Plant Physiology

Minorsky, Peter V.

doi: 10.1104/pp.900013pmid: N/A

NADP Malic Enzymes in C3 and C4Flaveria Species The genus Flaveriais unusual in containing C3, C4, and C3–C4 intermediate species. This diversity makes Flaveria an interesting subject for studying the molecular events that have accompanied the evolutionary transition from C3 to C4 photosynthesis. In the most common C4 pathway for C fixation, NADP-malic enzyme (NADP-ME) is involved in decarboxylating malate in the chloroplasts of bundle sheath cells. Isoforms of plastidic NADP-ME are encoded by two genes in all species of Flaveria, including C3, C4, and C3–C4 intermediate types. In this issue, Lai, Wang, and Nelson (pp. ) report that only one of these genes (ChlME1) encodes for the isoform involved in C4 photosynthesis. A comparison of the expression patterns of ChlMe1 andChlME2 genes in developing leaves of C3 and C4 Flaveriaspecies revealed that in C4 species,ChlMe1 is expressed non-specifically early in leaf development and becomes bundle sheath-specific as leaves mature (Fig.1). In C3 species, however, ChlMe1 is only transiently expressed early in leaf development. In contrast, ChlMe2 expression occurs only transiently during chloroplast development in both C3 and C4 species, possibly serving to provide a burst of NADPH and pyruvate for protein and lipid synthesis during chloroplast biogenesis. These results indicate that during the course of C4 evolution, the expression pattern of ChlMe2 remained constant, while the expression pattern of ChlMe1 changed markedly. Fig. 1. Open in new tabDownload slide Expression of one of the forms of chloroplastic NADP-ME in the bundle sheath cells of the mature leaves of a C4-type Flaveria species indicates its involvement in C4 photosynthesis. Fig. 1. Open in new tabDownload slide Expression of one of the forms of chloroplastic NADP-ME in the bundle sheath cells of the mature leaves of a C4-type Flaveria species indicates its involvement in C4 photosynthesis. A companion paper by Lai, Tausta, and Nelson (pp. )examines the role of cytosolic NADP-ME in Flaveria. They show that the gene CytMe encodes for cytosolic NADP-ME in all Flaveria species regardless of the species' mode of photosynthesis. Based on the expression pattern of CytMe, the authors propose that cytosolic NADP-ME has several distinct roles in plants, including the supplying of NADPH for cytosolic metabolism, the balancing of cellular pH in illuminated leaves, and in providing reducing agents and carbon metabolites during wound repair.CytMe transcripts of different size appear to be involved in these three different processes. Nitric Oxide: A Key Link in Abscisic Acid-Induced Stomatal Closure The process by which abscisic acid (ABA) induces stomatal closure has been intensively studied, but despite great effort, our understanding of the ABA signal transduction mechanism in guard cells is far from clear. Perhaps some important pieces of the puzzle are provided by Neill et al. (pp. ), who present pharmacological evidence that nitric oxide (NO) plays a critical role in ABA-induced stomatal closure in pea (Pisum sativum). NO causes stomatal closure, and both inhibitors of NO synthesis and NO scavengers block ABA-induced stomatal closure. Neill et al. also employed diaminofluorescein diacetate (DAF-2 DA), a fluorescent indicator probe to visualize NO levels in guard cells under various pharmacological treatments. The application of ABA increased DAF-2 DA fluorescence in pea guard cells, and this increase was prevented by pretreatment with either a NO scavenger or an inhibitor of NO synthesis (Fig. 2). Because NO signaling commonly involves the production of the second messengers cyclic GMP and cADP-Rib, the authors also studied the respective effects of a specific inhibitor of NO-sensitive guanylate cyclase and an antagonist of cADP-Rib on stomatal aperture. Neither of these inhibitors alone had an effect on stomatal aperture, but both inhibited ABA- and NO-induced stomatal closure. The authors propose that NO is a key link in ABA-induced stomatal closure, and that ABA- and NO-induced stomatal closures require the synthesis and action of cyclic GMP and cADP-Rib. Fig. 2. Open in new tabDownload slide The fluorescent indicator probe DAF-2 DA reveals NO synthesis in pea guard cells under different pharmacological conditions: a, control; b, ABA; c, ABA and NO scavenger; d, ABA and NO synthase inhibitor. Fig. 2. Open in new tabDownload slide The fluorescent indicator probe DAF-2 DA reveals NO synthesis in pea guard cells under different pharmacological conditions: a, control; b, ABA; c, ABA and NO scavenger; d, ABA and NO synthase inhibitor. New Light on the Functions of Phytochromes The perception of red (R) and far-red (FR) light by various phytochromes affects the growth and development of a plant throughout its life. In Arabidopsis, phytochrome is a small gene family consisting of five members, PHYA through PHYE. Individual phytochrome family members have both partially overlapping and distinct functions. In the case of the photoregulation of hypocotyl elongation, phyB mediates the classic R/FR photoreversible low fluence response (LFR). In contrast, phyA mediates two other types of responses: the high irradiance response (HIR) that requires sustained exposure to FR, and the very-low-fluence response (VLFR) that is mediated by brief exposures to radiation between 300 and 780 nm. In this issue, Luccioni et al. (pp. ) report on their studies of the relative magnitudes of HIR, VLFR and LFR responses in different accessions of Arabidopsis. Their analysis reveals a significant negative correlation between VLFR and LFR or HIR. The authors also provide tantalizing evidence that brassinosteroids may be part of the “switch” mechanism that adjusts plant sensitivity to light by means of these different phytochrome responses. A mutant that displays an enhanced VLFR but reduced HIR and LFR was found to be allelic to a brassinosteroid biosynthesis mutant. The enhancement of VLFR by this mutation was lost in seedlings not expressing functional phyA. The authors suggest that brassinosteroids may play a role in fine tuning a plant's repertoire of phytochrome-mediated responses to best suit the growth and development of the plant under the light conditions it encounters. In contrast to our insight into the functions of phyA and phyB, much less is known about the function of other phytochromes. In this issue,Hennig et al. (pp. ) report that phyE plays a role in controlling photo-induced seed germination in Arabidopsis. Previous studies have shown that both phyA and phyB mediate the photo-induction of seed germination by R light whereas the induction of seed germination by FR light is mediated only by phyA. However, a role for other phytochrome members in this process was indicated by the fact that phyA phyB double mutants still demonstrated R/FR-reversible induction of seed germination. Hennig et al. employed a set of photoreceptor mutants to test whether phyD or phyE or both can control photo-induced germination. Their results indicate that only phyB and phyE participate directly in R/FR reversible germination, but that phyE, unlike phyB, does not inhibit phyA-mediated germination. In fact, phyE is required for germination of Arabidopsis seeds in HIR conditions. This interaction of phyE with phyA, however, is not observed in other HIR responses, including the induction of cotyledon unfolding or agravitropic growth. A Papain Ortholog Expressed in Differentiating Xylem Elements Tonoplast rupture releases vacuolar contents into the cytoplasm of differentiating tracheary elements and is rapidly followed by cell death. Hydrolytic enzymes released during this process continue the post-mortem digestion of the cell. A Cys peptidase (XCP1) that is homologous to papain has previously been detected in Arabidopsis, and it is localized exclusively in the xylem. To determine whether XCP1 could be involved in tracheary element autolysis,Funk et al. (pp. ) investigated the localization of XCP1 using XCP1 promoters fused to β-glucuronidase and immunofluorescent confocal microscopy. Their results indicate that XCP1 is localized in the in the vacuole, consistent with it playing a role in tracheary element differentiation. The ectopic expression ofXCP1 resulted in a range of phenotypes, with the most severely affected lines exhibiting stunting, increased anthocyanin levels, and early leaf senescence. The authors also present an intriguing hypothesis that the differentiation of laticifers may simply be a variation of the emerging model of tracheary element differentiation. They point out that differentiating tracheary elements and laticifers have many features in common, including their occurrence in the xylem, their accumulation of high levels of hydrolytic enzymes, and their formation of intercellular connections through end-wall perforations. Within the laticifer protoplast, however, only vesicles of ER origin are retained as the rest of the internal organelles, including the vacuole, become broken down. In laticifers, papain is localized in the ER vesicles, not in the central vacuole. Perhaps because of this, the complete autolysis of laticifers is prevented, and enzymes identical or paralogous to those used to catalyze the final steps of tracheary element autolysis are employed in laticifers as part of a pressurized defense network that is poised for the quick release of defensive peptidases. Systemic Induction of a Ca2+-Dependent Protein Kinase (CDPK) Plants undergo systemic physiological changes in response to local injuries caused by insects, pathogen attack, or mechanical wounding. The systemic wound-induced response is regulated by chemical factors including abscisic acid, jasmonic acid, oligosaccharides and the octadecapeptide systemin, and by physical signals such as hydraulic variation potentials and electrical activation potentials. An important step in the signal transduction pathways of many of these chemical and physical factors is a transient increase in cytoplasmic Ca2+ levels, and the activation of CDPKs. In this issue,Chico et al. (pp. ) report upon their isolation of a cDNA clone (LeCDPK1) from tomato (Lycopersicon esculentum) that encodes for a CDPK. LeCDPK1 was rapidly and transiently enhanced in detached tomato leaves treated with pathogen elicitors or H2O2. Moreover, a systemic increase in LeCDPK1 mRNA was detected upon wounding, and this was correlated with an increase in the activity of a soluble CDPK. These results suggest that the up-regulation of LeCDPK1 is an integral part of tomato's defense against both biotic and abiotic attacks. Author notes www.plantphysiol.org/cgi/doi/10.1104/pp.900013. Copyright © 2002 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)

Cleland, Robert

doi: 10.1104/pp.900016pmid: N/A

Robert Cleland Growing up, I never considered becoming a biologist. That may seem rather strange, considering that my father was a botanist/geneticist. However, I rarely spent any time in his lab, and plants were almost never discussed at the dinner table. My mother, a chemist, had much more influence on me at that stage. I went to Oberlin College with no idea as to what I wanted to do. From living at a university all my life, I knew the advantages of an academic career, but I hadn't picked a field. At Oberlin, I took one botany course, which I didn't particularly like, and majored in organic chemistry. However, by the end of my junior year, I knew that I was not really excited about a career as an organic chemist. Then my father invited me to accompany him to the American Institute of Biological Sciences meeting that summer. It sounded like a lark, so I went. I certainly didn't expect it to change the direction of my career! I listened to a series of interesting symposium talks on plant biochemistry, and I was particularly entranced by F.C. Steward's talk on amino acid biochemistry. Seeing a spark of interest, my father introduced me to a number of plant biochemists. One of them, James Bonner, talked to me at length and urged me to apply to the California Institute of Technology, despite my deficiency in courses in life sciences. I did so, and to my surprise I was accepted. I really wonder how many biology departments now would admit a person with my deficient biology background, or with my lack of a clear vision as to my future. After my graduation from Oberlin, my father gave me a copy ofPrinciples of Plant Physiology, by Bonner and Galston. I started reading it, and I couldn't put it down until I had finished it. It read like a novel! It was not nearly as comprehensive as modern plant physiology textbooks, and it was even wrong about certain things. This book prepared me to be excited about the science I was about to embrace, which is what a really good textbook should do! The Biology Department at Cal Tech was the ideal place to motivate a graduate student. It was full of brilliant faculty, postdocs, visitors, and fellow graduate students, many of whom were leaders in their fields or would soon become leaders. At least seven of the people there then subsequently received Nobel Prizes. The most important aspect of the department for me was the positive atmosphere that existed. We thrived on the thrill of finding out new things, on learning how to give scientific talks and to teach, and on an ethos of hard work followed by hard play. The faculty constantly built up our self-esteem. We were made to feel that we were the “local world's authority” in our own area. With time, I have come to realize how unusual that is; in too many cases, graduate students are being belittled and made to feel unworthy. We left Cal Tech with a firm belief that we were going to make a mark in our field, and that we could do anything if we were willing to put in the time and effort. That belief sustained me during the difficult years as an Assistant Professor when I was trying to get my research program established. When I first started in Bonner's lab, James was convinced that auxins caused cell enlargement by promoting active water uptake. I was assigned to do an experiment that was going to prove this theory. I knew so little about the subject that when I showed the results to James, I was unaware that the data destroyed his theory. He had me repeat the experiments, and he then explained to me that the results indicated that auxin must, instead, be promoting cell elongation by causing cell wall loosening. That started me on a pursuit that has captivated me my whole career. It led to the studies on the mechanical properties of cell walls, which I initiated at Berkeley and continued during a sabbatical with Preston at Leeds. This then led to the “acid-growth theory” of auxin-induced cell enlargement, which was the focus of so much activity here at the University of Washington. Few graduate students or postdocs seem to appreciate the great difficulties that will face them when they become an Assistant Professor. I certainly was unprepared for what I would have to do when I joined the Botany Department at the University of California, Berkeley. There just never was enough time! Teaching, research, and grant writing were all full-time jobs. Then there were all the committees, meetings, and other jobs that had to be done. And finally, if possible, there had to be some time left over for the family. It was really an impossible task. I know that I had troubles reaching the proper balance of activities, and would have failed completely without the understanding and encouragement of my wife, Molly. Berkeley, in those days, was not a particularly research-friendly place for an Assistant Professor. For example, the Botany department had no ice machine. There was one next door to my lab, but it belonged to another department. It took me nearly 2 months and many interviews to get permission to use some of their ice, and then I was limited to not more than two buckets a day. It was a difficult decision to leave the “prestigious” Berkeley for the less well known University of Washington. Without a doubt, it was the best decision I could ever have made. When I arrived in Seattle, I found that there were no biology courses or programs, only separate botany and zoology departments. With a few other newly arrived faculty, I set up the first majors-level introductory biology course. This was to be an alternative to the traditional introductory botany and zoology courses, but within a few years, this biology course expanded and displaced the other courses. I have taught in that course every year, and find it just as fascinating to teach now as it was at the start (perhaps more so, because of all the advances in plant biology). We also set up a biology major at the same time, and I ran it for its first few years (as well as 7 years more recently). Our Interdisciplinary Biology Program still thrives, and biology is the fourth-largest major at this university. All of this focus on undergraduate teaching has taken a lot of time and certainly reduced my research productivity, but I wouldn't have done it any differently if I could do it over again. I realize that persons like myself, with academic positions, are among the luckiest people in the world. There is the constant thrill of finding out something new. That doesn't occur every day, or even every week or month, but it does occur often enough to keep life interesting. Then there is the joy of the association with the undergraduates. It is such fun to watch them learn and see their enthusiasm build as they start to understand how plants work. And they ask those difficult-to-answer questions that make you stop and think about established dogma. The association with the graduate students and postdocs tops it off. I keep being delighted at the new ideas they generate on a daily basis. And finally, there are the interactions with the scientists working on the same problems. I am amazed at how close and friendly those associations have been. In my areas of research, cooperation and sharing of data and ideas has been the norm, not the competition that exists in some other areas. I can't remember a time when anyone has ever refused to provide me with data that I sought, or held back advice that might have aided me. I wonder how many other professions there are where you can go through your whole career and make those statements. Finally, there are the opportunities to take sabbatical leaves, which have permitted me to live and work for a period of time at some other university in this country or abroad. These advantages far outweigh the disadvantages of being overworked, insufficient time with the family, and feeling that nothing is done as well as it could be done. If I had to do it over again, there is little I would change. Author notes www.plantphysiol.org/cgi/doi/10.1104/pp.900016. Copyright © 2002 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.900015pmid: N/A

Peter V. Minorsky By a curious twist of fate, I have returned this year to Vassar College, my undergraduate alma mater. I am 42 years old now, and if the actuarial tables are to be believed I shall live another 42 years. In short, it is a good time to reflect on one's fate and one's future. CHILDHOOD I was a typical American boy. Home from school, I would fortify myself with a bowl or two of heavily sweetened cereal and rush off to the adjoining woods to join my friends in our daily reenactments of the Battle of the Bulge. Besides giving me a certain proficiency in the slaughtering of phantasmic Wehrmacht soldiers, these exercises also gave me a singular but practical knowledge of the local flora. This vine adheres to trees; this one hangs free. This stick is too heavy for a sword, this one too light. Not only is this hollow tree a good place to hide, the tree is still alive! I did not know it then, but I was observing Nature up close, the first step to becoming a biologist. Vietnam came to the United States like an ideological whirlwind. One could not remain neutral in these polarized times. I became an anti-war hippy, or as much a hippy as I could be, living under my parents' roof. De rigeur for this new lifestyle was disdain for all things “unnatural.” One afternoon, while my parents were away at work, I plowed up half the side lawn and planted an “organic” vegetable garden. At first, they were aghast at this marring of their crab-grassed suburban splendor, but as children of the Great Depression, I think the idea of free vegetables eventually won them over. And I delivered; the vegetables were plentiful and delicious. My zeal for gardening and landscaping soon spread to the rest of the yard, a passion of mine that has not faded. SCHOOL DAYS I enrolled at Vassar College with vague plans of becoming a physician. Thumbing through the photographs in my introductory biology textbook, I tried to imagine myself taking the rectal temperature of the elephantitis victim or breaking the sad news to the parents of the kids with progeria. My imagination failed me. There's more to doctoring I realized than removing splinters from the fingers of children and pocketing $250,000 a year. I switched majors to History and then to Literature. Because I enjoy the smells of greenhouses, I took a botany course my junior year, and to my amazement found that reading about phytochrome was infinitely more fascinating than reading Spenser's The Faerie Queen. My senior year I switched back to Biology, and took seven biology courses my senior year. I began my graduate studies at Cornell University in the laboratory of Roger Spanswick. It was a fortunate choice because Roger, an Englishémigré, carried with him the laissez-faireattitude that characterizes the British style of education. I had at my disposal a well-equipped laboratory and a fantastic library. The idea for a thesis topic, however, I had to provide myself. One of the best attributes of a liberal arts education in general is its emphasis on thinking and challenging the status quo. This background I believe enabled me take on a bigger problem than do most graduate students. After three solid years in the library, I wrote two radical re-syntheses of the literature pertaining to the effects of cold temperatures on plants. In the first, I proposed that chilling injury arises from a loss of calcium homeostasis; in the second, that plants respond to rapid cooling by a transient increase in cytoplasmic calcium. Regrettably, the technology of the time was not sufficiently developed to measure changes in cytoplasmic calcium, but I believe that is was me who, in a visit to Edinburgh in 1987, inspired Tony Trewavas to begin the process of transgenically engineering the calcium-sensitive photoprotein aequorin into plants. As a result, my electrophysiological experiments concerning calcium and rapid-cooling stimulation have been complemented by this elegant technique. Ironically, far from being a boon to my career, my reviews, having earned me the not quite accurate reputation of being a “theorist,” have actually hobbled it. For example, an anonymous reviewer, in torpedoing my last grant proposal, wrote, “Minorsky has made some important theoretical contributions to the study of low temperature biology in plants, perhaps even more important than the average experimental paper, but theorists don't need money.” Such are the rewards for challenging the status quo! Even in the purely pedagogical sphere, these “theoretical” papers have been detrimental. I almost didn't get one teaching job because the selection committee divined, based on the fact that most of my articles were single-authored, that I didn't get along well with others! HAVE PhD, WILL TRAVEL After Cornell, I signed up as a post-doctoral fellow on aSaccharomyces project. From my physiologist's perspective,Saccharomyces proved to be an inscrutable little organism. I began to appreciate what Barbara McClintock meant by having a “feeling for the organism.” The truth is, I didn't care aboutSaccharomyces. Unbeknownst to me, my health was also failing. My pituitary had ceased its dialogue with my thyroid, and a crushing depression was settling upon me. Moreover, I felt as if I was getting to know more and more about less and less. My instincts told me to escape to the salt mines of institutional research and to embark upon the insouciant life of an undergraduate professor. Things haven't worked out quite as I imagined. A tenure-track position has remained elusive and the life of the nomadic visiting professor is discouraging, particularly if one has familial or social entanglements. I will soon be beginning my fifth stint as a visiting assistant professor at Mercy College. Since visiting professors generally have little or no opportunity to do research, it can be a slippery slope to oblivion. Fortunately, my other career as a scientific writer has kept me up-to-date and productive. I take great satisfaction in being a scientific writer. I recently had the privilege of revising and updating the Plant Form and Function unit of Campbell and Reece's Biology 6thedition, the leading introductory biology textbook in the English-speaking world. It turned out to be one of the greater challenges of my life. The problem in writing textbooks, I discovered, is not so much deciding what material to include as what to exclude. There is also the challenge of being clear and concise, and engaging but scientifically rigorous. Perhaps the most difficult part is to think like a 19-year-old who is confronting the material for the first time. I have also immensely enjoyed my first year as the Science Writer for Plant Physiology. This position has enabled me, indeed in a few instances, forced me, to continue learning. Every month I read every abstract in Plant Physiology, and about 30% of the articles in their entirety. What a marvelous opportunity to keep up on cutting edge research! AM I HAPPY? There is not much lucre or glory in being an undergraduate professor. Its main selling points are the broad intellectual stimulation and freedom it provides. For example, Vassar was recently the site for the filming of a new version of H.G. Wells's The Time Machine, and to mark this event, I organized a multi-disciplinary symposium concerning Wells and his literary works. As I lectured on the life and literature of Wells, I could not help but wonder whether my peers in the large research institutions have such intellectual freedom? What could be better than the freedom to pursue one's interests wherever they lead? If there be a better life, I would need more time to think about it. Right now, however, I must grade 30 freshman laboratory reports before tomorrow (OK, so every job has its downside!). Author notes www.plantphysiol.org/cgi/doi/10.1104/pp.900015. Copyright © 2002 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.900014pmid: N/A

