown fluence before the second spectrum was recorded. If these parameters were “correct,” then the detected reversibility could have been due to phytochrome A. This situation probably reenforces an old rule; i.e. when theory and data are in conflict, one should usually trust the data and alter one's theoretical interpretation. Our understanding of pigment localization has been even further transformed by very recent observations from the laboratory of Steve Kay (Mas et al., 2000). Recalling that many plant responses depend on interactions between multiple photoreceptors, Kay and his colleagues have found that cooperation between phyB and cry2 in control of flowering, circadian rhythms, and hypocotyl elongation in Arabidopsis depends upon their joint presence in nuclear “speckles” that are formed in a light-dependent fashion. Not only does phyB come down with cry2 in co-immunoprecipitation experiments, but the two pigments are able to transfer energy between them by a quantum-mechanical process of resonance energy transfer. Such a non-radiative mechanism can occur only if the two pigments are closely appressed, so that photoexcitation of one pigment can lead to fluorescence of the other. The authors conclude: “Together, these results demonstrate the light-dependent colocalization of phyB and cry2 in specific nuclear speckles.” What Kay et al. are describing might thus be characterized as a higher plant “eyespot.” The function of this pigment complex may well involve the binding of Ca2+ to a protein recently found by Guo et al. (2001) to be enriched at the periphery of the nucleus, near the nuclear envelope. The similarity to my description of the spectrophotometric localization of phytochrome is striking. Some of my other earlier spectrophotometric data are also brought forward by these observations. Recently, the Cashmore (Ahmad and Cashmore, 1993; Cashmore et al., 1999) and Briggs (Christie et al., 1998; Briggs and Huala, 1999) laboratories have used molecular biological techniques to establish that the blue light photoreceptors cryptochrome and phototropin, respectively, are flavoproteins. This confirms a suggestion I made more than a half century ago (Galston, 1949, 1950) partially on the basis of spectrophotometric evidence. At that time, virtually all informed opinions on phototropic receptors favored carotenoids, rather than flavins, as the relevant photoreceptors. When I discovered that photoactivated riboflavin could cause the oxidative destruction of indoleacetic acid (Galston, 1949), I suggested that this mechanism might be responsible for the well-known asymmetry in auxin distribution in unilaterally illuminated coleoptiles. This proposal had to be discarded in view of Briggs' quantitative support (Briggs et al., 1957) of Went's suggestion (Went, 1928) that there was no change in total diffusible auxin during phototropic curvature. This indicated that lateral auxin translocation, rather than auxin destruction, was responsible for the asymmetries in auxin and growth patterns, and led to a rejection of the significance of the riboflavin-indole-3-acetic acid reaction and thus of riboflavin's involvement in phototropism. However, I had also pointed out (Galston, 1950) that photoactivated flavins could catalyze oxidation of several amino acids like His and Trp, as well as peptides, enzymes, and even bacteriophages containing these amino acids. Thus, it was a mistake to discard the flavin hypothesis of photoreception on the basis of auxin data alone. I also pointed out (Galston, 1959) that action spectrum data from the blue region of the spectrum could not be used to discriminate between flavins and carotenoids. So, in this instance as well, modern genetic and molecular techniques have validated hypotheses derived from spectral data that could not resolve an old problem concerning photoreceptors. ACKNOWLEDGMENTS I thank my Yale colleague, Xing-Wang Deng, for urging me to write this article and for suggesting changes in the initial draft. 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