PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 c:/CIRC/DateDue.p65-p.15 WOODY PLANTS PQHD'THE APPLICATION OF MOLECULAR TOOLS BY Anne Edith Plovanich A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 2001 ABSTRACT WOODY PLANTS AND THE APPLICATION OF MOLECULAR TOOLS BY Anne Edith Plovanich Using various molecular biology tools three woody plant genera, Populus, Quercus, and Mbntanoa were chosen as experimental systems to elucidate heretofor unanswered questions. To elucidate genetic mechanisms of control of vascular cambial differentiation, hybrid poplar clone 47—174 (Populus deltoides x trichocarpa) was used as a model to examine gene expression in wood forming tissue and as a result of wind stress or mechanical perturbation (MP). RNA isolated from stressed and non-stressed poplar stems was examined using differential display of PCR products. One hundred thirty differential display gel bands from stem tissue were cloned and sequenced. Northern analysis and BLAST searches confirmed that more than seventy of these clones represent expressed sequence tags (ESTs) for poplar genes expressed in wood forming tissue. An abundance of representation of stress related genes indicate the ESTs are from abiotic induced mechanical perturbation. In the second study, DNA fingerprinting techniques were used for the identification of cultivars of Quercus x hispanica, the Lucombe oak. Using inter-simple sequence repeat (ISSRs) primers, 66 Q. x hispanica (and related) samples were analyzed and compared for band pattern differences to establish identities. DNA evidence revealed that some named cultivars had identical banding patterns, which then aided in identification of other named or unnamed cultivars with the same DNA banding patterns. A phylogeny of the genus Mbntanoa based on the Internal Transcribed Spacer (ITS) and the External Transcribed Spacer (ETS) was created. The combined dataset supports the monophyly of the genus and an early evolutionary split that coincides with geographic distribution. One lineage is composed mostly of central and southern Mexican species whereas the other lineage contains those species endemic to Mesoamerica and South America. The relationships of Mantanoa to other genera in the Heliantheae are briefly discussed. DEDICATION Morrie Schwarz Roland Barthes Red Dinsmore A.M.D.G. iv ACKNOWLEDGMENT S At the beginning and at the end are my roots, my beloved parents, Edith Ann Forst Plovanich, who loved all plants and Joseph Paul Plovanich, who loved trees. They left me a legacy and inspiration I cannot define. John Scott-Craig has been and continues to be my mentor, my guide, my inspiration and my friend. His patient support, encouragement and instruction have been the foundation for this dissertation and the accomplishment of my experiments. Without John, none of this would have happened. I have also had the privilege of the support and guidance of an extraordinary committee. Frank Telewski and I plotted our course of collaboration based on one strong bond, our love of trees. From the Aeschylus parviflora of our first meeting, through the Pinus ponderosa, the Pinus albicaulis, and the Populus trichocarpa x deltoides, it has always been the trees that helped us through the trials, successes and failures of this dissertation project. I have been truly honored and blessed to have his mentorship and friendship . John Ohlrogge has always been my support, my encouragement, my mentor and my friend. Knowing that John has always believed that this degree was going to happen has given me the strength to persevere. His assistance in the database searching and presentation of this EST collection was invaluable. Alan Prather stepped in to an ongoing dissertation project and provided the advice, knowledge and guidance to publish the Oak chapter and help prepare the Montanoa chapter for this dissertation. He helped fill in all the gaps in my background about systematics and taxonomy, particularly helpful for my reviewers from the Royal Horticultural Society on the Oak work. Michael Thomashow has been an inspiration to me, always. I was honored to have him serve on my committee, and he did not disappoint. His thoughtful evaluation of the poplar chapter has helped prepare for the publication of this project. Mike always challenges me to think more clearly, and to do better than my best. My committee has always believed in me, encouraged and supported me and has been there every step of the way for guidance when I needed it. I knew that from every one of them, the words, “I will help you out with this” were vi always there. Without any member of this committee, my degree would not have happened. Many people were