TRIGONELLINE: A DIVERSE REGULATOR IN PLANTS Trigonelline (N-methyl nicotinamide) first gained the attention of plant biologists because it proved to be very effective in inducing G2 arrest in the root apices of many plant species. Although trigonelline, a metabolite of nicotinamide, was put forth as a possible plant hormone over 20 years ago (Evans and Tramontano, 1981), this idea was to gain little acceptance in the ensuing years. Meanwhile, however, evidence from diverse quarters has slowly been accumulating that indicates that the effects of trigonelline are not just limited to cell cycle regulation: Trigonelline appears to be a regulator of sundry other functions in plants. This month's The Hot and the Classic summarizes the state of knowledge concerning the regulatory functions of trigonelline in plants. Cell Cycle Regulation Evans et al. (1979) identified trigonelline as the substance in pea (Pisum sativum) cotyledons that promoted G2 arrest in root and shoot meristems. Concentrations of trigonelline as low as 10−7 m were found to be effective. Trigonelline is present in ungerminated seeds and is transported to other parts of the seedling during early germination. The fact that added trigonelline can replace cotyledons in promoting G2 arrest, and that the trigonelline levels in planta were highly correlated with the proportion of cells arrested in G2, led to the suggestion that trigonelline may be a natural plant hormone (Evans and Tramontano, 1981). More recent evidence suggests that trigonelline may act as a cell cycle regulator by preventing the ligation of replicons during the S-phase of the cell cycle (Mazzuca et al., 2000). Mean replicon size was determined to be about 2.5-fold longer in lettuce (Lactuca sativa) seedlings treated with 3 mm trigonelline than in controls. Trigonelline also resulted in a lengthening of both the S-phase and the cell cycle and to a decrease in primary root elongation. Hence, Mazzuca et al. (2000) proposed that replicon inactivation may underlie the protracted S-phase and inhibition of growth. Trigonelline treatment also resulted in a 1.6-fold increase in fork rate compared with the control. The faster fork rate in the larger replicons is in accord with the highly significant positive relationship that has been established between fork rate and replicon size for various unrelated higher plants. Mazzuca et al. (1997) also noted that trigonelline treatments caused the nucleoli of plant cell nuclei to become very large and to undergo heavy labeling with radioactive thymidine. These changes were clearly related to the presence of trigonelline as the size of the nucleoli rapidly diminished following a recovery period in water. Since the modifications of the nucleoli detected in treated roots were accompanied by changes in the protein pattern, the results indicate that trigonelline may exert its role through synthesis of new specific proteins. Nodulation In addition to the flavonoids exuded by many legumes as signals to their rhizobial symbionts, alfalfa (Medicago sativa) releases trigonelline into the rhizosphere. Trigonelline specifically activates the expression in Rhizobium meliloti of a class of genes (trc genes) that are apparently involved in trigonelline catabolism (Boivin et al., 1990). The authors suggest that trigonelline may be used as a nutrient source by the bacteria during all stages of the symbiotic relation, including in the rhizosphere, and in the nodules of the host plant. Althoughtrc mutants produce normal appearing nodules under laboratory conditions, the authors speculate that under field conditions, the presence of these catabolic genes may confer upon certain strains a selective advantage for the colonization of the rhizosphere or in the development of the plant infection or both. Oxidative and UV Stresses Berglund (1994) has put forth the interesting hypothesis that nicotinamide is an important part of the signal transduction chain involved in the response of plant cells to conditions that cause DNA strand breakage, especially in connection with oxidative stress. In stressed cells, nicotinamide is released as a result of the activity of the nuclear enzyme poly(ADP-Rib) polymerase (PADPRP). PADPRP is activated by various types of stress that cause DNA strand breakage, including oxidative stress, UV stress, and mutagens. The activation of PADPRP does not occur at the level of the gene, but through the interaction of PADPRP with damaged DNA. PADPRP synthesizes polymers of ADP-Rib that become attached to various DNA-associated proteins. The ADP-Rib consumed in this process comes from NAD, which is thus degraded with the release of nicotinamide. Thus, Berglund proposes that during those types of stress that cause an increased frequency of strand breaks in DNA, there is a rapid increase in nicotinamide (and consequently trigonelline) levels within plant cells. Berglund proposes that nicotinamide and trigonelline may serve as potent inducers of defensive metabolism in plants, including glutathione metabolism, and the accumulation of secondary defense compounds. Indeed, Kalbin et al. (1997) found that strong UV-B irradiation caused marked increases in the levels of nicotinamide, trigonelline, and total oxidized glutathione in pea leaves. They concluded that elevated levels of nicotinamide and trigonelline do occur in response to UV-B, but only at UV-B doses high enough to cause oxidative stress (see also Berglund et al., 1996). Thus, nicotinamide and/or its metabolites (including trigonelline) may function as signal transmitters in the response of plants to oxidative stress, and poly(ADP-Rib) polymerase may play an important role in the induction of defensive metabolism. DNA Methylation It has been suggested that the physiological effects of trigonelline and other quaternary ammonium compounds in plants could occur at the level of DNA methylation (Kraska and Schönbeck, 1993). Trigonelline, choline, and betaine show a hypo-methylating effect in plants. In the case of trigonelline,Berglund (1994) proposes that the deamidation of nicotinamide to nicotinic acid followed by the methylation of nicotinic acid to trigonelline, may consume S-adenosyl-Met, which is the methyl donor employed when DNA is methylated. Because DNA methylation is generally linked to DNA replication, DNA demethylation may play a role in mediating the effects of trigonelline on the cell cycle. Salt Stress In response to excess salt, many plants accumulate osmoregulators such as Gly betaine, Pro, and trigonelline to prevent water loss. Tramontano and Jouve (1997) found that alfalfa plants undergo a 5-fold increase in Pro and a 2-fold increase in trigonelline after salt-stress. Further experiments examined whether other known osmoregulators (e.g. Pro and Gly betaine) could affect the cell cycle parameters in cultured root meristems of peas in a manner similar to trigonelline. At concentrations of 10−4 to 10−7 m, trigonelline induced an accumulation of G2 nuclei, whereas Pro was ineffective and Gly betaine only slightly effective in promoting G2 nuclei accumulation. These results confirm that the cell cycle effects of trigonelline are indeed specific and that trigonelline may play a role as an osmoregulator in salt-stressed plants, an idea strengthened by the observation by Shomerilan, Jones, and Paleg (1991) that trigonelline, like Pro, increases the in vitro thermal and salt stability of pyruvate kinase. Nyctinasty The laboratory of M. Ueda has been extremely prolific in identifying factors from leaf extracts that are effective in inducing leaf closure in various species that undergo pronounced sleep movements. Trigonelline was isolated from Aeschynomene indica as a bioactive substance for nyctinasty (Ueda, Niwa, and Yamamura, 1995). The compound was quite effective for leaf closing of this species at 0.1 μm in the daytime, but not for the nyctinastic species Cassia mimosoides and Mimosa pudica. It competed with indole-3-acetic acid, which is effective in leaf opening. These results suggest that trigonelline may be involved in the circadian rhythm of A. indica. LITERATURE CITED 1 Berglund T Nicotinamide, a missing link in the early stress-response in eukaryotic cells—a hypothesis with special reference to oxidative stress in plants. FEBS Lett 351 1994 145 149 Google Scholar Crossref Search ADS PubMed WorldCat 2 Berglund T Kalbin G Strid A Rydstrom J Ohlsson AB UV-B- and oxidative stress-induced increase in nicotinamide and trigonelline and inhibition of defensive metabolism induction by poly(ADP-ribose) polymerase inhibitor in plant tissue. FEBS Lett 380 1996 188 193 Google Scholar Crossref Search ADS PubMed WorldCat 3 Boivin C Camut S Malpica CA Truchet G Rosenberg C Rhizobium meliloti genes encoding catabolism of trigonelline are induced under symbiotic conditions. Plant Cell 2 1990 1157 1170 Google Scholar Crossref Search ADS PubMed WorldCat 4 Evans LS Almeida MS Lynn DG Nakanishi N Chemical characterization of a hormone that promotes cell arrest in G2 in complete tissues. Science 203 1979 1122 1123 Google Scholar Crossref Search ADS PubMed WorldCat 5 Evans LS Tramontano WA Is trigonelline a plant hormone pea seedlings? Am J Bot 68 1981 1282 1289 Google Scholar Crossref Search ADS WorldCat 6 Kalbin G Ohlsson AB Berglund T Rydstrom J Strid A Ultra-violet-B-radiation-induced changes in nicotinamide and glutathione metabolism and gene expression in plants. Eur J Biochem 249 1997 465 472 Google Scholar Crossref Search ADS PubMed WorldCat 7 Mazzuca S Bitonti MB Innocenti AM Francis D Inactivation of DNA replication origins by the cell cycle regulator, trigonelline, in root meristems of Lactuca sativa. Planta 211 2000 127 132 Google Scholar Crossref Search ADS PubMed WorldCat 8 Mazzuca S Bitonti MB Pranno S Innocenti AM Nuclear metabolic changes in root meristem of Lactuca sativa induced by trigonelline treatment. Cytobios 89 1997 39 50 Google Scholar OpenURL Placeholder Text WorldCat 9 Kraska T Schönbeck FJ About changes in the chromatin structure after resistance induction in Hordeum vulgareL. J Phytopathol 137 1993 10 14 Google Scholar Crossref Search ADS WorldCat 10 Shomerilan A Jones GP Paleg LG In vitro thermal and salt stability of pyruvate-kinase are increased by proline analogs and trigonelline. Aust J Plant Physiol 18 1991 279 286 Google Scholar OpenURL Placeholder Text WorldCat 11 Tramontano WA Jouve D Trigonelline accumulation in salt-stressed legumes and the role of other osmoregulators as cell cycle control agents. Phytochemistry 44 1997 1037 1040 Google Scholar Crossref Search ADS WorldCat 12 Ueda M Niwa M Yamamura S Trigonelline, a leaf-closing factor of the nyctinastic plant Aeschynomene indica. Phytochemistry 39 1995 817 819 Google Scholar Crossref Search ADS WorldCat Author notes www.plantphysiol.org/cgi/doi/10.1104/pp.900014. Copyright © 2002 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)

Coe, Edward; Cone, Karen; McMullen, Michael; Chen, Su-Shing; Davis, Georgia; Gardiner, Jack; Liscum, Emmanuel; Polacco, Mary; Paterson, Andrew; Sanchez-Villeda, Hector; Soderlund, Cari; Wing, Rod

doi: 10.1104/pp.010953

Neill, Steven J.; Desikan, Radhika; Clarke, Andrew; Hancock, John T.

doi: 10.1104/pp.010707pmid: N/A

Stomatal closure in response to the hormone abscisic acid (ABA) is mediated by a complex signaling network involving both calcium-dependent and calcium-independent pathways (Assmann and Shimazaki, 1999; Webb et al., 2001), activated by several signaling intermediates (Schroeder et al., 2001) that include hydrogen peroxide (Miao et al., 2000; Pei et al., 2000;Zhang et al., 2001) and lipids such as sphingosine-1-phosphate (Ng et al., 2001). Here, we provide evidence that nitric oxide (NO) is also a signaling component of ABA-induced stomatal closure. Our data show that NO synthesis is required for ABA-induced closure and that ABA enhances NO synthesis in guard cells. Exogenous NO induces stomatal closure, and ABA and NO-induced closure require the synthesis and action of cGMP and cyclic ADP Rib (cADPR). ABA-INDUCED STOMATAL CLOSURE REQUIRES NO SYNTHESIS NO is a key signaling molecule in plants, mediating responses to various abiotic and biotic stresses (Delledonne et al., 1998; Durner et al., 1998; Clarke et al., 2000; Beligni and Lamattina, 2001). The recent reports that treatment with a fungal elicitor induced the rapid synthesis of NO in tobacco (Nicotiana tabacum) epidermal cells (Foissner et al., 2000) prompted us to determine any involvement of NO in ABA-regulated stomatal movements. Epidermal peels from pea (Pisum sativum L. Argenteum) were incubated in ABA in the presence of 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a specific NO scavenger previously shown to block NO effects (Delledonne et al., 1998; Clarke et al., 2000), orN G-nitro-l-Arg-methyl ester (l-NAME), an inhibitor of NO synthase (NOS) in mammalian cells that also inhibits plant NOS (Barroso et al., 1999). Pretreatment with either l-NAME or PTIO largely suppressed stomatal responses to ABA (Fig.1a), indicating the requirement for NO synthesis and action during ABA-induced stomatal closure. Exogenous NO also induced stomatal closure. Both sodium nitroprusside (SNP) andS-nitrosoglutathione (GSNO), two chemically different NO donors previously shown to induce defense responses in plants (Delledonne et al., 1998; Durner et al., 1998; Clarke et al., 2000;A.-H.-Mackerness et al., 2001) induced stomatal closure, which was readily inhibited by pretreatment with PTIO (Fig. 1a). SNP effects were determined in more detail; the dose response and kinetics of SNP-induced stomatal closure are shown in Figure 1, b and c. At the concentrations tested, SNP did not reduce the viability of guard cells, and in wash-out experiments the stomata reopened fully, indicating that the effects of SNP were fully reversible (not shown). Fig. 1. Open in new tabDownload slide Effects of ABA and NO on stomatal closure in pea. a, Epidermal peels, prepared from Argenteum pea (Burnett et al., 2000), were incubated in the light in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (0.01m MES-KOH, 0.05 m KCl, pH 6.15) to induce stomatal opening and then: incubated for 2 h in buffer alone (light), 10 μm ABA (A), ABA + 200 μm PTIO (A+P), ABA + 25 μm l-NAME (A+L), 100 μm SNP (S), SNP + 200 μm PTIO (S+P), 500 μmGSNO (G), and GSNO + 200 μm PTIO (G+P). b, Dose response for SNP, after incubation for 2 h. c, Kinetics of SNP-induced stomatal closure (100 μm SNP). Bars = se (n = 180). Fig. 1. Open in new tabDownload slide Effects of ABA and NO on stomatal closure in pea. a, Epidermal peels, prepared from Argenteum pea (Burnett et al., 2000), were incubated in the light in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (0.01m MES-KOH, 0.05 m KCl, pH 6.15) to induce stomatal opening and then: incubated for 2 h in buffer alone (light), 10 μm ABA (A), ABA + 200 μm PTIO (A+P), ABA + 25 μm l-NAME (A+L), 100 μm SNP (S), SNP + 200 μm PTIO (S+P), 500 μmGSNO (G), and GSNO + 200 μm PTIO (G+P). b, Dose response for SNP, after incubation for 2 h. c, Kinetics of SNP-induced stomatal closure (100 μm SNP). Bars = se (n = 180). The effects of ABA on NO synthesis were determined using the cell-permeable fluorescent NO probe diaminofluorescein diacetate (DAF-2 DA), recently used to visualize NO synthesis in tobacco (Foissner et al., 2000), and Taxus brevifolia and Kalanchoe daigremontiana (Pedroso et al., 2000). Autofluorescence was observed associated with the inner walls of the guard cells in control samples, with low-level, diffuse fluorescence also apparent in a small number of the guard cells (Fig. 2a). Exposure to 10 μm ABA induced a rapid and striking increase in the fluorescence of guard cells that was evident after 5 min and substantial after 30 min (Fig. 2b). Fluorescence was apparent in the cytosol and particularly intense in chloroplasts. Average fluorescence intensity increased by 52% in epidermal cells and by 120% in guard cells (n = 21). After 30 min, 35% of the guard cells fluoresced brightly (n = 250) compared with 8% for control cells (n = 247), and within 60 min, 80% (n = 362) were fluorescing (17% for control,n = 216). ABA-induced DAF-2 DA fluorescence in guard cells was largely prevented by PTIO (14% cells fluorescing,n = 105; Fig. 2c). Pretreatment withl-NAME also substantially suppressed ABA-induced DAF-2 DA fluorescence (11% of cells fluorescing, n = 54; Fig. 2d), suggesting that pea guard cells possess a NOS-like enzyme. Interestingly, NOS enzyme activity and a partial NOS cDNA clone have been isolated from pea leaves (Barroso et al., 1999; Corpas et al., 2001). It has been reported recently that DAF-2 DA fluorescence is amplified in the presence of Ca2+, although still absolutely dependent on the presence of NO (Broillet et al., 2001). Because the stimulation by ABA of both the uptake and intracellular release of Ca2+ is well known, we repeated the experiments in the presence of 2 mm EGTA-AM, the membrane-permeable form of the Ca2+ chelator EGTA (Wu et al., 1997). This treatment had no effect on ABA-induced DAF-2 DA fluorescence (60 min, 87% of guard cells fluorescing,n = 326). NO synthesis by epidermal peels was also estimated using the hemoglobin assay (Clarke et al., 2000). Peels were incubated for 60 min and NO release over this period subsequently determined. Constitutive NO release was estimated as 93 ± 7 nmol g−1 (n = 7). This increased significantly (t test, P < 0.05) to 125 ± 7 nmol g−1 (n = 7), an increase of 35%, following treatment with 10 μm ABA. This increase was prevented by co-incubation with 25 μm l-NAME (99 ± 11 nmol g−1 [n = 5]). Fig. 2. Open in new tabDownload slide ABA induces NO synthesis in pea guard cells. Epidermal peels were floated in MES buffer in the light for 1 h and then loaded with DAF-2 DA (Calbiochem, Nottingham, UK; 10 μm in MES, 10 min in the dark, 20 min wash in MES). Following treatments, peels were observed with a laser confocal scanning microscope (Nikon PCM2000, Nikon Europe B.V. Badhoevedorp, The Netherlands; excitation 495 nm, emission 515–560 nm). Acquired images were processed using Adobe Photoshop (Adobe Systems, Mountain View, CA) and relative pixel intensities determined using EZ2000 version 2.1 software (Coord, Amsterdam). Images are shown after a 30-min treatment. PTIO and l-NAME treatments reduced both the intensity and the number of guard cells visibly fluorescing; figure shows those cells in which fluorescence was still visible. a, Control (buffer only). b, 10 μm ABA. c, ABA + 200 μm PTIO. d, ABA + 25 μm l-NAME. Scale bar = 7 μm. Fig. 2. Open in new tabDownload slide ABA induces NO synthesis in pea guard cells. Epidermal peels were floated in MES buffer in the light for 1 h and then loaded with DAF-2 DA (Calbiochem, Nottingham, UK; 10 μm in MES, 10 min in the dark, 20 min wash in MES). Following treatments, peels were observed with a laser confocal scanning microscope (Nikon PCM2000, Nikon Europe B.V. Badhoevedorp, The Netherlands; excitation 495 nm, emission 515–560 nm). Acquired images were processed using Adobe Photoshop (Adobe Systems, Mountain View, CA) and relative pixel intensities determined using EZ2000 version 2.1 software (Coord, Amsterdam). Images are shown after a 30-min treatment. PTIO and l-NAME treatments reduced both the intensity and the number of guard cells visibly fluorescing; figure shows those cells in which fluorescence was still visible. a, Control (buffer only). b, 10 μm ABA. c, ABA + 200 μm PTIO. d, ABA + 25 μm l-NAME. Scale bar = 7 μm. ABA AND NO SIGNALING DURING STOMATAL CLOSURE NO signaling commonly involves the second messenger cGMP, generated via the enzyme guanylate cyclase (Wendehenne et al., 2001), and previous work has provided data consistent with cGMP involvement in plant NO signaling (Durner et al., 1998; Clarke et al., 2000). Consequently, we pretreated epidermal peels with 1H-(1,2,4)-oxadiazole-[4,3- a]quinoxalin-1-one (ODQ), an inhibitor of NO-sensitive guanylate cyclase (Durner et al., 1998; Clarke et al., 2000). ODQ by itself had no effect on stomatal aperture (not shown), but it was a potent inhibitor of both ABA- and SNP-induced stomatal closure (Fig. 3). Furthermore, treatment with 8-bromo-cGMP (8-Br-cGMP), a cell-permeable analog of cGMP known to be active in plant cells (Durner et al., 1998;Clarke et al., 2000), reversed the inhibitory effects of ODQ on ABA- and NO-induced stomatal closure (Fig. 3). Treatment with 8-Br-cGMP alone had no effect (not shown). These data indicate that cGMP is required, but not sufficient, for ABA- and NO-induced stomatal closure. One downstream signaling response to NO and cGMP is intracellular generation of cADPR, a Ca2+-mobilizing molecule (Wendehenne et al., 2001). cADPR involvement in ABA responses has already been demonstrated (Wu et al., 1997; Leckie et al., 1998; MacRobbie, 2000). Consequently, we determined the effects of nicotinamide, an antagonist of cADPR production (Leckie et al., 1999; MacRobbie, 2000), on ABA- and NO-induced stomatal closure (Fig. 3). Nicotinamide inhibited the effects of both ABA and NO, suggesting that inhibition of ABA responses by nicotinamide is, at least partly, due to inhibition of cADPR synthesis following NO generation. Fig. 3. Open in new tabDownload slide ABA- and NO-induced stomatal closure requires cGMP and cADPR. Epidermal peels were incubated in the light to induce stomatal opening and then incubated for 2 h in buffer alone (light), 10 μm ABA (A), ABA + 2 μm ODQ (A+O), ABA + ODQ + 50 μm 8-Br-cGMP (A+O+8Br), ABA + 5 mm nicotinamide (A+Nic), SNP (100 μm), SNP + 2 μm ODQ (S+O), SNP + ODQ + 100 μm8-Br-cGMP (S+O+8Br), and SNP + 5 mm nicotinamide (S+Nic). Bars = se (n = 180). Fig. 3. Open in new tabDownload slide ABA- and NO-induced stomatal closure requires cGMP and cADPR. Epidermal peels were incubated in the light to induce stomatal opening and then incubated for 2 h in buffer alone (light), 10 μm ABA (A), ABA + 2 μm ODQ (A+O), ABA + ODQ + 50 μm 8-Br-cGMP (A+O+8Br), ABA + 5 mm nicotinamide (A+Nic), SNP (100 μm), SNP + 2 μm ODQ (S+O), SNP + ODQ + 100 μm8-Br-cGMP (S+O+8Br), and SNP + 5 mm nicotinamide (S+Nic). Bars = se (n = 180). In summary, the results presented here demonstrate that NO is a novel component of ABA signaling in stomatal guard cells. They show that guard cells generate NO in response to ABA via NOS-like activity, and that such NO production is required for full stomatal closure in response to ABA; that exogenous NO induces stomatal closure; and that cGMP and cADPR are both required for NO- and ABA-induced stomatal closure. Cyclic nucleotide-gated ion channels have recently been cloned and characterized in Arabidopsis (Kohler et al., 1999; Leng et al., 1999). Modulation of the activity of such channels by cGMP may be one mechanism by which NO effects stomatal closure. It will clearly be important to quantify accurately NO production in guard cells and other cell types in a range of species and to determine whether other ABA responses similarly involve NO, particularly as wilting can result in elevated NO production (Lesham and Haramaty, 1996). Very recently, Mata and Lamattina (2001) have reported that NO induces stomatal closure in fava bean (Vicia faba), Salpichroa organifolia, and Tradescantia spp., although a requirement for NO in ABA-induced stomatal closure was not determined. However, our preliminary data indicate that ABA-induced stomatal closure in Arabidopsis also requires NO, as in pea (not shown). These data are important because they point the way to molecular and genetic analyses, which will include studies of the ABA-insensitive and ABA-deficient abi and aba mutants. Moreover, the involvement of NO signaling during stomatal responses to ABA provides a new opportunity to manipulate plant water relations in order to increase agricultural productivity. LITERATURE CITED 1 A.-H.-Mackerness S John F Jordan B Thomas B FEBS Lett 489 2001 237 242 Crossref Search ADS PubMed 2 Assmann SM Shimazaki K-I Plant Physiol 119 1999 809 815 Crossref Search ADS PubMed 3 Barroso JB Corpas FJ Carreras LM Valderrama R Palma JM Lupianez JA del Rio LA J Biol Chem 274 1999 36729 36733 Crossref Search ADS PubMed 4 Beligni MV Lamattina L Plant Cell Environ 24 2001 267 278 Crossref Search ADS 5 Burnett EC Desikan R Moser RC Neill SJ J Exp Bot 51 2000 197 205 Crossref Search ADS PubMed 6 Broillet M-C Randin O Chatton J-Y FEBS Lett 491 2001 227 232 Crossref Search ADS PubMed 7 Clarke A Desikan R Hurst R Hancock JT Neill SJ Plant J 24 2000 667 677 Crossref Search ADS PubMed 8 Corpas FJ Barroso JB del Rio LA Trends Plant Sci 6 2001 145 150 Crossref Search ADS PubMed 9 Delledonne M Xia Y Dixon RA Lamb C Nature 394 1998 585 588 Crossref Search ADS PubMed 10 Durner J Wendehenne D Klessig DF Proc Natl Acad Sci USA 95 1998 10328 10333 Crossref Search ADS PubMed 11 Foissner I Wendehenne D Langebartels C Durner J Plant J 23 2000 817 824 Crossref Search ADS PubMed 12 Kohler C Merkle T Neuhaus G Plant J 18 1999 97 104 Crossref Search ADS PubMed 13 Leckie CP McAinsh MR Allen GJ Sanders D Hetherington AM Proc Natl Acad Sci USA 95 1998 15837 15842 Crossref Search ADS PubMed 14 Leng Q Mercier RW Yao W Berkowitz GA Plant Physiol 121 1999 753 761 Crossref Search ADS PubMed 15 Lesham YY Haramaty E J Plant Physiol 148 1996 258 263 Crossref Search ADS 16 MacRobbie EAC Proc Natl Acad Sci USA 97 2000 12361 12368 Crossref Search ADS PubMed 17 Mata CG Lamattina L Plant Physiol 126 2001 1196 1204 Crossref Search ADS PubMed 18 Miao Y-U Song C-P Dong F-C Wang X-C Acta Phytophysiologia Sinica 26 2000 53 58 19 Ng CK-Y Carr K McAinsh MR Powell B Hetherington AM Nature 410 2001 596 599 Crossref Search ADS PubMed 20 Pedroso MC Magalhaes JR Durzan D J Exp Bot 51 2000 1027 1036 Crossref Search ADS PubMed 21 Pei Z-M Murata Y Benning G Thomine S Klusener B Allen G Grill E Schroeder J Nature 406 2000 731 734 Crossref Search ADS PubMed 22 Schroeder JI Kwak JM Allen GJ Nature 410 2001 327 330 Crossref Search ADS PubMed 23 Webb AAR Larman MG Montgomery LT Taylor JE Hetherington AM Plant J 26 2001 351 361 Crossref Search ADS PubMed 24 Wendehenne D Pugin A Klessig DF Durner J Trends Plant Sci 6 2001 177 183 Crossref Search ADS PubMed 25 Wu Y Kuzma J Marechal E Graeff R Lee HC Foster R Chua N-C Science 278 1997 2126 2130 Crossref Search ADS PubMed 26 Zhang X Zhang L Dong F Gao J Galbraith DW Song C-P Plant Physiol 126 2001 1438 1448 Crossref Search ADS PubMed Author notes * Corresponding author; e-mail [email protected]; fax 00–44–117–3442904. www.plantphysiol.org/cgi/doi/10.1104/pp.010707. Copyright © 2002 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)

Kusaba, Makoto; Tung, Chih-Wei; Nasrallah, Mikhail E.; Nasrallah, June B.

doi: 10.1104/pp.010790pmid: N/A

Genetic dominance and recessiveness are most commonly addressed in the context of mutated alleles that confer aberrant phenotypes but have rarely been explained for functional variants. An opportunity to gain a mechanistic understanding of interactions between naturally occurring functional allelic variants is presented by the self-incompatibility (SI) system of crucifers. This intraspecific mating barrier, which allows the epidermal cells of the stigma to recognize and reject self-related pollen, is based on the activity of a large number of haplotypes of the S-locus complex. Each haplotype encodes highly divergent allelic variants of the S-locus receptor kinase (SRK), a transmembrane protein of the stigma epidermis that determines SI specificity in the stigma (Stein et al., 1991; Takasaki et al., 2000), and the S-locus Cys-rich protein (SCR), a pollen coat-localized ligand for SRK (Kachroo et al., 2001), which determines SI specificity in pollen (Schopfer et al., 1999; Takayama et al., 2000). Self-pollination is proposed to trigger an S haplotype-specific receptor-ligand interaction between SRK and SCR, which leads to the arrest of self-related pollen at the stigma surface (Nasrallah, 2000). In self-incompatible crucifers, including Brassicaspecies and Arabidopsis lyrata, a wild, self-incompatible relative of Arabidopsis, pollen SI specificity is determined by the diploid genotype of the pollen-producing parent rather than by the genotype of individual haploid pollen grains (Bateman, 1954; Thompson and Taylor, 1966; Kusaba et al., 2001; Schierup et al., 2001). Consequently, genetic interactions between S haplotypes occur in the specification of SI phenotype in pollen as well as in stigmas. Allelic interactions of codominance, dominance, incomplete dominance, or mutual weakening occur, and these interactions can differ in stigma and pollen, consistent with the activity of distinct determinants of SI specificity in these two tissues. Recessiveness in pollen confers an advantage on an Shaplotype by allowing pollen of the recessive genotype to elude theS haplotype-specific stigmatic surveillance mediated by SRK. In fact, “pollen-recessive” alleles attain high frequencies in populations (Uyenoyama, 2000). Elucidation of the molecular basis ofS haplotype recessiveness in pollen is important for understanding the mechanism of SI and the evolution and maintenance ofS haplotypes in a population. Here, we examine the genetic interaction of two S haplotypes of A. lyrata and elucidate the molecular basis of their dominant/recessive relationship in pollen. We recently isolated the SRK and SCR genes from two A. lyrata S haplotypes designated Sa andSb (Kusaba et al., 2001). Reciprocal crosses ofSaSb to SaSa and SbSb revealed that, in the stigma, Sa and Sb exhibit a codominant interaction with “weakening” of Sa. In pollen,Sa is recessive to Sb, and pollen grains fromSaSb plants exhibit Sb specificity. These interactions imply that SRKa and SRKb are both active in heterozygotes, although the SRKa allele exhibits somewhat lower activity. In contrast, the activity of SCRais completely masked in SaSb heterozygotes. We found significant differences in the temporal and spatial distribution of SCRa and SCRb transcripts inSa and Sb homozygotes, respectively. On RNA gel blots (Fig. 1A) and by reverse transcriptase-PCR, SCRa transcripts were detected in early stage anthers, which contain a tapetum (a cell layer derived from diploid cells of the sporophyte that serves as nurse tissue for the developing haploid microspores and that degenerates before anther dehiscence), but were not detectable in late-stage anthers, which lack tapetal cells (Fig. 1A). In contrast, SCRb transcripts were detected at early and late stages of anther development (Fig. 1A) as previously described (Kusaba et al., 2001). In situ hybridization of SbSb anthers demonstrated thatSCRb is expressed sporophytically in the tapetal cell layer and gametophytically in microspores (Fig.2), as described for allBrassica SCR alleles examined to date (Schopfer et al., 1999; Schopfer and Nasrallah, 2000; Takayama et al., 2000; Shiba et al., 2001). In contrast, SCRa, which is the only “pollen-recessive” allele isolated to date, exhibits strict sporophytic expression (Fig. 2). Thus, functional SCRalleles can vary dramatically in their expression pattern, and expression of SCR in the tapetal cell layer is sufficient for SI. The additional gametophytic expression exhibited bySCRb and all known Brassica SCRalleles might be redundant or serve to boost SCR levels in individual pollen grains. Fig. 1. Open in new tabDownload slide Differential expression of S-locus genes in A. lyrata. A, Developmental regulation ofSCRa and SCRb expression in A. lyrataanthers. Total RNA (15 μg per lane) was isolated from SaSaand SbSb leaves (L), pistils (P), and anthers at three stages of development: −3 anthers (with intact tapetum) and −1 anther (after degeneration of the tapetum) were collected at 3 d and 1 d before flower opening, respectively. Mature anthers (containing mature pollen grains) were collected from open flowers (0). Blots were probed with SCRa or SCRb cDNAs, which, being only 35% similar, serve as allele-specific probes. Hybridization with actin served as a loading control. Hybridization signals were quantitated using a PhosphorImager and the ImageQuant program (Molecular Dynamics, Sunnyvale, CA). B, Expression ofSRKa in A. lyrata stigmas. poly(A+) RNA (2 μg per lane) was isolated fromSaSa (aa) and SaSb (ab) stigmas (−1 stage) and probed with the SRKa ectodomain and with actin as a loading control. C, Expression of SCRa and SCRb inSaSa (aa), SbSb (bb), and SaSb (ab) anthers. Total RNA (15 μg per lane) was isolated from anthers (A) and microspores (M) collected from −3-stage buds. Probes are as in A. Fig. 1. Open in new tabDownload slide Differential expression of S-locus genes in A. lyrata. A, Developmental regulation ofSCRa and SCRb expression in A. lyrataanthers. Total RNA (15 μg per lane) was isolated from SaSaand SbSb leaves (L), pistils (P), and anthers at three stages of development: −3 anthers (with intact tapetum) and −1 anther (after degeneration of the tapetum) were collected at 3 d and 1 d before flower opening, respectively. Mature anthers (containing mature pollen grains) were collected from open flowers (0). Blots were probed with SCRa or SCRb cDNAs, which, being only 35% similar, serve as allele-specific probes. Hybridization with actin served as a loading control. Hybridization signals were quantitated using a PhosphorImager and the ImageQuant program (Molecular Dynamics, Sunnyvale, CA). B, Expression ofSRKa in A. lyrata stigmas. poly(A+) RNA (2 μg per lane) was isolated fromSaSa (aa) and SaSb (ab) stigmas (−1 stage) and probed with the SRKa ectodomain and with actin as a loading control. C, Expression of SCRa and SCRb inSaSa (aa), SbSb (bb), and SaSb (ab) anthers. Total RNA (15 μg per lane) was isolated from anthers (A) and microspores (M) collected from −3-stage buds. Probes are as in A. Fig. 2. Open in new tabDownload slide In situ localization of SCRa andSCRb transcripts. Paraffin-embedded sections were prepared from SaSa, SbSb, and SaSb −3-stage anthers and hybridized with dioxigenin-labeled RNA probes transcribed in vitro essentially according to protocols athttp://www.Arabidopsis.org/cshl-course. The probes were: αs-a, antisense SCRa; αs-b, antisense SCRb. Negative controls: Sense SCRa (s-a) and sense SCRb (s-b) RNA probes. T, Tapetum; M, microspores. Some microspores inSaSb anthers did not hybridize with the αs-b probe, in keeping with the expected segregation of Sa andSb microspores. Magnification, 450×. Fig. 2. Open in new tabDownload slide In situ localization of SCRa andSCRb transcripts. Paraffin-embedded sections were prepared from SaSa, SbSb, and SaSb −3-stage anthers and hybridized with dioxigenin-labeled RNA probes transcribed in vitro essentially according to protocols athttp://www.Arabidopsis.org/cshl-course. The probes were: αs-a, antisense SCRa; αs-b, antisense SCRb. Negative controls: Sense SCRa (s-a) and sense SCRb (s-b) RNA probes. T, Tapetum; M, microspores. Some microspores inSaSb anthers did not hybridize with the αs-b probe, in keeping with the expected segregation of Sa andSb microspores. Magnification, 450×. We examined the expression of the SRK and SCRalleles in SaSb heterozygotes. We found no difference in the level of SRKa transcripts beyond that expected from reduced gene dosage in heterozygous stigmas relative to homozygous stigmas (Fig. 1B). Thus, the weakening of Sa activity in heterozygous stigmas, like dominant/recessive relationships in theBrassica stigma (Hatakeyama et al., 2001), is not related to differences in SRK expression levels. It may be based on interference between receptor or ligand isoforms either in the SRK-SCR interaction or in the recruitment of downstream effectors of the SI response. In contrast, the SCR alleles were differentially regulated in heterozygotes. SCRb transcripts were detected inSaSb anthers (Fig. 1C) and were localized to tapetum and microspores as in Sb homozygotes (Fig. 2). However,SCRa transcripts were drastically reduced in heterozygotes relative to Sa homozygotes (Figs. 1C and 2), with average reductions of approximately 80-fold and 30-fold estimated from long exposures of RNA gel blots and quantitative reverse transcriptase-PCR, respectively. In contrast, SCRb transcripts were reduced by only approximately 10% in heterozygotes relative to Sbhomozygotes. Importantly, comparison of eight SaSa and eightSaSb plants generated by forced selfing of anSaSb plant in which SCRa was “silent” showed that SCRa was expressed in SaSa progeny and “silenced” in their SaSb sibs. Thus, the low-expression state of SCRa is not heritable and is probably not due to an unlinked modifier gene influencing SCRa transcription or the stability of its transcripts. Why is expression of the SCRa allele suppressed inSaSb heterozygotes? This effect might be due to direct interference from the SCRb allele or from other sequences within the Sb haplotype, or it might result fromSCRa-specific properties. Several eukaryotic genes exhibit monoallelic expression, with selection of the expressed allele occurring either stochastically, according to parental origin (genomic imprinting), or based on allele-inherent characteristics (Rothenburg et al., 2001). Furthermore, severe down-regulation or silencing has been documented for a number of eukaryotic genes in the form of transgene effects (Kooter et al., 1999) and other trans-sensing phenomena, such as paramutation in maize (Zea mays) and transvection in Drosophila melanogaster (Tartof and Henikoff, 1991). Many of these examples are associated with increased DNA methylation (Martienssen and Colot, 2001) or with RNA degradation effected by aberrant small (21–25 nt) interfering RNAs (Hamilton and Baulcombe, 1999; Mallory et al., 2001; Matzke et al., 2001a). We compared the DNA of leaves and anthers of SaSa andSaSb plants by methylation-sensitive restriction enzyme digestion and by genomic bisulfite sequencing (Clark et al., 1994). We detected no consistent differences between SaSa andSaSb plants in the methylation state of SCRawithin the two exons and one intron of the gene and within approximately 500 bp of sequence 5′ of the initiating codon. We also failed to detect SCRa degradation products or smallSCRa-related RNA species in small RNA-enriched fractions isolated from SaSb anthers at two stages of development. Nevertheless, neither phenomenon can be categorically ruled out, because modifications restricted to tapetal cells, which constitute only a small proportion of anther cells, might not be detected by current methods. An alternative explanation for the differential expression ofSCRa and SCRb in homozygotes and heterozygotes is suggested by the approximately 65% sequence divergence ofSCRa and SCRb and by the extensive structural heteromorphism that distinguishes the Sa and Sbhaplotypes (Kusaba et al., 2001), two features that are likely to interfere with chromosome pairing. It is possible that expression of the SCRa allele, but not that of the SCRb allele, is dependent on homolog pairing. Such dependence has been described for some eukaryotic genes (Aramayo and Metzenberg, 1996; Goldsborough and Kornberg, 1996; Matzke et al., 2001b), with expression being affected even by transient pairing of homologous chromosomes in some cases (LaSalle and Lalande, 1996). Interestingly, chromosome pairing has been described in tapetal cells (Aragon-Alcaide et al., 1997). The possibility that SCRa is expressed only in Sahomozygotes (S-locus homozygotes can occur naturally in the case of recessive alleles) or in heterozygous combinations that allowS haplotype pairing is at least consistent with the absence of SCRa transcripts in haploid microspores. However, further analysis of SCRa expression in the presence of differentS haplotypes is required to test this hypothesis. Irrespective of the underlying mechanism(s) for gene silencing, the recessive/dominant interaction exhibited by the SCRa andSCRb alleles in pollen is explained by the severe down-regulation of the recessive SCRa allele in the tapetum of SaSb heterozygotes, which, together with the lack ofSCRa expression in microspores, results effectively in monoallelic expression of the dominant SCRb allele. The observed reduction in SCR concentration of approximately 30-fold or more results in loss of the corresponding SI specificity in pollen because too few SCR molecules are delivered to the stigma surface by any individual pollen grain for SRK activation to occur. We propose that this unusual feature of allelic differences in the temporal and spatial pattern of SCR gene expression, as well as allele-specific differences in susceptibility to silencing, may explain many, if not all, cases of dominant/recessive interactions and mutual weakening of S haplotypes in the pollen of crucifers. ACKNOWLEDGMENTS We thank V. Vance and A. Mallory for advice on small RNA isolation, and M. Wofner, T. Fox, and U. Grossniklaus for helpful comments. 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GM5752) and the National Science Foundation (grant no. IBN–0077289). 2 Present address: Institute of Radiation Breeding, National Institute of Agrobiological Science, Ohmiya-machi, Naka-gun, Ibaraki 319-2293, Japan. * Corresponding author; e-mail [email protected]; fax 607–255–5407. www.plantphysiol.org/cgi/doi/10.1104/pp.010790. Copyright © 2002 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)

Koh, Serry; Wiles, Amy M.; Sharp, Joshua S.; Naider, Fred R.; Becker, Jeffrey M.; Stacey, Gary