there to assist in my science with teaching of techniques, discussions of results, experimental design or lab space and equipment. I will only elaborate on one, Ann Greene. My dear beloved and departed friend, “my Ann with no ‘e’”. Ann taught me how to do my first plasmid mini—preps, make my first solutions and encourage me when molecular experiments don’t work! She is missed. Richard Allison, Kyrstoff Szyzglowski, Dirk Hamburger, Frans DeBruijn and the DeBruijn lab, Jonathon Walton, John Pitkin, Kerry Pedley, John Tonukari and the Walton lab, ShengYang He and the He lab, Tom Newman, Sue Stoltzfus, Barb Christian, Ambo VanHoof, Anna Wiese, Li Ping, Neal Dittmer, Ken Keegstra (whose leadership and generous spirit inspires and guides the department), and in Sweden Anneli Stendberg, Bjorn Sundberg and Magnus Hertzeberg. This has been my science support group. Data Basing and Graphics for my figures, publications and defense seminar include Paula Lee Hauck, Julie Zwiesler- vii Vollick, Abe Koo, Diem T. Hoa, Uwe Rossbach, Kurt Stepnitz and Marlene Cameron. Those who have provided academic encouragement and inspiration have been Richard Allison, Ken Nadler, Dr. Phu Nguyen, Dr. Jim Flore, Hannah Mathers, Sarah Gilmour and Christoff Benning. Each of them were there at crucial points to foster inspiration. My financial support has come from Frank Telewski, ShengYang He, Jose Panero, the National Science Foundation, Michigan State University AURIG, the MSU Graduate School and the Horticulture Department. I cannot fail to mention the incredible moral support of my friends. When one does not have a family, friends achieve a greater importance in one’s life. These friends have been and are my family: Julie Zwiesler-Vollick, Anita Davelos, Michele Pruyn, Paula Lee Hauck, Ray Pacovsky, Glen Jones, Karen Bird, John Scott-Craig, Linda Danhoff, The PRL Office Staff, M.O.M., my beloved other mother and my spirit guide, Judy Ferris. And for those who understand, A.M.D.G. viii TABLE OF CONTENTS LIST OF TABLES ........................................... xi LIST OF FIGURES ......................................... xii INTRODUCTION .............................................. 1 CHAPTER 1 OVERVIEW OF FOREST BIOTECHNOLOGY. ...................... 5 CURRENT TRENDS IN FORESTRY BIOTECHNOLOGY ............... 5 POPULUS(POPLAR) AS A MODEL SYSTEM ..................... 16 THE EARLY DAYS OF TRANSGENIC POPLARS .................. 20 CURRENT TREND FOR THE USE OF TRANSGENIC POPLARS ....... 20 LIGNIN MODIFICATION AND WOODY PLANTS .................. 22 MODIFIED POPLARS AND THE ENVIRONMENT .................. 34 WIND STRESS IN POPLARS ................................ 36 GENE EXPRESSION IN RESPONSE TO WIND STRESS OR MP ...... 41 WIND STRESS AND OTHER ABIOTIC STRESSES ................ 43 WIND STRESS AND POPULUS (POPLAR) ...................... 45 CONCLUSION ............................................ 46 REFERENCES ............................................ 47 CHAPTER 2 GENE EXPRESSION IN WOOD FORMING TISSUE OF HYBRID POPULUS (POPLAR) ......................... 65 ABSTRACT .............................................. 65 INTRODUCTION .......................................... 66 The importance of wood ............................. 66 Wood development and the function of xylem ......... 67 Methods of wood plant modification ................. 68 Gene expression in developing cambium .............. 70 Wind stress and mechanical flexure ................. 73 Gene expression and mechanosensory Perception ...... 76 MATERIALS AND METHODS ................................. 80 Plant material and growth conditions ............... 81 Mechanical perturbation and harvesting ............. 81 RNA Isolation and differential display ............. 83 Cloning and Sequencing of differential Display bands ................................... 89 Northern analysis .................................. 91 Database searches .................................. 92 RESULTS AND DISCUSSION ................................ 94 Results from differential display .................. 94 Sequencing results ................................. 94 ix Results from database searches ..................... 95 Northern analysis ................................. 103 Wimps of particular interest ...................... 111 RNA extraction .................................... 123 Limits of differential display .................... 126 SUMMARY .............................................. 130 FUTURE DIRECTIONS .................................... 131 REFERENCES ........................................... 133 CHAPTER 3 CULTIVARS OF QUERCUS CERRIS X QUERCUS SUBER: Q.X HISPANICA, THE LUCOMBE OAK AND INTER-SIMPLE SEQUENCE REPEATS (ISSRS) ............................. 