doi: 10.1104/pp.010332pmid: N/A

Abstract We have identified nine oligopeptide transporter (OPT) orthologs (AtOPT1 to AtOPT9) in Arabidopsis. These proteins show significant sequence similarity to OPTs of Candida albicans (CaOpt1p),Schizosaccharomyces pombe (Isp4p), andSaccharomyces cerevisiae (Opt1p and Opt2p). Hydrophilicity plots of the OPTs suggest that they are integral membrane proteins with 12 to 14 transmembrane domains. Sequence comparisons showed that the AtOPTs form a distinct subfamily when compared with the fungal OPTs. Two highly conserved motifs (NPG and KIPPR) were found among all OPT members. The identification of multiple OPTs in Arabidopsis suggests that they may play different functional roles. This idea is supported by the fact that AtOPTs have a distinct, tissue-specific expression pattern. The cDNAs encoding seven of the AtOPTs were cloned into a yeast vector under the control of a constitutive promoter. AtOPT4 expressed in S. cerevisiae mediated the uptake of KLG-[3H]L. Similarly, expression of five of the seven AtOPT proteins expressed in yeast conferred the ability to uptake tetra- and pentapeptides as measured by growth. This study provides new evidence for multiple peptide transporter systems in Arabidopsis, suggesting an important physiological role for small peptides in plants. Peptide transport involves the translocation of peptides (2–6 residues in length) across the cellular membrane in an energy-dependent manner and has been well documented in bacteria, fungi, and mammals (Payne and Smith, 1994; Becker and Naider, 1995; Meredith and Boyd, 2000). After uptake, the internalized peptides are rapidly hydrolyzed by peptidases and used as a source of amino acids, nitrogen, or carbon. Compared with prokaryotes and animals, peptide transport in higher plants has received little attention. There are few published reports dealing with small peptides in plants.Higgins and Payne (1982) reported that significant levels of peptides were found in phloem and xylem exudates. These included the non-protein-derived peptides (e.g. alanylaminobutyric acid and glycylketoglutaric acid). The xylem contains lower levels of nitrogen than the phloem, yet peptides were reported in the xylem exudates of several species, such as the vegetative organs and berries of grape (Khachidze, 1975) and the sap of corn (Fejer and Konay, 1958). Higgins and Payne (1982) suggested that the transport of peptides is a more efficient means of nitrogen distribution than the transport of individual amino acids. This would be especially true for long-distance transport during the bulk movement of protein-degradation products (e.g. leaf senescence, seed germination). As nitrogen carriers, peptides may also protect amino acids from catabolism by enzymes known to be present in the phloem during transport within the plant (Higgins and Payne, 1980). Glutathione (GSH), a modified tripeptide (γ-Glu-Cys-Gly), has also been suggested as a carrier of reduced sulfur in the phloem and xylem (Higgins and Payne, 1982). γ-Glutamyl peptides frequently occur in considerable quantities, especially in seeds and storage organs (Higgins and Payne, 1982). For example, 34% of the non-protein amino nitrogen of a kidney bean seed is present as γ-glutamyl-S-methyl-l-Cys (Goore and Thompson, 1967). Upon seed germination, this peptide is degraded, suggesting that it plays an important role in the storage of nitrogen and/or sulfur. Plant growth factors, such as auxin and gibberellin, are frequently bound to small peptides and the peptide-hormone conjugates are present in many tissues, such as the vascular system and the endosperm of plant seeds (Salisbury and Ross, 1991). These conjugates may be involved in regulating hormone activity in response to plant growth and development, as well as facilitating hormone transport (Salisbury et al., 1991). In addition to endogenous plant peptides, several phytotoxins produced by plant pathogens are modified peptides (Walton, 1990). Gross (1991) suggested that plant peptide transport systems could be responsible for the recognition and transport of peptide phytotoxins into plant cells. Peptide transporters can be grouped into three distinct families based on sequence similarity and mechanism: 1) the ATP-binding cassette (ABC) superfamily (Higgins, 1992); 2) the peptide transporter (PTR) or the proton-dependent oligopeptide transporter (POT) family (Paulsen and Skurray, 1994; Steiner et al., 1995); and 3) the recently identified oligopeptide transporter (OPT) family (Lubkowitz et al., 1997; Hauser et al., 2001). Plant members of the ABC family of peptide transporters include the recently identified Arabidopsis GSH S-conjugate transporters, AtMRP1 to AtMRP4 (Lu et al., 1997,1998; Sánchez-Fernández et al., 1998). The first plant PTR-like peptide transporter, Arabidopsis AtPTR2, was identified by complementation of a yeast mutant defective in di/tripeptide transport (Steiner et al., 1994). Transgenic plants expressing antisense AtPTR2 exhibited a delayed flowering phenotype and an arrest in seed development, especially seed maturation (Song et al., 1997). In barley (Hordeumvulgare), peptide transfer occurs across the scutellum layer, a specialized absorptive tissue abutting the endosperm. The peptide transporter in barley seeds shares a number of similarities to members of the PTR family, including transport of di- and tripeptides (Sopanen et al., 1977; Higgins and Payne, 1978). A cDNA clone encoding a barley peptide transporter (HvPTR1) was recently isolated (West et al., 1998) and this protein was localized on the plasma membrane of the scutellar epithelium (Waterworth et al., 2000). Recent database comparisons revealed eight additional PTR orthologs in Arabidopsis, suggesting an important role for this family in plant growth and development (data not shown). Members of the OPT family of peptide transporters have 12 to 14 predicted transmembrane domains and show no sequence similarity to ABC or PTR transporters. Until now, members of the OPT family were only characterized from yeast (i.e. Candida albicans, CaOpt1p [Lubkowitz et al., 1997]; Schizosaccharomyces pombe, Isp4p [Lubkowitz et al., 1998]; Saccharomyces cerevisiae, Opt1p and Opt2p; [Hauser et al., 2000]). These proteins were shown to mediate the uptake of tetra- and pentapeptides (e.g. KLGL, KLLG, or KLLLG). S. cerevisiae Opt1p was shown to transport Met-enkephalin (YGGFM) and Leu-enkephalin (YGGFL) with aK m of 310 μm for YGGFL (Hauser et al., 2000). In contrast, Opt1p did not transport various amino acids or di-/tripeptides tested (e.g. Tyr, L-L, and G-G-F; Hauser et al., 2000). Interestingly, Bourbouloux et al. (2000)reported that Opt1p was a high affinity GSH transporter, whereas Opt2p was not. The discovery of the OPT family of transporters in yeast (Lubkowitz et al., 1997) led to a search of the sequence database for possible orthologs in Arabidopsis. We report here the identification of nine putative OPT family members that exhibit 49% to 53% sequence similarity to the yeast OPTs. Expression of seven of these proteins in yeast confirmed the ability of some to mediate the uptake of tetra- and pentapeptides. RESULTS Arabidopsis OPT Orthologs Can Be Identified by Sequence The GenBank database was searched using the TBLASTN 2.1.1 algorithm (Altschul et al., 1990, 1997) with the complete sequence of the C. albicans OPT, CaOpt1p (Lubkowitz et al., 1997). This analysis led to the identification of nine possible Arabidopsis OPT orthologs (Fig. 1) that exhibited 49% to 53% sequence similarity to CaOpt1p. The Arabidopsis OPTs formed a distinct subgroup when compared with the yeast OPT members and showed 61% to 85% sequence similarity when compared with each other (Fig.1A). AtOPTs were positioned on the Arabidopsis genetic map based on the position of their corresponding BAC or P1 clones used for genome sequencing. AtOPT2 is located on chromosome 1,AtOPT3, 5, 6, and 7 are located on chromosome 4, whereas AtOPT1, 4, 8, and 9are located on chromosome 5 (Fig. 1A). Fig. 1. Open in new tabDownload slide Comparison of OPTs. A, Dendogram showing a sequence comparison of the known members of the OPT family. Analysis was performed using the CLUSTAL method in MegAlign (DNASTAR, Madison, WI) using default parameters. Accession numbers are as follows: AtOPT1, AB026659 GI:9758213; AtOPT2, AAB60748 GI:2160185; AtOPT3, Z97341 GI:2244994; AtOPT4, AB008268 GI:9759417; AtOPT5,AL078465 GI:4938497; AtOPT6, AL035602.1 GI:4469024; AtOPT7, AF080119GI:3600039; AtOPT8, BAB09728.1 GI:9759191; AtOPT9, AB015476 GI:9759190; Opt1p, Z49487; CaOpt1p, U60973; Isp4p, P40900; Opt2p, U25841. The mapped positions of each AtOPT are indicated. B, Hydrophilicity plots of AtOPT1–9 and CaOpt1p as predicted by Kyte and Doolittle (1982). The size (amino acids) of the each protein is shown below the name of the gene. Analysis was performed using Protean (DNASTAR) under default parameters. The bars over each sequence show the location of the two conserved motifs (NPG and KIPPR motifs) (i.e. NPG[P/A]F[N/T/S]XKEH[V/T/A][L/I/V][I/V]I[T/S/V][I/V/M] [F/M][A/S][N/S/A] and K[L/F][G/A][H/M/T]YMK[I/V/L][P/D/S]PR). Fig. 1. Open in new tabDownload slide Comparison of OPTs. A, Dendogram showing a sequence comparison of the known members of the OPT family. Analysis was performed using the CLUSTAL method in MegAlign (DNASTAR, Madison, WI) using default parameters. Accession numbers are as follows: AtOPT1, AB026659 GI:9758213; AtOPT2, AAB60748 GI:2160185; AtOPT3, Z97341 GI:2244994; AtOPT4, AB008268 GI:9759417; AtOPT5,AL078465 GI:4938497; AtOPT6, AL035602.1 GI:4469024; AtOPT7, AF080119GI:3600039; AtOPT8, BAB09728.1 GI:9759191; AtOPT9, AB015476 GI:9759190; Opt1p, Z49487; CaOpt1p, U60973; Isp4p, P40900; Opt2p, U25841. The mapped positions of each AtOPT are indicated. B, Hydrophilicity plots of AtOPT1–9 and CaOpt1p as predicted by Kyte and Doolittle (1982). The size (amino acids) of the each protein is shown below the name of the gene. Analysis was performed using Protean (DNASTAR) under default parameters. The bars over each sequence show the location of the two conserved motifs (NPG and KIPPR motifs) (i.e. NPG[P/A]F[N/T/S]XKEH[V/T/A][L/I/V][I/V]I[T/S/V][I/V/M] [F/M][A/S][N/S/A] and K[L/F][G/A][H/M/T]YMK[I/V/L][P/D/S]PR). The hydrophilicity plots of the various AtOPTs were quite similar to that of CaOpt1p (Fig. 1B). The size of proteins predicted for each of the AtOPT varied from 696 amino acids (i.e. AtOPT3) to 766 amino acids (i.e. AtOPT7; Fig. 1B). With the exception of AtOPT3, which has shorter N and C terminus regions, all AtOPTs were similar in size to CaOpt1p (783 amino acids) and Opt1p (799 amino acids). Interestingly, sequence comparisons revealed two domains that were strongly conserved among all of the nine OPT family members. Both of these motifs (i.e. NPG motif, NPG[P/A]F[N/T/S]XKEH[V/T/A][L/I/V][I/V]I[T/S/V] [I/V/M][F/M][A/S][N/S/A] and KIPPR motif, K[L/F][G/A][H/M/T]YMK[I/V/L][P/D/S] PR; Fig.2A) were found in regions of the protein predicted to be hydrophilic (Fig. 1B). As reported previously byLubkowitz et al. (1998), none of the OPT family members showed any significant sequence similarity to the known ABC or PTR peptide transporters (data not shown). The putative transmembrane regions of the various AtOPTs were predicted either by hydrophilicity plots or the PRED-TMR algorithm (version 1.0,http://o2.db.uoa.gr/PRED-TMR), and the predicted transmembrane domains of AtOPT1 (as example) are shown in Figure 2B. Fig. 2. Open in new tabDownload slide Analysis of the OPT sequences. A, Two conserved motifs (NPG and KIPPR motifs) among the OPT members, including fungal OPTs, were determined based on the consensus of their sequences after analysis using the CLUSTAL method in the MegAlign (DNASTAR). Shaded area represents the consensus. B, The putative transmembrane domains of the AtOPTs were determined by the PRED-TMR algorithm. Predicted transmembrane regions of AtOPT1 are shown in bold and the underlined sequences represents the two conserved motifs found in all OPTs. Fig. 2. Open in new tabDownload slide Analysis of the OPT sequences. A, Two conserved motifs (NPG and KIPPR motifs) among the OPT members, including fungal OPTs, were determined based on the consensus of their sequences after analysis using the CLUSTAL method in the MegAlign (DNASTAR). Shaded area represents the consensus. B, The putative transmembrane domains of the AtOPTs were determined by the PRED-TMR algorithm. Predicted transmembrane regions of AtOPT1 are shown in bold and the underlined sequences represents the two conserved motifs found in all OPTs. Tissue-Specific Expression of AtOPTs Full-length cDNAs for the Arabidopsis OPT genes AtOPT1to AtOPT7 were amplified by reverse transcriptase (RT)-PCR using gene-specific primers deduced from the DNA sequence in the database (see “Materials and Methods”). These cDNAs were completely sequenced to confirm that full-length cDNAs were obtained. The sequences of AtOPT8 and ATOPT9 only recently appeared in the database and, therefore, were not included in this study. The tissue-specific expression pattern of each of theAtOPTs was determined by quantitative RT-PCR using gene-specific primers. This was deemed necessary because of the likelihood of cross-hybridization among the different family members. As an internal control, Actin2 mRNA was also amplified. The data shown in Figure 3 indicate that those OPTs that showed the greatest sequence similarity (Fig. 1A) also exhibited similar patterns of expression. For example, the levels ofAtOPT2 and AtOPT4 mRNA were evenly expressed in all tissues. In contrast, the levels of AtOPT6 andAtOPT7 mRNA were highest in flower and root tissues but showed relatively low expression in leaf and stem. AtOPT1was highly expressed in flower, and moderately expressed in leaf and stem. AtOPT5 showed the highest sequence similarity to AtOPT1 but its expression pattern was much more specific, being expressed predominantly in flowers. However, counts of 32P activity of AtOPT5in the blot shown in Figure 3 confirmed very low expression ofAtOPT5 in leaf and root (data not shown). AtOPT3showed a unique pattern of expression being strongest in flower, leaf, and root. These data suggest that proteins with similar sequence, as indicated in the CLUSTAL analysis (Fig. 1A), may have similar physiological function, at least with regard to tissue specificity. The various expression patterns clearly suggest that theAtOPTs are likely playing a variety of physiological roles. Fig. 3. Open in new tabDownload slide Tissue-specific mRNA expression ofAtOPT1–7. RT-PCR analysis was performed as described in “Materials and Methods” using gene-specific primers. The level ofActin2 mRNA (right panel) was measured as an internal control. F, Flower; L, leaf; S, stem; and R, root. Fig. 3. Open in new tabDownload slide Tissue-specific mRNA expression ofAtOPT1–7. RT-PCR analysis was performed as described in “Materials and Methods” using gene-specific primers. The level ofActin2 mRNA (right panel) was measured as an internal control. F, Flower; L, leaf; S, stem; and R, root. The AtOPTs Are Functional Peptide Transporters To test the biochemical function of the various AtOPT proteins, their cDNAs were cloned behind the constitutive ADH (alcohol dehydrogenase) promoter in the pDB20 vector (Becker et al., 1991) and transformed into S. cerevisiae strain BY4730. Transport activity was measured by the ability of various Leu-containing peptides to support prototrophic growth of this strain, which is a Leu auxotroph. In this assay system, S. cerevisiae strain BY4730 will only grow if the cells can transport and utilize the peptides provided as the sole source of Leu. Oligopeptides are not hydrolyzed extracellularly and remain intact until transported into the cell cytoplasm (Perry et al., 1994; Steiner et al., 1994; Song et al., 1996;Lubkowitz et al., 1997, 1998; Hauser et al., 2000). After transformation with each of the AtOPT plasmids, theS. cerevisiae BY4730 transformants SK101 to SK107 were tested for their ability to grow in the presence of the tetra-, and pentapeptides KLLG, KLGL, KLLLG, or YGGFL. As shown in Figure4A and TableI, AtOPT1, 4, 5, 6, and 7 were able to support prototrophic growth in the presence of 200 μm KLLLG. In addition, AtOPT4 also mediated uptake of KLGL (at 200 μm) and KLLG (at 100 μm; Fig. 4B). The growth obtained was comparable with that shown by the positive control (i.e. BY4730 expressing CaOpt1p). These data show that AtOPTs (excluding AtOPT2 and AtOPT3) are functional tetra- and pentapeptide transporters. Fig. 4. Open in new tabDownload slide Peptide growth assays. S. cerevisiaeBY4730 strain transformed with pDB20 (vector alone), or expressing CaOpt1p, Opt1p, or AtOPT1–7 were tested for their ability to use Leu-containing peptides (i.e. KLLG, KLGL, KLLLG) to fulfill the auxotrophic requirement for Leu. A, 200 μmKLLLG. The cell number added to each well is shown. B, 200 μm KLGL (left) and 100 μm KLLG (right). Fig. 4. Open in new tabDownload slide Peptide growth assays. S. cerevisiaeBY4730 strain transformed with pDB20 (vector alone), or expressing CaOpt1p, Opt1p, or AtOPT1–7 were tested for their ability to use Leu-containing peptides (i.e. KLLG, KLGL, KLLLG) to fulfill the auxotrophic requirement for Leu. A, 200 μmKLLLG. The cell number added to each well is shown. B, 200 μm KLGL (left) and 100 μm KLLG (right). Table I. Peptide growth assay Yeast Transformants1-a . Peptides . −Leu . +Leu . KLLG . KLGL . KLLLG . YGGFL . pDB20 −1-b + 1-c − − − − Opt1p − + − − + + SK101 (AtOPT1) − + − − + − SK102 (AtOPT2) − + − − − − SK103 (AtOPT3) − + − − − − SK104 (AtOPT4) − + + + + − SK105 (AtOPT5) − + − − + − SK106 (AtOPT6) − + − − + − SK107 (AtOPT7) − + − − + − Yeast Transformants1-a . Peptides . −Leu . +Leu . KLLG . KLGL . KLLLG . YGGFL . pDB20 −1-b + 1-c − − − − Opt1p − + − − + + SK101 (AtOPT1) − + − − + − SK102 (AtOPT2) − + − − − − SK103 (AtOPT3) − + − − − − SK104 (AtOPT4) − + + + + − SK105 (AtOPT5) − + − − + − SK106 (AtOPT6) − + − − + − SK107 (AtOPT7) − + − − + − F1-a  S. cerevisiae BY4730 (Met−, Leu−, Ura−). F1-b  −, No growth. F1-c  +, Growth. Open in new tab Table I. Peptide growth assay Yeast Transformants1-a . Peptides . −Leu . +Leu . KLLG . KLGL . KLLLG . YGGFL . pDB20 −1-b + 1-c − − − − Opt1p − + − − + + SK101 (AtOPT1) − + − − + − SK102 (AtOPT2) − + − − − − SK103 (AtOPT3) − + − − − − SK104 (AtOPT4) − + + + + − SK105 (AtOPT5) − + − − + − SK106 (AtOPT6) − + − − + − SK107 (AtOPT7) − + − − + − Yeast Transformants1-a . Peptides . −Leu . +Leu . KLLG . KLGL . KLLLG . YGGFL . pDB20 −1-b + 1-c − − − − Opt1p − + − − + + SK101 (AtOPT1) − + − − + − SK102 (AtOPT2) − + − − − − SK103 (AtOPT3) − + − − − − SK104 (AtOPT4) − + + + + − SK105 (AtOPT5) − + − − + − SK106 (AtOPT6) − + − − + − SK107 (AtOPT7) − + − − + − F1-a  S. cerevisiae BY4730 (Met−, Leu−, Ura−). F1-b  −, No growth. F1-c  +, Growth. Open in new tab Previously, Opt1p from S. cerevisiae was reported to transport Leu-enkephalin (YGGFL), Met-enkephalin (YGGFM; Hauser et al., 2000), and GSH (Bourbouloux et al., 2000). As shown in Table I, our experiments showed that BY4730 expressing Opt1p grew well in the presence of YGGFL. However, no growth was observed with YGGFL when cells were expressing any of the various AtOPTs (Table I). Uptake studies using [3H]GSH failed to reveal any uptake when yeast cells were expressing any of the AtOPTs, whereas, under similar conditions, Opt1p supported uptake of GSH (data not shown). Other studies using various di-and tripeptides (e.g.L-L, G-G-F) as sources of Leu showed no growth of yeast expressing the various AtOPTs (data not shown). AtOPT4 Mediates the Uptake of KLG-[3H]L Few of the possible 160,000 (204) tetra- and 3,200,000 (205) pentapeptides containing the 20 naturally occurring amino acids are commercially available. Therefore, based on the results in Table I, we synthesized KLG-[3H]L and tested the ability of AtOPT4 to uptake this tetrapeptide when expressed in yeast. As shown in Figure5, yeast cells expressing AtOPT4 accumulated KLG-[3H]L to a level significantly above the controls. However, this level of uptake was much less than that mediated by CaOpt1p from C. albicans. Transport of substrate at 0°C by both CaOpt1p and AtOPT4 was attenuated and similar to that of the negative control (empty vector, pDB20). These data indicate that AtOPT4 does mediate the uptake of KLG-[3H]L to a level significantly above background and confirm the results from the growth assays (Fig. 4; Table I). However, this peptide appears to be a poor substrate. Given the large number of possible tetrapeptides and the few available to test, it is would have been surprising to find that KLGL is an ideal substrate. Fig. 5. Open in new tabDownload slide Uptake of KLG-[3H]L byS. cerevisiae BY4730 transformants. Uptake of KLG-[3H]L after 30 min at 30°C or 0°C for cells transformed with pSK104 (AtOPT4), pCaOPT1 (CaOpt1p from C. albicans), and 30°C for cells containing the empty vector pDB20. Uptake is expressed in nanomoles per milligram dry weight on the lefty axis, and fraction of uptake as compared with the positive control of CaOpt1p at 30°C on the right y axis. Data shown represent the average of three replicates ±sd. Fig. 5. Open in new tabDownload slide Uptake of KLG-[3H]L byS. cerevisiae BY4730 transformants. Uptake of KLG-[3H]L after 30 min at 30°C or 0°C for cells transformed with pSK104 (AtOPT4), pCaOPT1 (CaOpt1p from C. albicans), and 30°C for cells containing the empty vector pDB20. Uptake is expressed in nanomoles per milligram dry weight on the lefty axis, and fraction of uptake as compared with the positive control of CaOpt1p at 30°C on the right y axis. Data shown represent the average of three replicates ±sd. DISCUSSION Searching of the available Arabidopsis sequences using the CaOpt1p sequence identified nine possible OPT orthologs (AtOPT1–AtOPT9). Both the ability of AtOPT4 to take up KLG-[3H]L and the ability of AtOPT1, 4, 5, 6, and 7 to promote growth of a yeast strain defective in peptide transport demonstrates that these are bonafide OPT transporters. Sequence comparisons suggest that the Arabidopsis proteins comprise a distinct subfamily of OPT peptide transporters. All OPT family members (plant and fungus) appear to be integral membrane proteins with 12 to 14 predicted transmembrane domains. The availability of these AtOPT sequences allowed us to identify two highly conserved sequence motifs found in all OPT members. These motifs are found in regions predicted to be hydrophilic suggesting that they are probably critical to function. As is the case for the yeast OPTs, no significant sequence similarity was found between the AtOPTs and members of the ABC or PTR families of peptide transporters. Analysis of the expression of the various AtOPTs suggests that these proteins likely play distinct roles in the plant. Those proteins showing the highest sequence similarity also appeared to have comparable tissue-specific expression patterns, suggesting that they have related function. Unfortunately, very few peptide substrates applicable to the yeast strain utilized are commercially available. Therefore, we synthesized the majority of the substrates used in this study, none of which appeared to be excellent substrates for transport. The nature of the physiological substrates for the various AtOPTs remains an important, unanswered question. Of all of the substrates tested (including di-and tripeptides), the AtOPTs were only able to mediate the uptake of selected tetra-and pentapeptides. Therefore, the data would suggest that their physiological substrates are likely small peptides, larger than a tripeptide. The presence of several OPT transporters in Arabidopsis suggests that these proteins are important and, therefore, small peptides may play an important physiological role in plants. Although the function of these transporters may be strictly nutritional, it is interesting to speculate that they could also mediate the transport of important regulatory molecules (e.g. hormone-peptide conjugates). MATERIALS AND METHODS Sequence Comparisons The Arabidopsis orthologs were identified by comparison of the CaOpt1p sequence (Lubkowitz et al., 1997) to the data in the GenBank database using the TBLASTN 2.1.1 algorithm (Altschul et al., 1990,1997). These orthologs were named AtOPT1 toAtOPT9. The map positions of these Arabidopsis orthologs were determined based on the genomic sequence information of the BAC clones or P1 clones that harbor the Arabidopsis orthologs (Fig.1A). All of the OPT sequences were compared using MegAlign (DNASTAR). This led to the identification of two conserved motifs found in all OPT proteins. A dendogram comparing the various OPT sequences was generated using the CLUSTAL method in MegAlign under the default parameters. Hydrophilicity plots for AtOPT1 through -7 were generated based on theKyte and Doolittle (1982) method using Protean sequence analysis software (DNASTAR) under default parameters. The putative transmembrane domains were predicted by the PRED-TMR algorithm (version 1.0; Pasquier et al., 1999). Analysis of Tissue-Specific Expression Total RNA was isolated from 2- to 5-week-old whole Arabidopsis (Landsberg erecta) plants grown under 16-h light/8-h dark at 21°C. RNA was isolated using the Trizol reagent (Invitrogen, San Diego) as described by the manufacturer. Gene-specific primers from the 5′-untranslated region of each AtOPT were designed by analyzing the DNA sequence of each gene using Primer3 (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). The gene-specific primers used for full length cDNA cloning in this study are as follows: AtOPT1-RC3, 5′-GCCAGGACGGGAAAAAGGAGCTTGAAGAC-3′; AtOPT2-RC2, 5′-TGACGTCGTCTCCTTCACCAAATTCCA-3′; AtOPT3-RC2, 5′-GGCCAACAACGCAACTTTGTCTGGTACTTCA-3′; AtOPT4-RC1, 5′-TCACACACAAACTAAACCGGAATGG-3′; AtOPT5-RC2, 5′-AATGTCAGGTCATTAACACAGGTTGCT-3′; AtOPT6-RC1, 5′-GGCCCCAAAGAACGGAACACTCACTCT-3′; AtOPT7-RC1, 5′-GGCCGTCAGATTCACATCTCCCCAAAA-3′. First strand cDNA synthesis was performed using M-MLV reverse transcriptase (Promega, Madison, WI) using the AP adapter primer (Invitrogen), according to the manufacturer's protocol. The products of this reaction were then used in PCR reactions containing the gene-specific primers and the Abridged Universal Amplification Primer (Invitrogen) primer under the following conditions: 96°C for 5 min (1 cycle), 94°C for 15 s, 65°C for 30 s, 72°C for 2 min (36 cycles), and 72°C for 4 min (1 cycle). The PCR products obtained were cloned into the pCR2.1 TOPO cloning vector following the instructions provided in the TOPO TA cloning kit (Invitrogen). The complete DNA sequence (both strands) was determined for each of the cDNA clones and checked to confirm the presence of both the translational start and stop codons. Because of the sequence similarity among the variousAtOPTs, RT-PCR, using gene-specific primers was used to measure mRNA levels in various tissues. Total RNA from 3- to 5-week-old plant flower, leaf, bolting stem, and root was isolated using the Trizol reagent. The PCR conditions were identical to those described above. However, as an internal control, a 629-bp cDNA fragment ofActin2 was also amplified using specific primers (Actin2-for, 5′-GTTGGTGATGAAGCACAATCCAAG-3′, and Actin2-rev, 5′-CTGGAACAAGACTTCTGGGCATCT-3′). Amplification of theActin2 and AtOPT cDNA was done in the same tube. After PCR, the products were electrophoresed in agarose gels and then blotted. AtOPT expression was visualized by Southern hybridization using a 32P-labeled probe made from the cDNA clone of the respective AtOPT gene. Likewise, the level of Actin2 expression was visualized by hybridizing to a labeled actin gene probe. The CPM/mm2values for each hybridization were obtained with an instant imager (Packard Instrument Co., Meriden, CO) and calculated using the following equation: relative gene expression = cpm/mm2of AtOPTs/the cpm/mm2 ofActin2. Synthesis of Peptides KLGL, KLLG, and KLLLG were prepared by conventional automated solid phase peptide synthesis on an synthesizer (model 433A, Applied Biosystems, Foster City, CA). Peptides were cleaved from the resin with trifluoroacetic acid and purified using a C18 reversed phase column (19 × 300 mm) to >99% homogeneity with a 5% to 20% linear gradient of acetonitrile in water over 60 min. TheM r and amino acid composition were verified using mass spectrometry. Growth Assays Full-length cDNAs of AtOPT1 toAtOPT7 were cloned into the pDB20 vector, under the control of the constitutive ADH promoter (Becker et al., 1991) using either the in vivo ligation method (Gietz et al., 1991) or in vitro cloning method. AtOPT1 and AtOPT2 full-length cDNAs including the translation start and stop codons were amplified with the respective forward and reverse gene-specific primers including the intact NotI sites, and cotransformed with theBstXI digested pDB20 (URA3) vector intoSaccharomyces cerevisiae BY4730 (MATα leu2Δ0 met15Δ0 ura3Δ0). Yeast transformants with in vivo ligated plasmids (pSK101 [AtOPT1] and pSK102 [AtOPT2]) were selected on 0.2% (w/v) casamino acid medium lacking uracil. Plasmids were isolated from cells that grew on this medium and re-transformed into Escherichia coli. NotI enzyme digestions of re-isolated plasmids from E. coli were used to confirm the cloning of AtOPT1and AtOPT2 in pSK101 and pSK102, respectively. Plasmids pSK101 and pSK102 were transformed into S. cerevisiaeBY4730 to create strains SK101 and SK102, respectively. Full-length cDNAs of AtOPT3, 4,5, 6, and AtOPT7 were cloned into the pDB20 vector using a normal in vitro cloning method.AtOPT3, AtOPT6, and AtOPT7full-length cDNAs including the translation start and stop codons were amplified using the respective gene-specific primers with theNotI sites. The resulting PCR products were digested with NotI and ligated into the NotI site of the pDB20 vector. AtOPT4 and AtOPT5full-length cDNAs were also PCR amplified using 5′-untranslated region gene-specific primers and the AUAP-NotI primer as 3′ primer and also cloned into the NotI site of the pDB20 vector. The orientation of each gene in the pDB20 vector was determined by digestion with various restriction enzymes. The resulting clones were transformed into S. cerevisiae BY4730 and transformants were selected on 0.2% (w/v) casamino acid medium lacking uracil. The resulting transformants were named SK103 to SK107, corresponding to AtOPT3 to AtOPT7, respectively. Growth assays were performed as described previously (Hauser et al., 2000). SK101 to SK107 were grown overnight in a Pro liquid medium containing yeast nitrogen base without amino acids and ammonium sulfate, 2% (w/v) Glc, 0.1% (w/v) Pro, 228 μmLeu, and 191 μm Met (Hauser et al., 2000). The cells were harvested by centrifugation, washed twice with sterile distilled water, and resuspended in sterile distilled water to 2 × 107cells/mL or 2 × 106 cells/mL. Five microliters of each suspension (1 × 106 cells or 1 × 105 cells, respectively) was applied as a small spot to a solid growth medium supplemented with a specific tetra- and pentapeptides (i.e. KLLG, KLGL, KLLLG, or YFGGL) instead of Leu and incubated at 30°C for 110 h. Growth was scored every 24 h as uniform colony formation compared with both negative (pDB20 vector only) and positive controls (CaOpt1p and Opt1p). The medium for the growth assay used Pro (0.1%) as a nitrogen source and was supplemented with either 100 or 200 μm of a specifically synthesized tetra-or pentapeptide (KLLG, KLGL, YFGGL, or KLLLG) as indicated. 191 μm Met was included in the medium to fulfill the auxotrophic requirement of the strain BY4730 for this amino acid. Leu-enkephalin (YGGFL) was purchased from Sigma (St Louis). Met-enkephalin (YGGFM) from Sigma was also purchased but found to contain a high level of Met amino acid contamination that prevented its use. Radiolabeled Peptide Uptake Assays Radioactive uptake assays with KLGL or GSH were initiated by combining equal volumes of prewarmed cells in 2% (w/v) Glc (30°C) and 2× uptake assay medium (2% [w/v] Glc, 40 mm sodium citrate/potassium buffer, pH 5.5, and 500 μm KLGL or 500 μm GSH [Sigma; New England Nuclear, Boston] at 0.5 μCi/mL of [3H]labeled substrate). Cells were incubated either with medium at 30°C or 0°C for 30 min with KLGL or 12 min with GSH. Medium containing GSH was constantly kept under nitrogen until it was mixed with cells, to prevent its oxidation. At the end of the incubation period, aliquots (90 μL) were removed, and placed on a membrane filter (HAWP, Millipore, Bedford, MA). The filter was immediately washed four times by vacuum filtration with 1 mL of ice water. Filters were counted by liquid scintillation spectrometry, and results were reported as nanomoles per milligram dry weight. KLGL uptake assays were done in triplicate, whereas GSH uptake was measured in quadruplicate. ACKNOWLEDGMENTS We acknowledge the contribution of Dr. Chengdong Zhang for cloning of the AtOPT1 gene. LITERATURE CITED 1 Altschul SF Gish W Miller W Myers EW Lipman DJ Basic local alignment search tool. J Mol Biol 215 1990 403 410 Google Scholar Crossref Search ADS PubMed WorldCat 2 Altschul SF Madden TL Schäffer AA Zhang J Zhang Z Miller W Lipman DJ Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. 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Kleinkauf H Dohren HV 1990 179 203 Walter de Gruyter New York 35 Waterworth WM West CE Bray CM The barley scutellar peptide transporter: biochemical characterization and localization to the plasma membrane. J Exp Bot 51 2000 1201 1209 Google Scholar Crossref Search ADS PubMed WorldCat 36 West CE Waterworth WM Stephens SM Smith CP Bray CM Cloning and functional characterization of a peptide transporter expressed in the scutellum of barley grain during the early stages of germination. Plant J 15 1998 221 230 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the U.S. Department of Agriculture (grant no. 99–35304–8194). 2 Present address: Carnegie Institution of Washington, 260 Panama Street, Stanford, CA 94305–1297. * Corresponding author; e-mail [email protected]; fax 865–974–4007. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.010332. Copyright © 2002 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)

Wu, Keqiang; Tian, Lining; Hollingworth, Jamie; Brown, Daniel C.W.; Miki, Brian

doi: 10.1104/pp.010696pmid: N/A

Abstract Pti4 is a tomato (Lycopersicon esculentum) transcription factor that belongs to the ERF (ethylene-responsive element binding factor) family of proteins. It interacts with the Pto kinase in tomato, which confers resistance to the Pseudomonas syringae pv tomato pathogen that causes bacterial speck disease. To study the function of Pti4, transgenic Arabidopsis plants were generated that expressed tomatoPti4 driven by the strong constitutive promoters, cauliflower mosaic virus 35S and tCUP. Global gene expression analysis by Affimetric GeneChip indicated that expression of Pti4 in transgenic Arabidopsis plants induced the expression of GCC box-containing PR genes. We also demonstrated that Pti4 enhanced GCC box-mediated transcription of a reporter gene. The data suggests that tomato Pti4 could act as a transcriptional activator to regulate expression of GCC box-containing genes. Furthermore, we show that the expression of tomatoPti4 in transgenic Arabidopsis plants produced a phenotype similar to that seen in plants treated with ethylene, thus providing evidence that the Pti4 gene is involved in the regulation of a subset of ethylene-responsive genes containing the GCC box. The ERF (ethylene-responsive element binding factor) proteins (formerly known as EREBPs [ethylene-responsive element binding proteins]) were first isolated as GCC box binding proteins from tobacco (Nicotiana tabacum; Ohme-Takagi and Shinshi, 1995). The GCC box contains a conserved AGCCGCC sequence, which was first indentified from the promoters of ethylene-inducible genes in tobacco (Ohme-Takagi and Shinshi, 1990). ERF proteins contain a highly conserved DNA binding domain (ERF domain) consisting of 58 or 59 amino acids (Ohme-Takagi and Shinshi, 1995; Hao et al., 1998). Although the ERF domain shares some sequence homology with the AP2 domain found in the Arabidopsis protein APETALA2 (Jofuku et al., 1994; Riechmann and Meyerowitz, 1998), the ERF and AP2 domain proteins belong to distinct families (Fujimoto et al., 2000). A number of ERF proteins have been identified from different plant species (Ohme-Takagi and Shinshi, 1995; Buttner and Singh, 1997;Stockinger et al., 1997; Zhou et al., 1997; Liu et al., 1998; Solano et al., 1998; Menke et al., 1999; Fujimoto et al., 2000; Ohta et al., 2000). For example, CBF1, DREBP1A, and DREB2A have been shown to bind to a C-repeat/dehydration-responsive element that is involved in drought and cold stress (Stockinger et al., 1997; Liu et al., 1998). The ERFs, Pti4/5/6, AtERP, Arabidopsis ERF1, AtERFs, and ORCA2 bind to the GCC box in the ethylene-responsive element that is essential for the responsiveness of some promoters to ethylene (Ohme-Takagi and Shinshi, 1995; Buttner and Singh, 1997; Zhou et al., 1997; Solano et al., 1998; Menke et al., 1999; Fujimoto et al., 2000; Gu et al., 2000;Ohta et al., 2000). In addition, a tobacco ERF protein, Tsi1, that could bind both the GCC and the C-repeat/dehydration-responsive element sequences was also identified (Park et al., 2001). Studies on the tomato (Lycopersicon esculentum) resistance (R) gene, Pto, provided evidence that linked the ERF genes to the defense response. Pto is a protein kinase that confers resistance toPseudomonas syringae pv tomato, a bacteria that expresses the avirulence gene avrPto. Pto was shown to directly interact in two-hybrid assays with the tomato ERF proteins, Pti4/5/6 (Zhou et al., 1997). Pti4/5/6 proteins have been shown to bind the GCC box cis-element, which is present in the promoter region of many ethylene-regulated pathogenesis-related (PR) genes (Ohme-Takagi and Shinshi, 1995; Zhou et al., 1997; Gu et al., 2000). It has been proposed that the Pti4/5/6 proteins may activate PR gene expression by binding to the GCC box of the PR gene promoters (Zhou et al., 1997; Gu et al., 2000). Ethylene has been implicated in the regulation of basic-type PR genes during the defense responses of plants attacked by pathogens. Infection by a pathogen and treatment with an elicitor both promote the synthesis of ethylene and ethylene activates the transcription of basic-type PR genes (Ecker, 1995; Yamamoto et al., 1999). The differential expression of Pti4, Pti5, and Pti6 in various tomato tissues implies that they may have distinct roles in plants (Thara et al., 1999; Gu et al., 2000).Pti4 is particularly interesting because its expression is induced by ethylene and salicylic acid, and its product is phosphorylated by the Pto kinase (Gu et al., 2000). We have generated transgenic Arabidopsis plants transformed with binary vectors carrying fusions of the tCUP or cauliflower mosaic virus (CaMV) 35S promoter to the tomato Pti4cDNA. Overexpression of Pti4 in Arabidopsis induces the expression of the GCC box-containing PR genes. EtiolatedPti4 transgenic seedlings show inhibition of hypocotyl elongation, which is a typical characteristic of plants treated with ethylene (Ecker, 1995). In addition, Pti4 transgenic plants also display a dwarf phenotype similar to that of constitutive ethylene-responsive mutants. Our study provides direct evidence that the Pti4 gene product is involved in the regulation of the ethylene-responsive genes containing the GCC box. RESULTS Pti4 Protein Activates GCC Box-Mediated Transcription of a Reporter Gene To test whether the tomato Pti4 protein can interact with the GCC box, Pti4 effector plasmids were constructed in which the Pti4 cDNA was driven by a strong constitutive promoter, CaMV 35S or tCUP (Fig. 1A). The reporter plasmids, GCC::GUS andmGCC::GUS, were constructed using a β-glucuronidase (GUS) reporter gene. Two GCC boxes or mutated GCC boxes (mGCC; Ohme-Takagi and Shinshi, 1995) were fused to a minimal promoter, -62tCUP (Wu et al., 2001) to drive the GUS reporter gene expression. The effector plasmids were cobombarded into tobacco leaves together with a reporter plasmid. As shown in Figure 1B, cotransfection of the reporter plasmid GCC::GUSwith a effector plasmid resulted in a 3- to 4-fold increase in GUS expression, indicating that Pti4 protein can interact with the GCC boxes in the promoter of the reporter construct to activate transcription. Transcription of the reporter gene that had a mutated GCC box was not activated by Pti4 (data not shown). Fig. 1. Open in new tabDownload slide Activation of the GCC box-mediated transcription of GUS reporter gene by Pti4 protein in transient expression assays. A, Schematic diagram of the effector and reporter constructs used in cobombardment experiments. The effector constructs contain thePti4 cDNA fused to the Nos terminator driven by the 35S or tCUP promoter. The reporter construct contains two GCC boxes fused to the -62tCUP minimal promoter-GUS construct. B, Activation of theGCC::GUS fusion gene by Pti4. The reporter plasmidGCC::GUS was cobombarded with each effector plasmid or the control plasmid pUC19. GUS activity was reported as picomoles of 4-methylumbelliferone per milligram of protein per minute. Bars indicate the se of three replicates. Fig. 1. Open in new tabDownload slide Activation of the GCC box-mediated transcription of GUS reporter gene by Pti4 protein in transient expression assays. A, Schematic diagram of the effector and reporter constructs used in cobombardment experiments. The effector constructs contain thePti4 cDNA fused to the Nos terminator driven by the 35S or tCUP promoter. The reporter construct contains two GCC boxes fused to the -62tCUP minimal promoter-GUS construct. B, Activation of theGCC::GUS fusion gene by Pti4. The reporter plasmidGCC::GUS was cobombarded with each effector plasmid or the control plasmid pUC19. GUS activity was reported as picomoles of 4-methylumbelliferone per milligram of protein per minute. Bars indicate the se of three replicates. Ectopic Expression of Tomato Pti4 Induces Resident Basic Chitinase Gene Expression Transgenic Arabidopsis plants were generated that expressedPti4 driven by a strong constitutive promoter, CaMV35S or tCUP (Foster et al., 1999). Southern-blot analysis was performed to determine whether the genomic DNA of the putative transformants contained the transgenic DNA (data not shown). Four of the transgenic lines (tCUP::Pti4-1,tCUP::Pti4-3 and tCUP::Pti4-4, andtCUP::Pti4-5) contained the Pti4transgene driven by tCUP promoter and two transgenic lines (35S::Pti4-3 and 35S::Pti4-6) contained Pti4 transgene driven by CaMV 35Spromoter. The expression of Pti4 RNA in the transgenic lines was determined by northern analysis. The predicted 1-kb transcript was detected in five transgenic lines,tCUP::Pti4-1, tCUP::Pti4-3,tCUP::Pti4-4, 35S::Pti4-3 and35S::Pti4-6, using the Pti4 cDNA probe. It was absent from the wild-type plants as expected (Fig.2). One transgenic line,tCUP::Pti4-5, showed bands that were larger in size than the bands in the other lanes of the transgenic plants. This is most likely due to the downstream termination of transcription. Different levels of Pti4 transcript accumulation were detected in the transgenic lines, with the transgenic linetCUP::Pti4-1 having the lowest level ofPti4 expression. Fig. 2. Open in new tabDownload slide Northern-blot analysis of thePti4 transgenic plants. Total RNA was isolated from wild-type (WT) and transgenic lines (1–6). Lanes 1 to 6 correspond to transgenic lines tCUP::Pti4-1,tCUP::Pti4-3, tCUP::Pti4-4,tCUP::Pti4-5, 35S::Pti4-3, and 35S::Pti4-6, respectively. Five micrograms of total RNA was probed with a Pti4 cDNA, a basic chitinase (BC), and an rDNA probe, respectively. Fig. 2. Open in new tabDownload slide Northern-blot analysis of thePti4 transgenic plants. Total RNA was isolated from wild-type (WT) and transgenic lines (1–6). Lanes 1 to 6 correspond to transgenic lines tCUP::Pti4-1,tCUP::Pti4-3, tCUP::Pti4-4,tCUP::Pti4-5, 35S::Pti4-3, and 35S::Pti4-6, respectively. Five micrograms of total RNA was probed with a Pti4 cDNA, a basic chitinase (BC), and an rDNA probe, respectively. Solano et al. (1998) reported that overexpression of another ERF protein, ERF1, in transgenic Arabidopsis plants induced basic chitinase gene expression. Basic chitinase is an ethylene-responsive gene, which contains the GCC box in its promoter (Samac et al., 1990). We therefore examined whether the expression of tomato Pti4 in Arabidopsis could induce the expression of the Arabidopsis basic chitinase gene. As shown in the Figure 2, the basic chitinase gene was expressed at a relative low level in the wild-type but was induced in the transgenic lines tCUP::Pti4-3,tCUP::Pti4-5, 35S::Pti4-3, and 35S::Pti4-6. The transgenic linetCUP::Pti4-1, which had the lowest level ofPti4 expression among the six transgenic lines, did not show the induction of chitinase expression. These data indicated that there was a general correlation between Pti4 expression and chitinase RNA accumulation, suggesting that Pti4 induced the expression of the basic chitinase gene in Arabidopsis. The expression of other GCC box-containing genes, PDF1.2, PR-1, and HOOKLESS1, however, was not induced by expression ofPti4 in transgenic plants (data not shown), suggesting that the expression of these genes and basic chitinase gene was regulated by different mechanisms. Pti4 Induces Expression of the GCC Box-Containing Genes To identify other genes regulated by Pti4, we compared global gene expression in tCUP::Pti4-3 and wild-type seedlings by cDNA hybridization to GeneChip (Affymetrix, Santa Clara, CA) containing 8,247 Arabidopsis genes. Of the 8,247 Arabidopsis genes, only 28 genes that exhibited greater than 2.5-fold expression intCUP::Pti4-3 compared with the wild-type (TableI). In comparison, the expression of the control genes such as actin, GAPDH, andUBQ4 did not show significant difference. Search for cis-elements in promoter regions of these 28 induced genes revealed that 18 of them contain GCC box related sequences in 5′ upstream sequences. Some of them encode well-known pathogen-related proteins. These include chitinase and β-1,3-glucanase that have antifungal activities; xyloglucan endo-transglycosylase and β-glucosidase and monooxygenase 1 involve in cell wall modification; and peroxidases, basic blue protein, and protein disulfide isomerase involve in oxidative burst. Table I. Genes induced by Pti4 in Arabidopsis Accession No. . Gene Product . −Fold Induction . GCC Motif1-b . X98453 Peroxidase ATPN 20.7 GCCGCC AC005662 Unknown 18.6 GCCGCC AL022604 Putative sugar transport protein 13.9 GCCGAC X98809 Peroxidase ATP5a 13.2 GCCACC AC004138 Putative basic blue protein 9.6 GCCACC AC002333 Putative endochitinase 7.8 GCCGCC Y14070 Heat shock protein 17.6A6.7 6.7 — AF088280 PAP3 4.9 GCCGNC X92975 Xyloglucan endo-transglycosylase 4.3 GCCACC AC003033 Putative protein disulfide isomerase 3.8 GCCACC AF082157 β-glucosidase 3.7 GCCGNC Z97340 β-1,3-glucanase precursor 3.7 GCCGCC AJ001809 Succinate dehydrogenase flavaprotein 3.7 GCCGAC AB023448 Basic endochitinase 3.6 GCCGCC AC006340 Unknown protein 3.6 — AC002335 Putative trypsin inhibitor 3.6 GCCACC AF002109 Putative anthocyanin 5-aromatic acyltransferase 3.4 — AF082299 AGP2 3.4 GCCGNC X79052 SRG1 3.3 — AB003280 Phosphoglycerate dehydrogenase 3.2 — L04637 Lipoxygenase 3.1 — AL034567 Putative ubiquinol-cytochrome c reductase 3.0 GCCGNC AC004561 Putative tropinone reductase 2.9 — AJ007587 Monooxygenase 1 2.8 GCCGNC AC002343 HSP90-like protein 2.7 GCCGCC AC003114 Calreticulin 2.6 — X75365 Sucrose-proton symporter 2.6 — AC005662 Putative embryo-abundant protein 2.6 GCCGNC U41998 Actin 1.0 — M64115 GAPDH 1.3 — U33014 UBQ4 1.1 — Accession No. . Gene Product . −Fold Induction . GCC Motif1-b . X98453 Peroxidase ATPN 20.7 GCCGCC AC005662 Unknown 18.6 GCCGCC AL022604 Putative sugar transport protein 13.9 GCCGAC X98809 Peroxidase ATP5a 13.2 GCCACC AC004138 Putative basic blue protein 9.6 GCCACC AC002333 Putative endochitinase 7.8 GCCGCC Y14070 Heat shock protein 17.6A6.7 6.7 — AF088280 PAP3 4.9 GCCGNC X92975 Xyloglucan endo-transglycosylase 4.3 GCCACC AC003033 Putative protein disulfide isomerase 3.8 GCCACC AF082157 β-glucosidase 3.7 GCCGNC Z97340 β-1,3-glucanase precursor 3.7 GCCGCC AJ001809 Succinate dehydrogenase flavaprotein 3.7 GCCGAC AB023448 Basic endochitinase 3.6 GCCGCC AC006340 Unknown protein 3.6 — AC002335 Putative trypsin inhibitor 3.6 GCCACC AF002109 Putative anthocyanin 5-aromatic acyltransferase 3.4 — AF082299 AGP2 3.4 GCCGNC X79052 SRG1 3.3 — AB003280 Phosphoglycerate dehydrogenase 3.2 — L04637 Lipoxygenase 3.1 — AL034567 Putative ubiquinol-cytochrome c reductase 3.0 GCCGNC AC004561 Putative tropinone reductase 2.9 — AJ007587 Monooxygenase 1 2.8 GCCGNC AC002343 HSP90-like protein 2.7 GCCGCC AC003114 Calreticulin 2.6 — X75365 Sucrose-proton symporter 2.6 — AC005662 Putative embryo-abundant protein 2.6 GCCGNC U41998 Actin 1.0 — M64115 GAPDH 1.3 — U33014 UBQ4 1.1 — F1-a  28 genes that exhibited greater than 2.5-fold expression in tCUP∷Pti4-3 compared with the wild type were shown. In addition, three control genes (actin, GAPDH, and UBQ4) that did not show significant difference in expression were also listed for comparison. F1-b  GCC-motifs were found between 100 and 1,500 bp upstream of the translation start site for a subset of genes up-regulated in tCUP∷Pti4-3transgenic plants. Open in new tab Table I. Genes induced by Pti4 in Arabidopsis Accession No. . Gene Product . −Fold Induction . GCC Motif1-b . X98453 Peroxidase ATPN 20.7 GCCGCC AC005662 Unknown 18.6 GCCGCC AL022604 Putative sugar transport protein 13.9 GCCGAC X98809 Peroxidase ATP5a 13.2 GCCACC AC004138 Putative basic blue protein 9.6 GCCACC AC002333 Putative endochitinase 7.8 GCCGCC Y14070 Heat shock protein 17.6A6.7 6.7 — AF088280 PAP3 4.9 GCCGNC X92975 Xyloglucan endo-transglycosylase 4.3 GCCACC AC003033 Putative protein disulfide isomerase 3.8 GCCACC AF082157 β-glucosidase 3.7 GCCGNC Z97340 β-1,3-glucanase precursor 3.7 GCCGCC AJ001809 Succinate dehydrogenase flavaprotein 3.7 GCCGAC AB023448 Basic endochitinase 3.6 GCCGCC AC006340 Unknown protein 3.6 — AC002335 Putative trypsin inhibitor 3.6 GCCACC AF002109 Putative anthocyanin 5-aromatic acyltransferase 3.4 — AF082299 AGP2 3.4 GCCGNC X79052 SRG1 3.3 — AB003280 Phosphoglycerate dehydrogenase 3.2 — L04637 Lipoxygenase 3.1 — AL034567 Putative ubiquinol-cytochrome c reductase 3.0 GCCGNC AC004561 Putative tropinone reductase 2.9 — AJ007587 Monooxygenase 1 2.8 GCCGNC AC002343 HSP90-like protein 2.7 GCCGCC AC003114 Calreticulin 2.6 — X75365 Sucrose-proton symporter 2.6 — AC005662 Putative embryo-abundant protein 2.6 GCCGNC U41998 Actin 1.0 — M64115 GAPDH 1.3 — U33014 UBQ4 1.1 — Accession No. . Gene Product . −Fold Induction . GCC Motif1-b . X98453 Peroxidase ATPN 20.7 GCCGCC AC005662 Unknown 18.6 GCCGCC AL022604 Putative sugar transport protein 13.9 GCCGAC X98809 Peroxidase ATP5a 13.2 GCCACC AC004138 Putative basic blue protein 9.6 GCCACC AC002333 Putative endochitinase 7.8 GCCGCC Y14070 Heat shock protein 17.6A6.7 6.7 — AF088280 PAP3 4.9 GCCGNC X92975 Xyloglucan endo-transglycosylase 4.3 GCCACC AC003033 Putative protein disulfide isomerase 3.8 GCCACC AF082157 β-glucosidase 3.7 GCCGNC Z97340 β-1,3-glucanase precursor 3.7 GCCGCC AJ001809 Succinate dehydrogenase flavaprotein 3.7 GCCGAC AB023448 Basic endochitinase 3.6 GCCGCC AC006340 Unknown protein 3.6 — AC002335 Putative trypsin inhibitor 3.6 GCCACC AF002109 Putative anthocyanin 5-aromatic acyltransferase 3.4 — AF082299 AGP2 3.4 GCCGNC X79052 SRG1 3.3 — AB003280 Phosphoglycerate dehydrogenase 3.2 — L04637 Lipoxygenase 3.1 — AL034567 Putative ubiquinol-cytochrome c reductase 3.0 GCCGNC AC004561 Putative tropinone reductase 2.9 — AJ007587 Monooxygenase 1 2.8 GCCGNC AC002343 HSP90-like protein 2.7 GCCGCC AC003114 Calreticulin 2.6 — X75365 Sucrose-proton symporter 2.6 — AC005662 Putative embryo-abundant protein 2.6 GCCGNC U41998 Actin 1.0 — M64115 GAPDH 1.3 — U33014 UBQ4 1.1 — F1-a  28 genes that exhibited greater than 2.5-fold expression in tCUP∷Pti4-3 compared with the wild type were shown. In addition, three control genes (actin, GAPDH, and UBQ4) that did not show significant difference in expression were also listed for comparison. F1-b  GCC-motifs were found between 100 and 1,500 bp upstream of the translation start site for a subset of genes up-regulated in tCUP∷Pti4-3transgenic plants. Open in new tab Pti4 Transgenic Plants Display an Ethylene-Responsive Phenotype To evaluate the involvement of Pti4 in the ethylene signaling pathway, Pti4 transgenic plant lines were examined for the ethylene-responsive phenotype. This is characterized by a