141 ABSTRACT ............................................. 144 INTRODUCTION ......................................... 145 MATERIALS AND METHODS ................................ 148 RESULTS .............................................. 151 DISCUSSION ........................................... 157 ACKNOWLEDGMENTS ...................................... 159 REFERENCES ........................................... 160 CHAPTER 4 A PHYLOGENY OF THE ITS AND ETS REGIONS FOR MONTANOA (ASTERACEAE: HELIANTHEAE) ................... 163 INTRODUCTION ......................................... 164 MATERIALS AND METHODS ................................ 168 RESULTS .............................................. 172 DISCUSSION ........................................... 176 ACKNOWLEDGMENTS ...................................... 185 REFERENCES ........................................... 186 CONCLUSION .............................................. 189 APPENDICES .............................................. 196 APPENDIX A ........................................... 197 APPENDIX B ........................................... 264 LI ST OF TABLES Table 1. Conference session - Paper abstracts ............. 8 Table 2. Conference poster abstracts ..................... 10 Table 3. Enzymes in the lignin pathway ................... 32 Table 4. Suggested model of tree response ................ 40 Table 5. ESTS from wood forming tissue ............... 99-102 Table 6. Wimps that hybridized to poplar RNA ........... 105 Table 7. Plant samples used for DNA microsatellite analysis ............................................. 149 Table 8. List of taxa used in phylogenetic studies ...... 169 xi LIST OF FIGURES Figure 1. Monolignol biosynthetic pathway ............. 29-30 Figure 2. Abiotic environmental stresses ................. 37 Figure 3. Plant abiotic environmental stresses ........... 73 Figure 4. Wimp 59 B-glucosidase ........................ 111 Figure 5. Wimp 68 dehydration response protein .......... 112 Figure 6. Different storage, transport and deglycosylation for monolignol alcohols ................................. 114 Figure 7. Alignment of B-glucosidases ................... 116 Figure 8. Plant B-glucosidase protein family ............ 117 Figure 9. Protein alignment of Wimp62 subtilisin ........ 118 Figure 10. Protein alignment of Wimp 68 dehydration response protein ........................................ 119 Figure 11. Banding pattern of UBC 834 ................... 152 Figure 12. Banding pattern of ‘Crispa’ .................. 153 Figure 13. Banding pattern UBC 834 for ‘Cana major’ ....154 Figure 14. Banding pattern of UBC 834 for nine samples of ‘Lucombeana’ .......................................... 155 Figure 15. Comparison of Kensington, ‘Lucombeana’ and ‘Fulhamensis’ ........................................... 156 Figure 16. Phylogeny of ITS and ETS of Genus Mbntanoa ..174 xii INTRODUCTION WOODY PLANTS AND MOLECULAR TOOLS? DO THEY HAVE A FUTURE TOGETHER? Plants that have woody stems (secondary growth or secondary xylem) have traditionally presented problems for scientists wishing to work with them. They take up a large amount of space, most of them grow slowly, are difficult to propagate and maintain in a greenhouse, many are slow to reproduce, flower, or bear fruit; and the woody stems themselves often present a formidable challenge to experimentation. These problems were difficult to overcome, but with the advent of molecular biology, the problems became compounded. In addition to the “normal” associated problems, add to that large genome size, (Populus 550 mph, Pinus taeda 25,000 mpb compared to A.thaliana 125 mbp), enormous challenges to transformability, and difficulty of extraction of DNA and RNA. There is also a general lack of funding for what may be considered high risk projects because of the aforementioned difficulties as well as the reluctance of forestry industries to fund projects that may take years to reach marketable products. Genetically modified trees may just not be cost effective. A further consideration in using molecular tools for woody plant modification is the growing opposition to genetically modified organisms (GMOs) by an increasing vocal and fear motivated public. Genetically modified trees, growing for years in the field versus short-term annual agricultural crops represent an even greater perceived threat to natural forests. Steve Strauss at Oregon State University has had two genetically modified poplar plantations cut down, while Toby Bradshaw at University of Washington, Seattle has had his lab (as well as others in the same building) bombed. This