triple response in Arabidopsis, which includes inhibition of root and hypocotyl elongation, radial swelling of the hypocotyl and root, and exaggeration in the curvature of the apical hook (Ecker, 1995; Chang and Shockey, 1999). The hypocotyls of the etiolated transgenic seedlings were measured 72 h after germination. As shown in Figures 3 and 4, the transgenic lines showed inhibition of hypocotyl elongation, a phenotype similar to those observed in the constitutive ethylene response-mutants or in wild-type plants exposed ethylene (Solano et al., 1998). The seedlings from the transgenic linetCUP::Pti4-3, which had a high Pti4expression, displayed strong inhibition of hypocotyl elongation. The seedlings from the transgenic line tCUP::Pti4-1, which had a lower level of Pti4 transgene expression, showed weak inhibition of hypocotyl elongation (Fig. 3). These data indicated that there was a correlation between the Pti4 expression and the inhibition of hyopcotyl elongation. Fig. 3. Open in new tabDownload slide Length of hypocotyl of transgenic Arabidopsis seedlings. Surface-sterilized seeds from wild-type (WT) and transgenic lines were planted in growth medium and cold treated at 4°C for 4 d before germination and growth in the dark at 23°C for 72 h in the presence (with ACC) or absence (without ACC) of 1-aminocyclopropane-1-carboxylic acid. The lengths of seedling hypocotyls were measured to the closest millimeter. Fourteen to 20 seedlings from each line were measured. Error bars correspond to these. Fig. 3. Open in new tabDownload slide Length of hypocotyl of transgenic Arabidopsis seedlings. Surface-sterilized seeds from wild-type (WT) and transgenic lines were planted in growth medium and cold treated at 4°C for 4 d before germination and growth in the dark at 23°C for 72 h in the presence (with ACC) or absence (without ACC) of 1-aminocyclopropane-1-carboxylic acid. The lengths of seedling hypocotyls were measured to the closest millimeter. Fourteen to 20 seedlings from each line were measured. Error bars correspond to these. Fig. 4. Open in new tabDownload slide Phenotype of Pti4 overexpression in transgenic seedlings. Each panel is composed of two etiolated Arabidopsis seedling. Surface-sterilized seeds were planted in growth medium and cold treated at 4°C for 4 d before germination and growth in the dark at 23°C for 72 h. A, Wild type incubated without aminocyclopropane carboxylic acid (ACC); B, wild type displaying the triple response in the presence of 10 μm ACC; C, tCUP::Pti4-3transgenic seedlings incubated without ACC; and D,tCUP::Pti4-3 transgenic seedlings incubated in the presence of 10 μm ACC. Fig. 4. Open in new tabDownload slide Phenotype of Pti4 overexpression in transgenic seedlings. Each panel is composed of two etiolated Arabidopsis seedling. Surface-sterilized seeds were planted in growth medium and cold treated at 4°C for 4 d before germination and growth in the dark at 23°C for 72 h. A, Wild type incubated without aminocyclopropane carboxylic acid (ACC); B, wild type displaying the triple response in the presence of 10 μm ACC; C, tCUP::Pti4-3transgenic seedlings incubated without ACC; and D,tCUP::Pti4-3 transgenic seedlings incubated in the presence of 10 μm ACC. The seedlings from the transgenic lines did not show strong curvature of the apical hook (Fig. 4), suggesting a partial seedling triple response phenotype. This is consistent with the observation that theHOOKLESS1 gene, a gene required for apical hook curvature (Lehman et al., 1996), was not expressed in the transgenic plants (data not shown). The adult plants from the transgenic lines also displayed reduced leaf size when compared with the wild-type plants (Fig.5). This is a phenotype similar to that of the constitutive ethylene-responsive mutants such as ctr1(Ecker, 1995). Fig. 5. Open in new tabDownload slide Phenotype of Pti4 overexpression in transgenic plants. The transgenic tCUP::Pti4-3plants (middle and right) displayed a reduced leaf size when compared with the wild-type plant (left). The photo was taken after plants were grown for 5 weeks in a growth chamber (16 h of light and 8 h of darkness at 23°C). Fig. 5. Open in new tabDownload slide Phenotype of Pti4 overexpression in transgenic plants. The transgenic tCUP::Pti4-3plants (middle and right) displayed a reduced leaf size when compared with the wild-type plant (left). The photo was taken after plants were grown for 5 weeks in a growth chamber (16 h of light and 8 h of darkness at 23°C). DISCUSSION The Pti4 protein belongs to the ERF-type proteins, which is a large family of plant transcription factors (Ohme-Takagi and Shinshi, 1995; Riechmann and Meyerowitz, 1998; Fujimoto et al., 2000). The binding of some ERF proteins to the GCC box in the ethylene-responsive element suggests a role for these proteins in the regulation of ethylene-responsive gene expression. In tomato, Pti4 transcripts rapidly accumulated in response to ethylene, before expression of the GCC box-containing GluB and Osm genes (Thara et al., 1999; Gu et al., 2000), further supporting a role in ethylene-regulated PR gene expression. Using a transient expression system, we have shown that Pti4 can function as a transcriptional activator of a GCC box-containing reporter gene. Furthermore, expression of Pti4 in Arabidopsis induced the expression of GCC box-containing genes and conferred a constitutive ethylene-responsive phenotype. These data suggested thatPti4 is involved in the regulation of ethylene-responsive genes containing the GCC box. The GCC box contains a conserved AGCCGCC sequence, which was first identified from the promoters of ethylene-inducible PR genes in tobacco (Ohme-Takagi and Shinshi, 1990; Eyal et al., 1993; Hart et al., 1993). It has been suggested that this sequence is a target in the ethylene signal transduction pathway because deletion of the GCC box eliminates ethylene responsiveness (Broglie et al., 1989; Meller et al., 1993;Vogeli-Lange et al., 1994; Shinshi et al., 1995). A search for plant promoter sequences containing the GCC box sequence uncovered a number of predominantly basic PR genes from bean (Phaesoleus vulgarus), tobacco, potato (Solanum tuberosum), Arabidopsis, Brassica sp., and tomato (Zhou et al., 1997;Jia and Martin, 1999), suggesting that these PR genes might be regulated by related ERF transcriptional factors. In Arabidopsis, the GCC box-containing genes include basic chitinase gene,PDF1.2, PR-1, and HOOKLESS1 (Samac et al., 1990; Lehman et al., 1996; Lebel et al., 1998; Manners et al., 1998). Overexpression of Pti4 in Arabidopsis induces the expression of the basic chitinase gene and other GCC box-containing genes. The expression of PDF1.2, PR-1, andHOOKLESS1, however, was not induced, suggesting that the expression of these genes might be regulated by different ERF proteins. The Arabidopsis ethylene-responsive factor 1 (ERF1) was induced rapidly by ethylene, and its role in regulating ethylene-inducible genes was demonstrated (Solano et al., 1998). The Arabidopsis ERF1 activates GCC box-containing PR genes such as the basic chitinase gene andPDF1.2 and confers constitutive ethylene response when overexpressed in Arabidopsis. Pti4 could be a tomato functional homolog of ERF1 in mediating ethylene-regulated expression of PR genes containing a GCC box. In Arabidopsis, expression of ERF1 is controlled by a novel DNA-binding protein encoded by theEIN3 gene, indicating that ERF1 acts downstream of EIN3 in ethylene signaling (Solano et al., 1998). It remains to be determined whether Pti4 gene expression is regulated by a transcriptional factor similar to EIN3 in tomato. In tobacco, at least four different ERF proteins, ERF1 through -4, have been identified (Ohme-Takagi and Shinshi, 1995). ERF2 and ERF4 enhance the GCC box-mediated transcription of a reporter gene in tobacco protoplasts, suggesting that they act as transcriptional activators (Ohta et al., 2000). In contrast to ERF2 and ERF 4, ERF3 reduces the transcription of the reporter gene in tobacco protoplasts, indicating that ERF3 functions as a repressor. Several Arabidopsis ERF-like genes,AtERF1 to -5, were also isolated from an Arabidopsis cDNA library by using tobacco ERFs as probes (Fujimoto et al., 2000). It has been shown that AtERF1, AtERF2, and AtERF5 act as transcriptional activators for GCC box-dependent transcription. AtERF3 and AtERF4, however, act as transcriptional repressors (Fujimoto et al., 2000). These studies indicate that GCC box-dependent transcription is controlled by a dynamic system utilizing antagonistic mechanisms in plants. Different ERF proteins also possess distinct DNA binding preferences, suggesting they could play different roles in the differential control of GCC box-containing gene expression (Hao et al., 1998; Fujimoto et al., 2000). In tomato, three ERF-like genes, Pti4/5/6, were identified by their interaction with Pto kinase in yeast two-hybrid screening (Zhou et al., 1997). Pti4/5/6 show differential expression patterns in various tomato tissues, implying that they may play distinct roles (Thara et al., 1999; Gu et al., 2000). Our study demonstrates that expression of the Pti4 gene in Arabidopsis induces the expression of GCC box-containing genes. In tomato, several GCC box-containing genes, such as GluB,Osm, and one 1-aminocyclopropane-1-carboxylic acid oxidase gene, have been identified (Jia and Martin, 1999). Pti4 transcripts rapidly accumulated in response to ethylene, before expression of the GCC box-containing GluB and Osm genes (Thara et al., 1999; Gu et al., 2000), suggesting that Pti4 may control the expression of a subset of GCC box-containing genes in tomato. In Arabidopsis, an ERF-like protein, AtEBP, was shown to interact with a basic Leu zipper transcription factor (Buttner and Singh, 1997), indicating that ERF proteins may interact with other transcriptional factors to regulate gene expression. Further research is required to investigate how subsets of GCC box-containing genes are regulated by different EFR proteins. In summary, we have demonstrated that Pti4 can act as a transcriptional activator to enhance GCC box-mediated gene transcription. Expression ofPti4 in transgenic Arabidopsis plants confers a constitutive ethylene phenotype and induces the GCC box-containing gene expression. Our study provides evidence that Pti4 gene product is involved in the regulation of a subset of ethylene-responsive genes containing the GCC box. MATERIALS AND METHODS Plant Material Arabidopsis (ecotype Columbia) was grown in a growth chamber (16 h of light and 8 h of darkness at 23°C) after a 2- to 4-d vernalization period. For growth under sterile conditions, seeds were surface sterilized (15-min incubation in 5% [v/v] sodium hypochlorite, and a three-time rinse in sterile distilled water) and sown on one-half-strength Murashige and Skoog salts (Sigma, St. Louis;Murashige and Skoog, 1962) supplemented with 1% (w/v) Suc, pH 5.7, and 0.8% (w/v) agar in petri dishes. To test the triple response of seedlings, surface-sterilized seeds were planted in Murashige and Skoog growth medium and cold treated at 4°C for 4 d. Seeds were then grown in the dark at 23°C for 72 h in the presence or absence of 1-aminocyclopropane-1-carboxylic acid, and the hypocotyl lengths of seedlings were measured. Southern- and Northern-Blot Analysis Total genomic DNA from Arabidopsis was extracted as described (Dellaporta et al., 1983). For Southern blots, Arabidopsis genomic DNA was digested with restriction enzymes, separated by agarose gel electrophoresis, and transferred to nylon membranes (Sambrook et al., 1989). For northern analysis, total RNA was isolated from 100 to 200 mg of Arabidopsis tissues using TriPure Reagent as described by the manufacturer (Boehringer Mannheim, Basel). Northern blots were prepared by electrophoresis of 5- to 10-μg samples of total RNA through agarose gels in the presence of formaldehyde (Strommer et al., 1993), followed by transfer to nylon membranes. Southern and northern blots were probed with 32P-labeled probes. Prehybridization and hybridization were performed at 65°C in 0.5 mNa2HPO4 (pH 7.2), 7% (w/v) SDS, and 1 mm EDTA. Filters were washed once for 15 min in 2× SSC with 0.1% (w/v) SDS at room temperature, then twice for 20 min in 0.1× SSC, 0.1% (w/v) SDS at 65°C. The damp filters were autoradiographed at −80°C using two intensifying screens. Filters were stripped in 5 mm Tris-HCl, pH 7.5, 1 mmEDTA, and 0.05% (w/v) SDS at 100°C for 2 min when reprobing was required. Synthesis of Biotin-Labeled cRNA The methods for preparation of cRNA directly from total RNA and subsequent steps leading to hybridization and scanning of the U95 GeneChip Arrays were provided by the manufacturer (Affymetrix). Briefly, first-stranded cDNA was synthesized from 20 μg of total RNA with a special oligo(dT)24 primer containing a T7 RNA polymerase promoter at its 5′ end in 20 μL of first-strand reaction mix at 42°C for 1 h. The second-strand was synthesized in second-strand reaction mix for 2 h at 16°C. After second-strand synthesis, biotin-labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction using BioArray RNA Transcript Labeling Kit (Enzo Diagnostics, New York) with biotin-labeled CTP and UTP. The labeled cRNA was purified by using RNeasy spin columns (Qiagen USA, Valencia, CA). Fifteen micrograms of each cRNA sample was fragmented at 94°C for 35 min in fragmentation buffer (40 mm Tris-acetate, pH 8.1, 100 mm potassium acetate, and 30 mmmagnesium acetate) and then used to prepare 300 μL of mixture. A biotinylated oligonucleotide, B2, was added that hybridizes to unique features at the center and four corners of each chip to map the probe sets on the chip. Oligonucleotide Array Hybridization and Scanning cRNA hybridization mix was heated to 94°C for 5 min, equilibrated to 45°C for 5 min, and clarified by centrifugation (14,000g) at room temperature for 6 min. Aliquots of each sample (10 mg of cRNA in 200 mL of the mixture) were hybridized GeneChip arrays at 45°C for 16 h in a rotisserie oven set at 60 rpm (GeneChip Hybridization Oven 640, Affymetrix). After this, the arrays were washed with SSPE, stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR) and washed again. The whole procedure of washing and staining was carried out in GeneChip Fluidics Station 400 (Affymetrix). Then the chip was scanned by GeneArray Scanner (HP and Affymetrix). Average difference and expression call for each features on the chip was computed using Affymetrix GeneChip Analysis Suite version with a default parameters. Plasmid Construction To prepare reporter constructs, the CaMV 35Spromoter of pBI221 (CLONTECH, Palo Alto, CA) was replaced with a truncated tCUP promoter, -394tCUP (Wu et al., 2001), to generate thepBI-BtCUP vector. The -394tCUP promoter of pBI-BtCUP was replaced with a minimaltCUP promoter, -62tCUP. DNA fragments containing two GCC boxes or mutated GCC boxes (Ohme-Takagi and Shinshi, 1995) were ligated into the PstI site located upstream of the -62tCUP promoter. To construct the effector plasmids, we replaced the 35Spromoter of pBI221 with the tCUP promoter (Foster et al., 1999) to generate the pBI-tCUP vector. The GUS gene in the pBI221 andpBI-tCUP was replaced with the Pti4coding region to generate 35S::Pti4 andtCUP::Pti4, respectively. To generate plasmid for Arabidopsis transformation, the35S::Pti4 andtCUP::Pti4 plasmids were digested withEcoRI and HindIII, and the resulting fragment containing the promoters and the Pti4 gene were then subcloned into the multicloning sites ofpCAMBIA2300 binary vector (Cambia, Canberra, Australia). Plant Transformation and Selection Plant transformation plasmids were electroporated intoAgrobacterium tumefaciens GV3101 as described by Shaw (1995). The A. tumefaciens-mediated transformation of Arabidopsis was performed as described (Clough and Bent, 1998), with the following modifications. Plants with immature floral buds and few siliques were dipped into a solution containing A. tumefaciens, 2.3 g L−1 Murashige and Skoog salts (Sigma), 5% (w/v) Suc, and 0.03% (w/v) Silwet L-77 (Lehle Seeds, Round Rock, TX) for 0.5 min. T1 seeds were collected, dried at 25°C, and sown on sterile media containing 40 μg mL−1 kanamycin to select the transformants. Surviving T1 plantlets were transferred to soil to set seeds (T2). Particle Gun Delivery Assays Tobacco (SR1; Nicotiana tabacum) plants were maintained in vitro in one-half-strength Murashige and Skoog medium (Murashige and Skoog, 1962) in Magenta containers (Magenta Corp., Chicago) in a growth chamber at 25°C. After transfer to fresh medium for 2 to 3 weeks, uniform-sized leaves (about 3 cm in width) were cut off from the plants and placed on a medium consisting of Murashige and Skoog salts, B5 vitamins (Gamborg et al., 1968), 1 mg L−16-benzyladenine, 0.1 mg L−1 naphthalene acetic acid, 3% (w/v) Suc, and 0.25% (w/v) Gelrite in a 20- × 15-mm petri dish. The leaves were preconditioned on this medium for 1 d before gene delivery. Plasmid DNA was isolated using the Qiagen Plasmid Midi Kit. The reporter plasmid was mixed with an effector plasmid at a 1:5 ratio (w/v). In the control, the reporter was mixed with the pUC19 plasmid. A modified particle inflow gun (Brown et al., 1994) was used for DNA delivery. DNA was precipitated onto tungsten particles using following protocol: 5 μg of DNA was added to 25 μL of tungsten particles (100 mg mL−1) and followed by the addition of 25 μL of 2.5m CaCl2 and 5 μL of 0.1 mspermidine. 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Plant J 20 1999 571 579 Google Scholar Crossref Search ADS PubMed WorldCat 40 Wu K Malik K Tian L Hu M Martin T Foster L Brown D Miki B Enhancers and core promoter elements are essential for the activity of a cryptic gene activation sequence from tobacco, tCUP. Mol Genet Genom 265 2001 763 770 Google Scholar Crossref Search ADS WorldCat 41 Zhou J Tang X Martin GB The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes. EMBO J 16 1997 3207 3218 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported in part by the Matching Investment Initiative Program at Agriculture and Agri-Food Canada. This paper is Eastern Cereal and Oilseed Research Centre contribution no. 001554. 2 ©Minister of Public Works and Government Services Canada 2002. For the Department of Agriculture and Agri-Food, Government of Canada. * Corresponding author; e-mail [email protected]; fax 613–759– 1701. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.010696. Copyright © 2002 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)