opposition to genetic engineering of trees may further impact funding potential. Why then, one could ask, would anyone wish to undertake the use of molecular tools to work on woody plants? For the same reason that one would use molecular tools for study of any other organisms: these tools furnish answers to questions that have not previously been able to be addressed, adding additional layers of knowledge to longstanding questions. And secondly, trees are merely a longer growing crop, planted and harvested like any other plant crop, such as wheat, corn, rice; however maintained in the field for longer periods of time. The world market for wood products, lumber, and paper has as much an increasing demand as the world market for edible resources. I propose to address some of these questions and furnish some limited answers. In this thesis I apply a number of molecular techniques to DNA and/or RNA extracted from three different woody plant genera, Quercus,.Mbntanoa, and Populus to furnish answers to problems that have previously not had resolution. The “Lucombe” oak, first named over two hundred and fifty years ago is grown throughout the UK and Europe. Enormous confusion exists over the true identity of this famous oak. In this dissertation I will look at molecular markers that make it possible to distinguish among cultivated varieties within a taxonomic group of named oak tree cultivars heretofore indistinguishable using traditional morphological characters. I also use molecular tools to examine the phylogeny of an unusual and spectacular woody genus of Mexico, the Mbntanoas, (“tree daisies”), shedding new light on the relationships of the twenty-five species, and the evolution and radiation of this genus. Previous studies of the genus have relied on morphological and histological studies to establish relationships between the twenty-five species, while failing to identify any related Composite species. And finally, molecular tools were used to elucidate a number of putative genes present in the cambial region of Populus (poplar) that contribute to growth and wood formation. A poplar cultivar (47-174 P. trichocarpa x P. deltoides) was used to find genes that may be expressed to due wind stress or mechanical perturbation. cDNAs have been cloned and sequenced that are found in stems of this poplar clone. CHAPTER 1 OVERVIEW OF FOREST BIOTECHNOLOGY THE IMPORTANCE OF POPULUS (Poplar) IN FOREST BIOTECHNOLOGY WIND STRESS IN POPLARS AND ITS SIGNIFICANCE IN WOODY PLANT MODIFICATION CURRENT TRENDS IN FORESTRY BIOTECHNOLOGY “Have you got a license for that tree (and can you afford to use it?)” is the title of a paper at the next forest biotechnology conference in Washington this July (Bryson, 2001). Research into genetic modification of woody plants has become a timely, controversial and much discussed topic. The current studies relating to genetic modification of trees are on the agenda at the upcoming meeting of the International Union of Forest Research Organization (IUFRO). Consider trees as crops that remain in the field for many seasons before harvest, rather than merely one. Then consider the many stresses that the crops have to endure to survive over these many seasons. In addition to survival, there are expectations for high yield and good wood quality for designated purpOses. All of the biotechnological improvements that science hopes to accomplish with single season crops are just as important, maybe more so, for a plantation of trees. Directed modification of many characteristics of forest trees, regarding wood quality, growth and development, resistance to pathogens and insect vectors, and response to environmental stresses is a direct challenge to scientists seeking to insure and improve yield. A single “crop” failure can cost the land owner years of investment. To respond to this challenge, it is necessary to have an understanding of the genes that affect these characteristics as well as an understanding of the genetic variation in desirable phenotypes of commercially or biologically important trees. By using genetic modification, improvements in plantation forestry can occur much more rapidly than in long—term field trials, so called “classical” breeding. Genetic tools can also aid in the conservation of forest species, preservation of biodiversity and better understanding of forest ecology. These genetic tools achieve even greater importance in the light of increasing land pressures and demands for wood products. The International Union of Forest Research Organizations (IUFRO) is a non-profit, non-governmental network of scientists involved in forestry and forest products research. The section on Molecular Biology (Genetics) of Forest Trees has convened meetings since 1985 (29 attendees) reflecting the early efforts of gene transfer into plants. The last IUFRO section meeting held in 1999 at Oxford, United Kingdom entitled “Forest Biotechnology ’99, A Working Party on Molecular Biology of Forest Trees” was attended by participants from thirty different countries (UK 168 attendees). An impressive array of sessions and posters was presented by investigators attempting to explore molecular applications and woody plants. Table 1 presents a summary of the topics and species being studied that were presented as papers at the meeting. Table 2 is a summary of the posters. Area of focus Topic Woody species Transformation and propagation Somatic embryogenesis Agrobacterium tumefaciens transformation Transgene stability/gene expression Norway spruce Eucalyptus globulus Theobroma cacao Pinus pinaster Acacia Liquidambar sytraciflua Teak (Tectona grandis) White pine Elm Aspen/Populus Loblolly pine Scots pine Casuarina glauca Directed wood modification Cellulose synthesis Lignin biosynthesis Aspen, and various non—woody species Poplar Spruce Yellow-poplar Eucalyptus gunnii Lodgepole pine Transgenic trees in the field Risks and controls Outcrossing Increased viral susceptibility Flower regulation Poplar Wild cherry Reduced lignin in tobacco Poplar Birch Conifers Eucalyptus Bambusa edulis Genomics Genome sequencing Loblolly pine projects; ESTs, Poplar RAPDs, QTLs Eucalyptus Japanese black pine White spruce Pinus sylvestris Maritime pine Larch Conservation Genomics and Norway spruce biodiversity Swietenia humilis (mahogany) Population studies Pedunculate oak Wild cherry Populus euphratica Table 1. Conference session — Paper abstracts Area of focus Topic Woody species Transformation and propagation Somatic embryogenesis Protoplast isolation Biolistic transformation Tissue specific transgene White spruce Maritime spruce Quercus suber Quercus robur Pinus elliottii x P.caribaea Eucalyptus Scots pine Pinus radiata Picea abies Passiflora species Prunus avium Sorbus aucuparia Elms Pinus radiata Apple Directed wood modification Lignin plasticity Lignin gene combinations Laccases CAD and Ozone COMT and reduced lignin Peroxidase/reduced lignin CAD-deficient pine Tobacco Arabidopsis Poplar Poplar Hybrid aspen Transgenic trees in the field Herbicide resistance Field trials Meristem activity Salicylic acid (increased disease resistance) Oxidative stress Wounding,drought, cold stress dehydrin Bacillus thuringensis ROLC transgene Mycorrizae Long distance pollinators Early flower induction Pinus radiata Picea abies Black locust Norway spruce (Syrs) Eucalyptus Poplar Poplar White spruce Hybrid larch Aspen/populus Transgenic poplar Eucalyptus Birch (MADS box) Genomics RAPDS DNA extraction Marker aided selection Genomic organization of repetitive DNA Avicennia marina Chimonanthus Maytenus ilicifolia Eucalyptus grandis Norway spruce Conservation Population studies Abies alba Picea rubens Pinus mariana Genetic diversity Betula pendula Norway maple (Finland) Gomortega keule Endangered species (Chile) Table 2. Conference poster abstracts The upcoming conference in July 2001 that will be held in Stevenson, Washington will address all aspects of molecular techniques applied to the study and manipulation of forest trees. Topics that are germane to this section include: use of molecular markers for ecogenetic studies, DNA marker-based breeding and selection, molecular and genomic studies of tree physiology and development, in—vitro culture and asexual gene transfer methods and silvicultural studies of genetically modified trees. The conference in Washington State has several topics that expand the scope of the meeting at Oxford. One area deals with the advent of high-throughput facilities for sequencing and microarrays that has led to an explosion of whole genome and Expressed Sequence Tag (EST) projects. The Swedish group plans to make publicly available 50,000 poplar ESTs at the meeting (Sandberg, 2001). The EST collections from this group as well as others are augmented by expression 10 data using other techniques such as Serial Analysis of Gene Expression (SAGE) and gene enhancer traps (Lorenz, 2001) (Groover, 2001). Of particular interest is the EST collection from mRNA from tension wood formation in poplar using Amplified Fragment Length Polymorphisms (AFLPS) (Leple, 1999). Jean-Charles LePle, Giles Pilate, and Florian Lafarguette have been working for three years to develop differentially expressed ESTs that are specific to wood tissue deposited in response to gravitropism; wood formed on the upper layer of stems and branches of angiosperms. They are