Tzafrir, Iris; McElver, John A.; Liu, Chun-ming; Yang, Li Jun; Wu, Jia Qian; Martinez, Audrey; Patton, David A.; Meinke, David W.

doi: 10.1104/pp.010911pmid: N/A

Abstract The titan mutants of Arabidopsis exhibit striking defects in seed development. The defining feature is the presence of abnormal endosperm with giant polyploid nuclei. SeveralTTN genes encode structural maintenance of chromosome proteins (condensins and cohesins) required for chromosome function at mitosis. Another TTN gene product (TTN5) is related to the ARL2 class of GTP-binding proteins. Here, we identify four additional TTN genes and present a general model for the titan phenotype. TTN1 was cloned after two tagged alleles were identified through a large-scale screen of T-DNA insertion lines. The predicted gene product is related to tubulin-folding cofactor D, which interacts with ARL2 in fission yeast (Schizosaccharomyces pombe) and humans to regulate tubulin dynamics. We propose that TTN5 and TTN1 function in a similar manner to regulate microtubule function in seed development. The titan phenotype can therefore result from disruption of chromosome dynamics (ttn3, ttn7, andttn8) or microtubule function (ttn1 andttn5). Three other genes have been identified that affect endosperm nuclear morphology. TTN4 andTTN9 appear to encode plant-specific proteins of unknown function. TTN6 is related to the isopeptidase T class of deubiquitinating enzymes that recycle polyubiquitin chains following protein degradation. Disruption of this gene may reduce the stability of the structural maintenance of chromosome complex. Further analysis of the TITAN network should help to elucidate the regulation of microtubule function and chromosome dynamics in seed development. Seed development in Arabidopsis requires coordinated differentiation of the embryo proper, suspensor, endosperm tissue, and seed coat. Interactions between these components have been explored in part through the analysis of embryo-defective mutants (Meinke, 1995). Some of these mutants have provided insights into the maintenance of cellular identity during seed development. Suspensor cell identity has been examined in twin mutants (Vernon and Meinke, 1994; Zhang and Somerville, 1997), meristem identity explored in stm mutants (Long et al., 1996), and cotyledon identity analyzed in lec mutants (Meinke, 1992;Lotan et al., 1998). Embryo-defective mutants have also been used to identify large numbers of genes with essential functions during seed development (McElver et al., 2001). Gene products identified to date include a variety of metabolic enzymes (Patton et al., 1998; Jang et al., 2000; Schrick et al., 2000; Boisson et al., 2001; Lukowitz et al., 2001), transcription factors (Long et al., 1996; Hardtke and Berleth, 1998; Li and Thomas, 1998; Lotan et al., 1998), chloroplast and mitochondrial proteins (Tsugeki et al., 1996; Uwer et al., 1998; Albert et al., 1999; Apuya et al., 2001), and proteins required for vesicle trafficking (Lauber et al., 1997; Assaad et al., 2001; Rojo et al., 2001). These essential genes represent an important subset of the minimal gene set needed to make a functional plant. Early endosperm development in Arabidopsis is characterized by specialized patterns of nuclear division, nuclear migration, and delayed cellularization (Brown et al., 1999; Otegui and Staehelin, 2000; Boisnard-Lorig et al., 2001; Olsen, 2001). Endosperm identity is therefore modulated to some extent by factors that regulate mitosis and cell division. The TITAN genes described in this report play an important role in this process of endosperm differentiation. Genetic analysis of endosperm development in Arabidopsis has focused in recent years on mutants with defects in gene imprinting and inappropriate endosperm development in the absence of fertilization (Grossniklaus et al., 1998; Luo et al., 1999; Ohad et al., 1999; Yadegari et al., 2000;Sorensen et al., 2001). These studies have underscored the importance of polycomb proteins and associated factors in regulating gene expression and nuclear division during early stages of endosperm development. Three titan mutants with striking defects in embryo and endosperm development were originally described by Liu and Meinke (1998). These mutants are characterized by dramatic enlargement of endosperm nuclei (Fig. 1). Embryo phenotypes depend on the locus involved: giant cells with enlarged nuclei (ttn1), small cells arrested early in development (ttn2), or viable cells that survive seed desiccation (ttn3). The taggedttn3 mutant is disrupted in a gene that encodes a chromosome scaffold protein (SMC2) related to structural maintenance of chromosome (SMC) proteins in Saccharomyces cerevisiae, which are required for normal chromosome function at mitosis (Liu et al., 2002). The weak embryo phenotype appears to result from expression of a duplicate gene with overlapping functions. Another tagged mutant (ttn5) with a phenotype similar to ttn1 was identified in a separate collection of insertion lines (McElver et al., 2001). This gene encodes a small GTP-binding protein (ARL2) related to ADP ribosylation factors (McElver et al., 2000). Related mutants (pilz) with large embryo cells and defects in microtubule organization have also been described by Mayer et al. (1999). Comparison of map locations suggests that hal corresponds tottn5 and that cho is ttn1. Fig. 1. Open in new tabDownload slide Phenotypic classes of titan mutants identified. Large black dots represent enlarged nucleoli. Small dots inttn3 endosperm correspond to condensed mitotic chromosomes. Arrow indicates continued embryo development in ttn3 seeds. An intermediate embryo phenotype is observed in ttn4 seeds late in development. Fig. 1. Open in new tabDownload slide Phenotypic classes of titan mutants identified. Large black dots represent enlarged nucleoli. Small dots inttn3 endosperm correspond to condensed mitotic chromosomes. Arrow indicates continued embryo development in ttn3 seeds. An intermediate embryo phenotype is observed in ttn4 seeds late in development. To establish a more complete picture of TITAN functions in seed development, we performed a forward genetic screen for additional knockouts within a large collection of insertion lines generated at Syngenta (McElver et al., 2001). This screen expanded the total number of titans to include at least 17 mutants defective in nine different genes. Two of these genes encode SMC1 and SMC3 cohesins, which are known to interact with condensins in other eukaryotes to regulate chromosome dynamics (Liu et al., 2002). We therefore have established a strong connection between loss of SMC function during seed development and the appearance of a titan endosperm phenotype. We describe in this report the identification of four additionalTITAN genes represented by tagged mutant alleles. One of these (TTN1) encodes a regulatory protein known as tubulin-folding cofactor D, which interacts with ARL2 in fission yeast (Schizosaccharomyces pombe; Radcliffe et al., 2000a,2000b) and humans (Homo sapiens; Bhamidipati et al., 2000) to modulate microtubule dynamics. This discovery makes it possible to explain much of the ttn1 phenotype (Liu and Meinke, 1998), clarify the role of TTN5 in seed development (McElver et al., 2000), and explain the loss of microtubules noted in pilz mutants (Mayer et al., 1999). A second gene (TTN6) encodes a deubiquitinating enzyme related to human isopeptidase T (Wilkinson, 1997). Knockouts in this gene (AtUBP14) have recently been noted to result in embryonic lethality (Doelling et al., 2001), but the titan phenotype was not identified. Thettn6 mutant establishes a connection between TITAN protein networks and the ubiquitin pathway. TTN4 corresponds to a senescence-associated gene (SAG18) that encodes a novel protein (Weaver et al., 1998; Miller et al., 1999) with an unknown function in seed development. Another gene (TTN9) with atitan endosperm phenotype also appears to encode a novel protein. These results are consistent with a model in whichtitan abnormalities result from disruption of either microtubule function or chromosome dynamics during seed development. The novel proteins may influence these central pathways indirectly through mechanisms unique to plants. Elucidation of additional TITAN functions should provide further insights into the regulation of mitosis and cytokinesis during endosperm development and the complex network of proteins required to differentiate endosperm from other parts of the seed. RESULTS Isolation of Tagged titan Mutants A forward genetic screen of T-DNA insertion lines produced at Syngenta was performed to identify tagged titan mutants amenable to gene isolation. This project was part of a large-scale effort to isolate tagged embryo-defective mutants and to identify essential genes in Arabidopsis (McElver et al., 2001). Two strategies were used to find titan mutants within this collection: screening immature siliques from heterozygous plants for glassy seeds indicative of a titan phenotype; and examining cleared seeds with Nomarski optics for the presence of enlarged endosperm nucleoli (Liu and Meinke, 1998). The second approach was generally reserved for tagged mutants with flanking sequence information. Tagging status was resolved by identifying mutant lines with a low ratio of resistant-to-sensitive seedlings, transplanting resistant seedlings to soil, and looking for linkage between the resistance gene and mutant phenotype (McElver et al., 2001). Results of this insertional mutagenesis project are summarized in TableI. Mutations in at least nine different genes have been identified that result in a strong titanphenotype. These mutants can be placed into four phenotypic classes as illustrated in Figure 1. Additional mutants with variable and intermediate titan phenotypes have also been recovered. Table I. Summary of titan mutants Mutant . Source . Class1-a . Linkage1-b . Gene . Putative Protein Function . ttn1-1 Feldmann A NA At3g60740 Tubulin-folding cofactor D ttn1-2 Syngenta A 103 /103 At3g60740 Tubulin-folding cofactor D ttn1-3 Syngenta A 105 /105 At3g60740 Tubulin-folding cofactor D ttn2 Feldmann B NA Unknown Gene identity unknown ttn3 Feldmann D 196 /196 At5g62410 SMC2 condensin ttn4 Syngenta B 123 /123 At1g71190 SAG18; unknown function ttn5-1 Syngenta A 206 /206 At2g18390 ARL2 GTPase ttn5-2 Lukowitz A NA At2g18390 ARL2 GTPase ttn6-1 Syngenta C 126 /126 At3g20625 Deubiquitinating enzyme ttn6-2 Syngenta C NA At3g20625 Deubiquitinating enzyme ttn6-3 Syngenta C 33 /33 At3g20625 Deubiquitinating enzyme ttn6-4 Syngenta C 102 /102 At3g20625 Deubiquitinating enzyme ttn7-1 Syngenta B 178 /178 At2g27170 SMC3 cohesin ttn7-2 Syngenta B 177 /177 At2g27170 SMC3 cohesin ttn8-1 Syngenta B 170 /170 At3g54670 SMC1 cohesin ttn8-2 Syngenta B 127 /127 At3g54670 SMC1 cohesin ttn9 Syngenta B 103 /103 At3g20070 Unknown function Mutant . Source . Class1-a . Linkage1-b . Gene . Putative Protein Function . ttn1-1 Feldmann A NA At3g60740 Tubulin-folding cofactor D ttn1-2 Syngenta A 103 /103 At3g60740 Tubulin-folding cofactor D ttn1-3 Syngenta A 105 /105 At3g60740 Tubulin-folding cofactor D ttn2 Feldmann B NA Unknown Gene identity unknown ttn3 Feldmann D 196 /196 At5g62410 SMC2 condensin ttn4 Syngenta B 123 /123 At1g71190 SAG18; unknown function ttn5-1 Syngenta A 206 /206 At2g18390 ARL2 GTPase ttn5-2 Lukowitz A NA At2g18390 ARL2 GTPase ttn6-1 Syngenta C 126 /126 At3g20625 Deubiquitinating enzyme ttn6-2 Syngenta C NA At3g20625 Deubiquitinating enzyme ttn6-3 Syngenta C 33 /33 At3g20625 Deubiquitinating enzyme ttn6-4 Syngenta C 102 /102 At3g20625 Deubiquitinating enzyme ttn7-1 Syngenta B 178 /178 At2g27170 SMC3 cohesin ttn7-2 Syngenta B 177 /177 At2g27170 SMC3 cohesin ttn8-1 Syngenta B 170 /170 At3g54670 SMC1 cohesin ttn8-2 Syngenta B 127 /127 At3g54670 SMC1 cohesin ttn9 Syngenta B 103 /103 At3g20070 Unknown function F1-a  Phenotype class as defined in Figure 1. F1-b  For tagged mutants, evidence of genetic linkage between T-DNA insert carrying resistance gene and mutant locus. Nos. represent plants heterozygous for mutation/total resistant plants screened. NA, Not applicable because the mutant is not tagged. Open in new tab Table I. Summary of titan mutants Mutant . Source . Class1-a . Linkage1-b . Gene . Putative Protein Function . ttn1-1 Feldmann A NA At3g60740 Tubulin-folding cofactor D ttn1-2 Syngenta A 103 /103 At3g60740 Tubulin-folding cofactor D ttn1-3 Syngenta A 105 /105 At3g60740 Tubulin-folding cofactor D ttn2 Feldmann B NA Unknown Gene identity unknown ttn3 Feldmann D 196 /196 At5g62410 SMC2 condensin ttn4 Syngenta B 123 /123 At1g71190 SAG18; unknown function ttn5-1 Syngenta A 206 /206 At2g18390 ARL2 GTPase ttn5-2 Lukowitz A NA At2g18390 ARL2 GTPase ttn6-1 Syngenta C 126 /126 At3g20625 Deubiquitinating enzyme ttn6-2 Syngenta C NA At3g20625 Deubiquitinating enzyme ttn6-3 Syngenta C 33 /33 At3g20625 Deubiquitinating enzyme ttn6-4 Syngenta C 102 /102 At3g20625 Deubiquitinating enzyme ttn7-1 Syngenta B 178 /178 At2g27170 SMC3 cohesin ttn7-2 Syngenta B 177 /177 At2g27170 SMC3 cohesin ttn8-1 Syngenta B 170 /170 At3g54670 SMC1 cohesin ttn8-2 Syngenta B 127 /127 At3g54670 SMC1 cohesin ttn9 Syngenta B 103 /103 At3g20070 Unknown function Mutant . Source . Class1-a . Linkage1-b . Gene . Putative Protein Function . ttn1-1 Feldmann A NA At3g60740 Tubulin-folding cofactor D ttn1-2 Syngenta A 103 /103 At3g60740 Tubulin-folding cofactor D ttn1-3 Syngenta A 105 /105 At3g60740 Tubulin-folding cofactor D ttn2 Feldmann B NA Unknown Gene identity unknown ttn3 Feldmann D 196 /196 At5g62410 SMC2 condensin ttn4 Syngenta B 123 /123 At1g71190 SAG18; unknown function ttn5-1 Syngenta A 206 /206 At2g18390 ARL2 GTPase ttn5-2 Lukowitz A NA At2g18390 ARL2 GTPase ttn6-1 Syngenta C 126 /126 At3g20625 Deubiquitinating enzyme ttn6-2 Syngenta C NA At3g20625 Deubiquitinating enzyme ttn6-3 Syngenta C 33 /33 At3g20625 Deubiquitinating enzyme ttn6-4 Syngenta C 102 /102 At3g20625 Deubiquitinating enzyme ttn7-1 Syngenta B 178 /178 At2g27170 SMC3 cohesin ttn7-2 Syngenta B 177 /177 At2g27170 SMC3 cohesin ttn8-1 Syngenta B 170 /170 At3g54670 SMC1 cohesin ttn8-2 Syngenta B 127 /127 At3g54670 SMC1 cohesin ttn9 Syngenta B 103 /103 At3g20070 Unknown function F1-a  Phenotype class as defined in Figure 1. F1-b  For tagged mutants, evidence of genetic linkage between T-DNA insert carrying resistance gene and mutant locus. Nos. represent plants heterozygous for mutation/total resistant plants screened. NA, Not applicable because the mutant is not tagged. Open in new tab Duplicate mutant alleles were expected to be found given the large number of insertion lines screened. Allelism was demonstrated through a combination of genetic complementation tests and flanking sequence information. The ethyl methanesulfonate-inducedttn5-2 allele obtained from Wolfgang Lukowitz (Carnegie Institution of Washington, Stanford, CA) was confirmed by direct sequencing of amplified DNA from heterozygotes (McElver et al., 2000). Allelism between the untagged ttn6-2 allele and the complexttn6-3 insertion allele was established by crossing heterozygotes. Approximately 22% of the 228 seeds produced fromttn6-1 × ttn6-2 crosses and 26% of the 235 seeds produced from ttn6-1 × ttn6-3 crosses appeared mutant. Similar crosses revealed allelism betweenttn1-1 and ttn1-2. In contrast, ttn2and ttn4 complemented when crossed, and the two genes mapped to different chromosomal regions. Analysis of F2plants produced from crosses with visible markers placedttn4 near the bottom of chromosome 1. A pilzmutant (pfi) with related phenotype has also been mapped to this region (Mayer et al., 1999). The genetic map position ofttn4, 12 cM below clv2 (180 F2 plants scored) and 15 cM above clv1(190 F2 plants), is consistent with the physical location based on sequence analysis. A composite genetic and physical map of TTN genes is shown in Figure2. Fig. 2. Open in new tabDownload slide Localization of TTN genes on a sequence-based chromosome map of Arabidopsis. Open rectangles correspond to centromeric regions as defined by genetic analysis (Arabidopsis Genome Initiative, 2000). Numbers indicate the estimated length of each chromosome in Megabases. The position of TTN2was estimated from genetic linkage data. Fig. 2. Open in new tabDownload slide Localization of TTN genes on a sequence-based chromosome map of Arabidopsis. Open rectangles correspond to centromeric regions as defined by genetic analysis (Arabidopsis Genome Initiative, 2000). Numbers indicate the estimated length of each chromosome in Megabases. The position of TTN2was estimated from genetic linkage data. Phenotypic Characterization of titan Mutants Three phenotypic classes of titan mutants were recognized by Liu and Meinke (1998). Differences were found in embryo morphology, seed viability, chromosome condensation, nucleolar appearance, and endosperm nuclear migration. Screening of the Syngenta collection yielded additional examples of the ttn1 (Fig. 1A) and ttn2 (Fig. 1B) classes. Another class (ttn6) characterized by a globular arrested embryo (Fig. 1C) was also identified. Our failure to recover mutants with a ttn3pattern (Fig. 1D) was not surprising given the subtle embryo phenotype. Nomarski images of ttn1, ttn4, andttn6 seeds at the heart-to-cotyledon stage of normal development are shown in Figure3. Fig. 3. Open in new tabDownload slide Phenotypes of mutant seeds examined with Nomarski optics. Embryo (E) and suspensor (S) cells, enlarged endosperm nucleoli (arrows), and endosperm cytoplasmic masses (CM) are visible in cleared mutant seeds from heterozygous siliques at the heart-to-cotyledon stages of normal development. A, ttn4 embryo; B,ttn6-1 embryo; C, ttn1-2 embryo; D, wild-type embryo; E, ttn1-2 endosperm; F, wild-type endosperm. Scale bar = 30 μm. Fig. 3. Open in new tabDownload slide Phenotypes of mutant seeds examined with Nomarski optics. Embryo (E) and suspensor (S) cells, enlarged endosperm nucleoli (arrows), and endosperm cytoplasmic masses (CM) are visible in cleared mutant seeds from heterozygous siliques at the heart-to-cotyledon stages of normal development. A, ttn4 embryo; B,ttn6-1 embryo; C, ttn1-2 embryo; D, wild-type embryo; E, ttn1-2 endosperm; F, wild-type endosperm. Scale bar = 30 μm. The ttn6 phenotype was examined in most detail because it defined a new titan class. Embryo cells often appeared rounded and disorganized. Endosperm cellularization was also disrupted. These abnormalities were confirmed in sectioned material, as shown in Figure 4. TableII documents developmental changes observed in cleared mutant seeds from tagged (ttn6-1) and untagged (ttn6-2) alleles. Defects visible at the heart stage of normal development included: increased size and reduced number of endosperm nuclei and nucleoli; and developmental arrest of the embryo proper. Endosperm nuclear enlargement was similar to that observed with other titans (Liu and Meinke, 1998; McElver et al., 2000). The average size of the embryo proper and largest endosperm nucleolus increased somewhat following the heart stage. A number of small nucleoli with a diameter of 5 to 6 μm were also found in the mutant endosperm, and their size remained constant between the heart and cotyledon stages. This variability in nuclear size within a single seed is a common feature of titan mutants. Mostttn6 seeds at the heart stage contained between 20 and 50 endosperm nuclei. This number did not increase later in development and remained far below the number found in wild-type seeds. Therefore, endosperm nuclear division is completed at about the same time in mutant and wild-type seeds. Fig. 4. Open in new tabDownload slide Light microscopy of ttn6-2 seeds. A through C, Stained sections of mutant seeds at the cotyledon stage of normal development. Abnormal cells of the embryo proper (E) and suspensor (S) are visible. Enlarged endosperm nuclei (EN) and nucleoli (arrows) are present. The image in B was rotated 90o counterclockwise. The vacuolated cell (right) is part of the suspensor. D, Wild-type embryo and cellularized endosperm from a seed at the equivalent time in development. Scale bar = 30 μm. Fig. 4. Open in new tabDownload slide Light microscopy of ttn6-2 seeds. A through C, Stained sections of mutant seeds at the cotyledon stage of normal development. Abnormal cells of the embryo proper (E) and suspensor (S) are visible. Enlarged endosperm nuclei (EN) and nucleoli (arrows) are present. The image in B was rotated 90o counterclockwise. The vacuolated cell (right) is part of the suspensor. D, Wild-type embryo and cellularized endosperm from a seed at the equivalent time in development. Scale bar = 30 μm. Table II. Analysis of ttn6 seeds at different stages of development Allele . Stage2-a . Diameter of Embryo Proper . Diameter of Largest Endosperm Nucleolus . Average . Range . Average . Range . μm ttn6-1 Heart 44 23–58 18 14–35 Linear cotyledon 52 32–64 21 12–41 Curled cotyledon 58 37–104 24 14–46 ttn6-2 Heart 40 30–51 14 9–20 Linear cotyledon 48 35–74 16 12–23 Curled cotyledon 69 69–108 23 14–32 Allele . Stage2-a . Diameter of Embryo Proper . Diameter of Largest Endosperm Nucleolus . Average . Range . Average . Range . μm ttn6-1 Heart 44 23–58 18 14–35 Linear cotyledon 52 32–64 21 12–41 Curled cotyledon 58 37–104 24 14–46 ttn6-2 Heart 40 30–51 14 9–20 Linear cotyledon 48 35–74 16 12–23 Curled cotyledon 69 69–108 23 14–32 F2-a  Developmental stage of normal seeds obtained from the same silique. No. of seeds analyzed:ttn6-1: heart (24), linear cotyledon (47), and curled cotyledon (40); ttn6-2: heart (31), linear cotyledon (38), and curled cotyledon (38). The diameter of a normal embryo cell is about 8 μm, and that of an endosperm nucleolus is 4 μm. Open in new tab Table II. Analysis of ttn6 seeds at different stages of development Allele . Stage2-a . Diameter of Embryo Proper . Diameter of Largest Endosperm Nucleolus . Average . Range . Average . Range . μm ttn6-1 Heart 44 23–58 18 14–35 Linear cotyledon 52 32–64 21 12–41 Curled cotyledon 58 37–104 24 14–46 ttn6-2 Heart 40 30–51 14 9–20 Linear cotyledon 48 35–74 16 12–23 Curled cotyledon 69 69–108 23 14–32 Allele . Stage2-a . Diameter of Embryo Proper . Diameter of Largest Endosperm Nucleolus . Average . Range . Average . Range . μm ttn6-1 Heart 44 23–58 18 14–35 Linear cotyledon 52 32–64 21 12–41 Curled cotyledon 58 37–104 24 14–46 ttn6-2 Heart 40 30–51 14 9–20 Linear cotyledon 48 35–74 16 12–23 Curled cotyledon 69 69–108 23 14–32 F2-a  Developmental stage of normal seeds obtained from the same silique. No. of seeds analyzed:ttn6-1: heart (24), linear cotyledon (47), and curled cotyledon (40); ttn6-2: heart (31), linear cotyledon (38), and curled cotyledon (38). The diameter of a normal embryo cell is about 8 μm, and that of an endosperm nucleolus is 4 μm. Open in new tab The most common titan embryo phenotype in the Syngenta collection was early lethality without dramatic cell enlargement (Fig.1B). This pattern was characteristic of knockouts in five differentTTN genes (Table I). Several of these mutants escaped our initial screen for glassy seeds and were identified astitans only after examination with Nomarski optics. Mutant embryos contained a few small cells and were often difficult to find in cleared seeds. Enlargement of endosperm nucleoli was pronounced but somewhat variable. Cellularization of the endosperm was also blocked. The ttn9 embryo, which contained at most four small cells, was typical of this class and resembled the cohesin (ttn7and ttn8) knockouts described by Liu et al. (2002). Thettn4 embryo was larger and more vacuolated late in development and therefore represented an intermediate class. In addition, embryo cells often accumulated wall thickenings that resulted in birefringence when viewed under Nomarski optics. These features are highlighted in Figure 5. Variations in titan seed phenotypes observed within each mutant are summarized in Table III. Typically, 10% to 20% of mutant seeds with an arrested embryo failed to exhibit atitan endosperm phenotype. The cellular basis for this variation remains to be explained. Globular embryos were found only inttn6 seeds. The tagged ttn1-2 allele (Fig. 3C) exhibited a seed phenotype identical to ttn1-1 (Liu and Meinke, 1998). Arrested embryos were found in 84% of 100 clearedttn1-2 seeds examined (Table III). Fifty-six percent of these embryos were composed of two cells (Fig. 3C). The remainder contained a single large cell. Embryo cell enlargement inttn1-3 was similar. Over 90% of these embryos contained two cells. Fig. 5. Open in new tabDownload slide Late phenotypes of ttn4 mutant embryos. A and B, Cell wall thickenings appear as bright regions on the surface of the embryo proper (E) and suspensor (S) in cleared seeds viewed with Nomarski optics. C and D, Embryo cells become enlarged and distorted in shape prior to seed desiccation. Scale bar = 30 μm. Fig. 5. Open in new tabDownload slide Late phenotypes of ttn4 mutant embryos. A and B, Cell wall thickenings appear as bright regions on the surface of the embryo proper (E) and suspensor (S) in cleared seeds viewed with Nomarski optics. C and D, Embryo cells become enlarged and distorted in shape prior to seed desiccation. Scale bar = 30 μm. Table III. Phenotypic variation observed in mutant seeds Mutant . No. Seeds Examined . Percentage of Seeds Observed with Specified Mutant Phenotype . Endospermtitanphenotype3-a . Arrested embryo phenotype . Strong . Moderate . Weak . Preglobular . Globular . ND3-b . ttn1-2 100 78 0 22 84 0 16 ttn1-3 100 67 9 24 96 0 4 ttn4 96 72 12 16 73 0 27 ttn6-1 100 81 14 5 28 70 2 ttn6-4 93 68 22 10 13 87 0 ttn9 100 73 14 13 50 0 50 Mutant . No. Seeds Examined . Percentage of Seeds Observed with Specified Mutant Phenotype . Endospermtitanphenotype3-a . Arrested embryo phenotype . Strong . Moderate . Weak . Preglobular . Globular . ND3-b . ttn1-2 100 78 0 22 84 0 16 ttn1-3 100 67 9 24 96 0 4 ttn4 96 72 12 16 73 0 27 ttn6-1 100 81 14 5 28 70 2 ttn6-4 93 68 22 10 13 87 0 ttn9 100 73 14 13 50 0 50 F3-a  Seeds with giant endosperm nucleoli were classified as strong, those with nucleoli of intermediate sizes were called moderate, and those with smaller nucleoli were considered weak. F3-b  ND, Not detected because the embryo was too small. Open in new tab Table III. Phenotypic variation observed in mutant seeds Mutant . No. Seeds Examined . Percentage of Seeds Observed with Specified Mutant Phenotype . Endospermtitanphenotype3-a . Arrested embryo phenotype . Strong . Moderate . Weak . Preglobular . Globular . ND3-b . ttn1-2 100 78 0 22 84 0 16 ttn1-3 100 67 9 24 96 0 4 ttn4 96 72 12 16 73 0 27 ttn6-1 100 81 14 5 28 70 2 ttn6-4 93 68 22 10 13 87 0 ttn9 100 73 14 13 50 0 50 Mutant . No. Seeds Examined . Percentage of Seeds Observed with Specified Mutant Phenotype . Endospermtitanphenotype3-a . Arrested embryo phenotype . Strong . Moderate . Weak . Preglobular . Globular . ND3-b . ttn1-2 100 78 0 22 84 0 16 ttn1-3 100 67 9 24 96 0 4 ttn4 96 72 12 16 73 0 27 ttn6-1 100 81 14 5 28 70 2 ttn6-4 93 68 22 10 13 87 0 ttn9 100 73 14 13 50 0 50 F3-a  Seeds with giant endosperm nucleoli were classified as strong, those with nucleoli of intermediate sizes were called moderate, and those with smaller nucleoli were considered weak. F3-b  ND, Not detected because the embryo was too small. Open in new tab Molecular Identification of TTN1 We first attempted to isolate the TTN1 gene through map-based cloning because the original ttn1-1 allele from the Feldmann collection was not tagged. Mapping with visible markers placed ttn1 below tt5, close tocer7 on chromosome 3 (Franzmann et al., 1995; Liu and Meinke, 1998). Rare recombinants obtained from crosses