using a poplar hybrid INRI #717-1- B4. This model system will potentially identify genes important for lignin non—deposition in the 53G layer, control of microfibril angle, and cambial activity (LePle personal communication). The genes found as important to tension wood formation may have many similarities to those found in response to mechanical perturbation. Reaction wood (formed in MP) has both tension and compression elements (Telewski, 1995). However the most striking difference between the two conferences is the attention given to the ecological and social issues that are everywhere the subject of much debate and analysis. The two principal organizers of the 11 Conference have been the victims of eco—terrorism. Toby Bradshaw, University of Washington, has had his laboratory burned in late May, while Steve Strauss, Oregon State, Corvallis, has had two transgenic poplar plantations cut down. Ironically Steve Strauss’ work has emphasis on the reduction or elimination of flowering in transgenic poplars to prevent environmental spillover while the bombing in Seattle destroyed the work of a scientist who has dedicated his research to conservation efforts. They are not alone as many countries report the eco-terrorism directed at genetic modification of woody plants. Since woody plants remain in the field for an extended period, their impact on the environment presents different issues than short rotation crops. Two papers that caught immediate attention Ware “A policy perspective on transgenic trees in Canada” (BOnfils, 2001); and the previously cited: “Have you got a license for that tree (and can you afford to use it)? which will be presented by a law firm from Washington, D.C. EC3C310gical, social, ethical and legal considerations of forest biotechnology have achieved paramount importance. The first two days of the conference are dedicated to an \\ eCEO—social” symposium to enlist dialogue on these issues. 12 mindful of the risks involved in woody plant modification, Scientists world-wide are continuing with their research because of inherent overall benefits. Woody plants share genetic features with herbaceous plants regarding growth and development, metabolic pathways and responses to biotic and abiotic stresses. However genomic approaches to important model tree species offer an understanding of the differences of woody plants from other model plant systems. Secondary growth and wood deposition cannot be understood using Arabidopsis thaliana as a model system. “Many aspects of tree development are radically different, and parallel genomics research for several commercial tree species will be required over the next decade.” (Robinson, 1999b) Other non—woody model plant systems which explore lignin deposition, such as Arabidopsis, Zinnia and Nicotiana can haVe a supporting role in hypothesis development and in 1:DIi-rainformatic searches of sequenced plant genomes. I“IQWever, trees must ultimately be used as the model systems for testing of such hypotheses. (Chaffey, 1999b) CcPrisequently studies of woody plants at the molecular leVel, despite difficulties, limitations, and risks are of majOr importance if plant scientists are to meet the chal lenges presented . 13 Trees as laboratory subjects present some difficulties and limitations. They are slow growing, many months before they reach experimental size, sometimes requiring years to achieve flowering and fruiting, and they require a significant amount of space when compared to the number of Arabidopsis that can be grown on a Petri dish. The woody stems present a formidable barrier to many laboratory experiments, and in many species, dormancy forces cessation of experimentation. These are the problems faced with traditional scientific research on trees. With molecular techniques the problems are compounded by often large and repetitive genomes: the plant model system Arabidopsis thaliana has a relatively small genome size of 125 mbp, , While poplar is 550 mbp, Pinus taeda 125,000 mbp. It is Suggested that it would take twenty two years to sequence given the techniques used in the Human Genome Project with a genome size of 600 mbp. (Bradshaw and Stettler, 1993; I{inlaw and Neale, 1997; Marie and Brown, 1993; Robinson, 1999a; Wakamiya et al., 1993). There are the added difficulties of extraction of DNA and RNA. complexity of genetic transformation and tissue culture, longevity and tissue specificity of the transgene 1“ long—lived plants, and the dearth of mutants that can 14 help elucidate genetic differences. Actually many potentially useful mutants are regularly discarded in traditional breeding programs or relegated as horticultural cultivars. Funding for