betweenttn1-1 heterozygotes (Wassilewskija [WS] ecotype) andtt5 or cer7 homozygotes (Ler ecotype) were analyzed with a series of linked molecular markers. From 1,852 F2 plants examined, 119 crossovers betweentt5 and ttn1 were obtained. The combined results, as summarized in Figure 6, enabled us to localize ttn1 below cer7 and likely on bacterial artificial chromosome (BAC) T4C21 within a region spanned by markers T22D23T7 and F26K11sp6. One gene in this region (T4C21.150) encodes a protein that resembles tubulin-folding cofactor D. This gene became a TTN1 candidate when we learned that ARL2 (TTN5) interacts with cofactor D to regulate microtubule assembly in yeast and humans (Bhamidipati et al., 2000; Radcliffe et al., 2000b). Two knockouts were later found in the Syngenta collection of embryo defectives. Allelism between these tagged mutants and ttn1-1was demonstrated through genetic complementation tests. Approximately 22% of the 510 seeds produced from reciprocal crosses between heterozygotes were mutant. These results confirmed that TTN1had been identified. Fig. 6. Open in new tabDownload slide Map-based localization of TTN1. TheTTN1 gene was localized to BAC T4C21 on chromosome 3 by analyzing recombinants produced from crosses with visible markers for the presence of linked molecular markers as described in the text. Fig. 6. Open in new tabDownload slide Map-based localization of TTN1. TheTTN1 gene was localized to BAC T4C21 on chromosome 3 by analyzing recombinants produced from crosses with visible markers for the presence of linked molecular markers as described in the text. TTN1 Resembles Tubulin-Folding Cofactor D The predicted structure for TTN1 (At3g60740) is shown in Figure 7. The gene is approximately 5.9 kb in length, contains 16 introns based on AGI gene models, and encodes a predicted protein of 1,249 amino acids. The ttn1-2allele contains a T-DNA insertion in exon 2 and lacks 12 bp around the insertion site. The ttn1-3 allele has an insertion in intron 9 and lacks 18 bp adjacent to the insertion site. The existence of two mutants with similar phenotypes and defined insertions in the same gene provides confirmation of gene identity. The location of the mutation inttn1-1 has not been determined but the strong phenotype is consistent with a null allele. Fig. 7. Open in new tabDownload slide Gene structures and T-DNA insertion sites forTTN1, TTN6, TTN4, and TTN9. Large black boxes designate exons, stippled boxes are introns, striped rectangles correspond to untranslated regions, and thin rectangles represent adjacent genomic DNA. Insertion sites and associated deletions are shown above the predicted gene structures. Gene structures for TTN6 and TTN4 have been confirmed by cDNA sequence analysis. Models for TTN1 andTTN9 intron and exon boundaries are based on the Arabidopsis Genome Initiative (2000). Fig. 7. Open in new tabDownload slide Gene structures and T-DNA insertion sites forTTN1, TTN6, TTN4, and TTN9. Large black boxes designate exons, stippled boxes are introns, striped rectangles correspond to untranslated regions, and thin rectangles represent adjacent genomic DNA. Insertion sites and associated deletions are shown above the predicted gene structures. Gene structures for TTN6 and TTN4 have been confirmed by cDNA sequence analysis. Models for TTN1 andTTN9 intron and exon boundaries are based on the Arabidopsis Genome Initiative (2000). From BLAST sequence analysis, TTN1 appears to be a single copy gene in Arabidopsis. Expression has been confirmed through identification of expressed sequence tags (ESTs) from vegetative structures (Asamizu et al., 2000), seedling hypocotyl (Newman et al., 1994), roots (Asamizu et al., 2000), and seedlings exposed to salt stress (Gong et al., 2001). BLASTP searches against all GenBank sequences revealed a high level of sequence identity to cofactor D from human (35% identity; e = 0.0), bovine (35% identity; 0.0), fruit fly (Drosophila melanogaster; 31% identity; −148),Caenorhabditis elegans (26% identity; −81), andS. pombe (27% identity; −48). A number of conserved protein domains were found when these sequences were compared. Results of this analysis are presented in Figure8. The high degree of sequence conservation observed in these domains is consistent with a critical cellular function for this protein in eukaryotes. Fig. 8. Open in new tabDownload slide Conserved protein domains identified in tubulin-folding cofactor D. Each segment corresponds to a conserved domain identified by BLOCKS (Henikoff et al., 1995). Bold letters represent amino acids conserved in at least five of the six sequences. Numbers mark the amino acid location within the protein. Species and GenBank accessions: Arabidopsis (CAB82678), human (NP005984),Bos taurus (AAB17537), fruit fly (AAF51300), C. elegans (T21018), and S. pombe (Q10197). Fig. 8. Open in new tabDownload slide Conserved protein domains identified in tubulin-folding cofactor D. Each segment corresponds to a conserved domain identified by BLOCKS (Henikoff et al., 1995). Bold letters represent amino acids conserved in at least five of the six sequences. Numbers mark the amino acid location within the protein. Species and GenBank accessions: Arabidopsis (CAB82678), human (NP005984),Bos taurus (AAB17537), fruit fly (AAF51300), C. elegans (T21018), and S. pombe (Q10197). TTN6 (AtUBP14) Resembles Isopeptidase T The predicted structure for TTN6 (At3g20625) is shown in Figure 7. This gene model was compiled from AGI sequence of two adjacent BACs (K10D20 and F3H11). The predicted gene is approximately 5.0 kb in length, contains 19 introns, and encodes a predicted protein of 797 amino acids. The protein sequence is based on the availability of a full-length cDNA (AF302664). The ttn6-1 allele contains a large deletion (approximately 2.7 kb) at the insertion site that removes 10 exons coding for the C-terminal half of the protein. Thettn6-4 allele has a smaller deletion (approximately 0.4 kb) that eliminates exons 6 and 7. The ttn6-3 allele appears to be tagged from genetic evidence, but it contains a complex T-DNA insert that remains to be resolved. Twenty-seven deubiquitinating enzymes (DUBs) of the ubiquitin-specific protease (UBP) class have been identified in Arabidopsis (Yan et al., 2000). TTN6 (AtUBP14) is most similar in protein sequence to the isopeptidase T class of enzymes (Wilkinson et al., 1995) from human (49% identity; e = 0.0), mouse (47% identity; 0.0), fruit fly (44% identity; 0.0), Dictyostelium discoideum (UbpA; 40% identity; −180), C. elegans (34% identity; −119), and S. cerevisiae (UBP14; 31% identity; −69). The most closely related protein is derived from genomic sequencing of rice (Oryza sativa; 65% identity; 0.0). Figure9 illustrates conserved protein domains identified by Pfam analysis (Bateman et al., 2000): a zinc finger UBP domain, ubiquitin carboxyl-terminal hydrolases (UCH)-1 domain with conserved “Cys” box, UBA domains, and C-terminal UCH-2 domain with conserved “His” box. The absence of an N-terminal extension in the rice protein may reflect an incorrect gene model. Sequence comparisons of these conserved motifs have recently been published by Doelling et al. (2001). Fig. 9. Open in new tabDownload slide Conserved Pfam domains identified in TTN6 (UBP14)-related proteins from different organisms. TTN6 contains all of the protein domains expected for DUBs of the isopeptidase T class. Pfam analysis (Bateman et al., 2000) revealed the presence of a zinc finger UBP domain (ellipse), UCH-1 domain (diamond) with conserved “Cys” box, two UBA domains (hexagons), and a C-terminal UCH-2 domain with conserved “His” box in the expected locations. Organisms and GenBank accession numbers: A, Arabidopsis, TTN6, AAG42755; B, rice,BAB17073; C, human, XP_006971; D, Mus musculus, NP_038728; E, S. cerevisiae, UBP14, NP_009614; F, D. discoideum, P54201; G, fruit fly, AAF47720. Fig. 9. Open in new tabDownload slide Conserved Pfam domains identified in TTN6 (UBP14)-related proteins from different organisms. TTN6 contains all of the protein domains expected for DUBs of the isopeptidase T class. Pfam analysis (Bateman et al., 2000) revealed the presence of a zinc finger UBP domain (ellipse), UCH-1 domain (diamond) with conserved “Cys” box, two UBA domains (hexagons), and a C-terminal UCH-2 domain with conserved “His” box in the expected locations. Organisms and GenBank accession numbers: A, Arabidopsis, TTN6, AAG42755; B, rice,BAB17073; C, human, XP_006971; D, Mus musculus, NP_038728; E, S. cerevisiae, UBP14, NP_009614; F, D. discoideum, P54201; G, fruit fly, AAF47720. TTN4 and TTN9 Appear to Be Plant-Specific Proteins Although the identities of TTN4 and TTN9 are each based on analysis of a single mutant allele, the genetic data summarized in Table I are consistent with tagging, and both sides of each insert were recovered and found to match a single locus. The isolation of single mutant alleles in contrast to duplicate alleles for other titans is also consistent with the small size of these genes. T-DNA insertion sites and predicted gene structures are presented in Figure 7. The TTN4 gene model predicted from the sequencing project (Arabidopsis Genome Initiative, 2000) was confirmed through isolation of a full-length cDNA. Two amino acid differences identified were attributed to errors in sequencing of the cDNA. The SAG18 partial cDNA sequence (AF053063) described by Weaver et al. (1998) in their screen for senescence-associated genes corresponds to the 3′ end of the full-length transcript. The T-DNA insert in ttn4 is located in the 3′-untranslated region. The predicted protein product contains 281 amino acids and lacks defined domains and sequence similarity to known proteins. BLASTP analysis revealed a related Arabidopsis gene (F14F18.40) with 47% identity (e = −60) and a corresponding EST. A similar gene has also been identified in rice (BAB56093; 47% identity; e = −23). No significant matches were found with any proteins identified from other organisms. TTN9 appears to be a single copy gene that is expressed in siliques based on EST data. The predicted protein is 282 amino acids in length and lacks known motifs. One BLASTP match was identified in GenBank: an EST (AF325722; 32% identity; e = −10) from pistils of an apomictic grass (Pennisetum ciliare). These results are consistent with the conclusion that TTN9 and TTN4 are plant-specific proteins of unknown function. DISCUSSION TITAN Proteins Have Diverse but Overlapping Functions in Seed Development We have identified two networks of TITAN proteins in Arabidopsis that regulate endosperm nuclear division and cellularization. A model illustrating the functions of these proteins is presented in Figure 10. One network involves chromosomal scaffold proteins known as condensins (SMC2 and SMC4) and cohesins (SMC1 and SMC3). These myosin-like ATPases play a central role in chromosome condensation, sister chromatid cohesion, dosage compensation, and recombination repair (Hirano, 2000). The importance of SMC proteins in endosperm development became apparent when TTN3 was identified as an SMC2 condensin and was later confirmed when additional titans were found to be disrupted in SMC cohesins (Liu et al., 2002). A second network of TITAN proteins involves the regulation of microtubule assembly. To our knowledge, the importance of this network in plants is described for the first time in this report. The titan phenotype therefore can result from disruption of either chromosomal proteins or cytoskeletal organization. This conclusion is consistent with the contrasting models of gene functions presented when titan mutants were first identified (Liu and Meinke, 1998). Fig. 10. Open in new tabDownload slide Model of TITAN gene functions in Arabidopsis. Nuclear division in the developing endosperm requires at least two networks of TITAN proteins. One modulates chromosome integrity through scaffold proteins known as cohesins (SMC1 and SMC3) and condensins (SMC2). Another regulates microtubule assembly through interactions between ARL2 and tubulin-folding cofactor D. Knockouts inAtSMC4, protein targets of TTN6 activity, and cellular functions of TTN4 and TTN9 remain to be identified. Fig. 10. Open in new tabDownload slide Model of TITAN gene functions in Arabidopsis. Nuclear division in the developing endosperm requires at least two networks of TITAN proteins. One modulates chromosome integrity through scaffold proteins known as cohesins (SMC1 and SMC3) and condensins (SMC2). Another regulates microtubule assembly through interactions between ARL2 and tubulin-folding cofactor D. Knockouts inAtSMC4, protein targets of TTN6 activity, and cellular functions of TTN4 and TTN9 remain to be identified. Two defining features of early endosperm development in angiosperms are nuclear migration and the suppression of phragmoplast formation following nuclear division (Olsen, 2001). These processes require appropriate coordination between cytoskeletal organization and cell-cycle progression. The complex networks of TITAN proteins described here perform an essential role in maintaining chromosome structure and function throughout the cell cycle and in regulating the establishment of the microtubule arrays required for chromosome movement and phragmoplast formation. TITAN proteins therefore can be viewed as central mediators in processes that help to distinguish endosperm tissue from adjacent parts of the seed. TTN1 and TTN5 Encode Proteins That Interact in Yeast and Humans Many proteins have been identified that regulate microtubule dynamics in eukaryotes (Nogales, 2000). The formation of α/β-tubulin heterodimers begins with the appearance of chaperonin complexes and proceeds through interactions with specialized folding cofactors (Tian et al., 1996; Radcliffe et al., 2000a). Cofactor D associates with β-tubulin subunits and is encoded byAlp1 in S. pombe (Hirata et al., 1998) andCIN1 (for chromosome instability) in S. cerevisiae (Fleming et al., 2000). Loss of Alp1activity is lethal and results in abnormal mitoses, destruction of microtubule structures, and defects in cell division (Hirata et al., 1998). In contrast, CIN1 mutations are not lethal (Stearns et al., 1990; Fleming et al., 2000). In addition to modulating assembly of tubulin heterodimers, cofactor D functions as a GTP-activating protein for hydrolysis of GTP by β-tubulin and subsequent release of free heterodimers (Nogales, 2000). Cofactor D can also interact with native tubulin, alter the ratio of free subunits by sequestering β-tubulin from GTP-bound heterodimers, and stimulate destruction of heterodimers (Bhamidipati et al., 2000; Martin et al., 2000). ARL2 interacts directly with human cofactor D in culture, prevents degradation of tubulin heterodimers, and reduces the GTP-activating protein activity of cofactor D in vitro (Bhamidipati et al., 2000). Deletion of the ARL2 homolog in S. pombe(Alp41) results in defects in cell division similar to those found in cofactor mutants (Radcliffe et al., 2000a, 2000b). Therefore, ARL2 (Alp41) and cofactor D (Alp1) are essential proteins in fission yeast. The subtle phenotype of CIN4 (ARL2) knockouts is consistent with the nonessential role of tubulin cofactors in budding yeast (Stearns et al., 1990; Fleming et al., 2000). Nuclear and cytoskeletal defects observed in ttn1 andttn5 seeds are consistent with known roles of ARL2 and cofactor D in regulating microtubule dynamics in fission yeast and humans. Enlargement of endosperm nuclei and nucleoli appears to result from microtubule-associated defects in chromosome mechanics and cell plate formation coupled with continued progression through the cell cycle. Defects in microtubule organization have been documented with fluorescence microscopy in the corresponding pilz mutants (Mayer et al., 1999). The dramatic changes in embryo cell morphology described here are consistent with known functions of microtubules in plants. These functions have been difficult to address from a genetic perspective in Arabidopsis because of redundancy in the tubulin gene family (Kopczak et al., 1992; Snustad et al., 1992). Several mutants defective in microtubule organization have nevertheless been identified, including ton2/fass (Torres-Ruiz and Jurgens, 1994; Traas et al., 1995), mor1 (Whittington et al., 2001),bot1 (Bichet et al., 2001), zwi (Oppenheimer et al., 1997), and fra2/AtKTN1 (Burk et al., 2001). Changes in cell morphology have also been noted following exposure of roots to microtubule inhibitors (Baskin et al., 1994). We describe here a genetic system for studying the consequences of a dramatic loss of microtubule function, demonstrate the importance of ARL2 and cofactor D in seed development, and clarify the connection between ARL2 function and microtubule dynamics in plants. The Ubiquitin Pathway Is Linked to TITAN Functions The ubiquitin pathway plays a key role in selective degradation of proteins in eukaryotic cells (Hershko and Ciechanover, 1998). Targeted proteins are modified through the formation of an isopeptide bond between the C terminus of ubiquitin and the ε-amino group of Lys on the target protein (Naviglio et al., 1998). DUBs are hydrolyzing proteases that process primary ubiquitin gene products, edit the ubiquitination state of cellular proteins, and recycle ubiquitin released following hydrolysis of proteins targeted for destruction via the proteasome. Isopeptidase DUBs have specificity for substrates containing ε-amide bonds to a side chain Lys (Wilkinson, 1997). Some isopeptidases can also disassemble specific ubiquitin-protein conjugates before proteolysis by the proteasome. This process is thought to have either a regulatory function for essential proteins or a salvaging function for incorrectly ubiquitinated proteins (Hershko and Ciechanover, 1998). Two general classes of DUBs that differ in sequence and substrate specificity have been identified: small UCH proteins and UBP proteins with conserved Cys and His boxes (Wilkinson, 1997). These DUBs have the ability to cleave ubiquitin linked to target proteins by either peptide or isopeptide bonds. TTN6 (AtUBP14) is a large protein with unknown substrate specificity but characteristic UBP domains. A number of DUB genes have already been identified by mutation. These include fruit fly fat facets, which is required for reproductive development and eye differentiation (Fischer-Vize et al., 1992) and is thought to act by preventing degradation of its target regulatory protein (Huang et al., 1995); S. cerevisiae DOA4and UBP3, which are required for a variety of cellular processes including control of DNA replication (see Wilkinson, 1997) and regulation of gene silencing (Moazed and Johnson, 1996); andD. discoideum UbpA, which is required for normal development but not for continued growth (Lindsey et al., 1998; Chung and Baek, 1999). The UBP family of Arabidopsis consists of at least 27 genes with the conserved protein domains expected for catalytic activity (Chandler et al., 1997; Rao-Naik et al., 2000; Yan et al., 2000). Knockouts in two of these genes (AtUBP1 andAtUBP2) exhibit increased sensitivity to the amino acid analog canavanine, which can increase the concentration of abnormal proteins produced during translation (Yan et al., 2000). Therefore, these family members appear to function in the removal of abnormal proteins from the cell. Although substrate specificities and cellular localizations of several Arabidopsis UPBs have been described (Chandler et al., 1997; Rao-Naik et al., 2000), much remains to be learned about the precise functions of specific UBP proteins in Arabidopsis. Doelling et al. (2001) recently described two allelic UBP14(TTN6) knockouts that resulted in embryonic lethality at the globular stage, demonstrated that mutant seeds accumulated multi-ubiquitin chains, consistent with a defect in ubiquitin cycling, and found that Arabidopsis UBP14 complements the corresponding yeast mutant. We demonstrate here the connection between UBP14 function and a titan seed phenotype. We propose two models to explain the relationship between ubiquitin pathways and a titan phenotype. These models are based on two observations: the absence of dramatic cell enlargement inttn6 embryos, which suggests that a disruption of microtubule function is not involved; and the connection between chromosome stability and protein degradation recently established for the SSC1 cohesin of yeast (Rao et al., 2001). According to the first model, accumulation of free multiubiquitin chains enhances the stability of a target protein that under normal circumstances modulates SMC function. An alternative model is that TTN6 removes ubiquitin directly from a target protein that influences chromosome dynamics in wild-type seeds and the resulting accumulation of this regulatory protein in mutant seeds is responsible for the mutant phenotype. This model could involve the same target protein as described for the first model but a different mechanism for altering the stability of this protein. Embryo Phenotypes Reflect Differences in TITAN Functions The titan endosperm phenotype is consistent with known functions of microtubules and SMC proteins in eukaryotes. Even the atypical ttn3 endosperm phenotype can be explained by the presence of a related gene with overlapping functions. Differences observed between titan embryo phenotypes, however, are more problematic. Two questions remain to be addressed: Why are giant cells found only in ttn1 and ttn5 seeds; and why do nuclei in many titan embryos fail to enlarge? With respect to the second question, we propose that different cell-cycle checkpoints are involved in the embryo and endosperm. Disruption of the SMC complex in the embryo interferes with essential cell functions and consequently results in cell abortion. DNA replication and nuclear enlargement continue in the endosperm because cellularization is not required. Disruption of the SMC complex may also be the cause of abnormalities seen in ttn2 and ttn9 seeds, which have similar embryo phenotypes. The intermediate ttn4 embryo phenotype is intriguing because the wall thickenings seen late in development are reminiscent of changes associated with programmed cell death and differentiation of tracheary elements (Fukuda, 2000; Roberts and McCann, 2000). The most dramatic embryo phenotype observed to date is the striking cell enlargement found in ttn1 andttn5 seeds. The continuation of DNA replication in these embryos indicates that the SMC-related checkpoint is bypassed. The progressive cell enlargement demonstrates that elimination of ARL2-cofactor D-mediated regulation of microtubule assembly is not immediately lethal. Whether a similar mechanism is used in the formation of giant feeding cells exposed to root-knot nematodes (Niebel et al., 1996) remains to be explored. MATERIALS AND METHODS Plant Materials and Growth Conditions The ttn1-1, ttn2, andttn3 mutants were generated through Agrobacterium tumefaciens-mediated seed transformation of the WS ecotype (Feldmann, 1991) and were identified and maintained as described (Liu and Meinke, 1998). The ttn5-2 mutant was isolated by Wolfgang Lukowitz (Carnegie Institution of Washington) in the Landsbergerecta ecotype following seed mutagenesis with ethyl methanesulfonate (McElver et al., 2000). The remainingtitan mutants described in this report were produced at Syngenta through A. tumefaciens-mediated plant transformation of the Columbia ecotype using the vacuum infiltration (Bechtold and Pelletier, 1998) and floral dip (Clough and Bent, 1998) methods. Seeds can be obtained through the Arabidopsis Biological Resource Center. Details of plant transformation, vector design, and screening of insertion lines for seed defects are presented in McElver et al. (2001). Additional information on mutants defective in SMC genes (ttn3, ttn7, and ttn8) can be found in Liu et al. (2002). Plants were grown in pots containing a mixture of vermiculite, soil, and sand, placed in a growth room at 24° ± 2°C under fluorescent lights on 16-h light/8-h dark cycles, and watered daily with a fertilizer solution (Heath et al., 1986). Heterozygotes were identified by screening immature siliques for the presence of 25% defective seeds following self pollination (Meinke, 1994). Genetic and Phenotypic Characterization T-DNA vectors used for transformation experiments conferred resistance to kanamycin (ttn3), hygromycin (ttn4), or Basta (ttn1-2,ttn1-3, ttn6-1, ttn6-3,ttn6-4, and ttn9). Linkage between the T-DNA insert and mutant phenotype was demonstrated by transplanting resistant seedlings from selection plates to soil and scoring the resulting plants for the presence of the seed mutation (McElver et al., 2001). Mapping of ttn4 with visible markers was performed as described by Franzmann et al. (1995). Complementation tests were performed by crossing heterozygotes and scoring the resulting siliques for 25% defective seeds with the expected phenotype. Mutant seeds cleared for observations were treated with Hoyer's solution (Meinke, 1994) and examined with a compound microscope (model E600; Nikon, Tokyo) equipped with Nomarski optics. Images were captured with a DXM1200 digital imaging system (Nikon). Sections of embedded mutant seeds were prepared as noted by Liu and Meinke (1998). Map-Based Localization of TTN1 Crosses were made between ttn1 heterozygotes (WS ecotype) and either dis1, clv2,er, tt5 homozygotes or er,gl1, cer7 homozygotes (Landsberg) to identify crossovers in the vicinity of TTN1. Known RFLP markers (CD2-12 and pCIT1210), cleaved-amplified polymorphic sequence markers (IMK2 and IMK3), and SSLP markers (nga6) were used to estimate the position of TTN1 on the physical map. Eight cleaved-amplified polymorphic sequence markers (TT5, T22D23T7, F26K11sp6, agl13, FUS6, 2A19E, ACS1, and 2A19B) based on the BAC contig and genomic sequences in this region were then used to initiate a walk toward the TTN1 gene. Sequence details, PCR primer sequences, cycling conditions, and information on restriction enzymes used can be obtained upon request from the authors. TTN Gene Identification and Sequence Analysis Plant sequences flanking T-DNA insertion sites in tagged mutants were obtained through plasmid rescue or thermal asymmetric interlaced-PCR and confirmed by direct PCR sequencing using a combination of genome-specific and T-DNA primers as described in detail by McElver et al. (2001). The TTN4 full-length cDNA was isolated and sequenced according to standard methods (McElver et al., 2000). Sequence comparisons were performed using the BLAST 2.0 algorithm (Altschul et al., 1997) with default settings and the low complexity filter removed. Conserved protein motifs were identified with Pfam (Bateman et al., 2000) and were subjected to CLUSTALW (Thompson et al., 1994) and BLOCKS (Henikoff et al., 1995) analyses through the Baylor College of Medicine (Houston; Smith et al., 1996; http://searchlauncher.bcm.tmc.edu). ACKNOWLEDGMENTS We thank the many members of the Patton laboratory at Syngenta, in particular George Aux, for assistance with the production of insertion lines, initial screening for seed mutations, and isolation of flanking plant sequences; Mike Rumbaugh and Mary Ann Cushman for assistance with map-based localization of TTN1; and Steven Hutchens, Cathy Sonleitner, Becky Rogers, and Shkelzen Shabani for assistance with phenotypic characterization of tagged mutants. LITERATURE CITED 1 Albert S Despres B Guilleminot J Bechtold N Pelletier G Delseny M Devic M The EMB506 gene encodes a novel ankyrin repeat containing protein that is essential for the normal development of Arabidopsis embryos. 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