molecular research on woody plants is scarce as forestry industries are reluctant to invest in technology that may or may not be economically beneficial, certainly not in the short term. Compounding these difficulties is the potential environmental contamination by transgenics (Greene, 1995; Strauss et al., 1999), the threat of eco—terrorism and public acceptance of genetically modified trees. All of these problems must be addressed by the scientists in the field of forest biotechnology. The history of molecular applications to forest trees has been a short 0118. The earliest efforts were directed at mapping of cieSirable traits for the purposes of “classical” tree breeding. Much early work had to do with attempts at gene transfer and of subsequent in vitro propagation. Twenty- eiSht speakers at the symposium entitled, “Genetic IVIariipulation of Woody Plants” held at Michigan State Uhiversity in June of 1987 focused on tissue culture and deVelopment of gene transfer systems (Hanover, 1987) . The first transgenic tree, a poplar, was created in 1987 15 (Fillatti, 1987) compared to the first transgenic plant, in 1980 (Hernalsteens et al., 1980) . Transformation and propagation of woody plants continues to be a considerable Challenge that remains elusive for many tree species. The genera that have been used in molecular studies, have been selected primarily because of their economic or conservation value. One genus comes to the forefront in molecular studies not only because of its socio—economic value, but also because of the relative ease with which it can be studied at the molecular level. That is. of course, POpulus, which may well be the first tree species designated for genome sequencing. POPULUS (POPLAR) AS A MODEL TREE SYSTEM For scientists who study trees, the diversity of woody plant genera under investigation using molecular tools is of amazing breadth as evidenced in the papers and posters presented at the IUFRO meetings. However one genus of WC30d}; plants appears in almost every category of anestigation excepting that of propagation and 16 transformation, the genus Populus (Poplar). The latter two categories have become fairly routine for poplar re s earchers . In the Northern hemisphere (Eucalyptus being the counterpart in the Southern) poplars are being used as the model system for non-molecular and molecular research pertaining to angiosperm wood formation (Stettler, 1996) . There are many reasons for this. Biologically they are easy to propagate from green or woody cuttings, and they are easy to maintain in a greenhouse. In winter under lights, growth will continue without dormancy. They are representative of many other woody plant species, pedigrees of various lines are available and many mutants responsive to a variety of conditions are being maintained. AS a cultivated species poplars have achieved important commercial, conservation, and remediation potential. Poplar can be used for biomass production, the wood used for pLllpwood, lumber, veneer, matchwood, and firewood. They can be used as shade trees, windscreens, and to stabilize Sites such as steep banks, landfills, spoil banks or borrow pits. Hybrid poplars exhibit different crown and leaf Shapes due to varied parentage (Demeritt, ?) . They are 17 rapid growing, amenable to use on marginal soils, and almost circumpolar in adaptation to a variety of climates. To cite a few of the newer uses of poplar worldwide: They provide a fast growing renewable energy source, in Hungary ongoing trials on marginal soils provide biomass energy wi 1:11 only a four year cutting cycle (Marosvolgyi et al., 1 9 99), in India poplar plantations intercropped with chamomile were successful on partially reclaimed marginal soil also for biomass production (Misra and Tewari, 1999) . Chinese use of flood tolerant poplar clones has proven successful for reforestation projects (Cao and Conner, 1999) . In New Zealand, poplar (which are exotic there) have been introduced successfully for erosion control Schemes (Wilkinson, 1999). An interesting concept has been the use of poplar clones that differ in resistance to air pollutants, SO; and 03, which would make the poplars effective bioindicators (Ballach, 1997) . Poplars have previously been shown to react: to ozone, varying from sensitive to tolerant (Cao and C"antler, 1999) (Wood and Coppolino, 1972) . Milt Gordon’s lab at the University of Washington in Seattle has studied many aspects of poplars applied to bioremediation. U“transformed Poplar hybrids will metabolize carbon 18 'I’ - D- v V.- tetrachloride (CT), perchloroethylene and trichloroethylene (TCE) . They have been shown to detoxify atrazine and TNT. potential has been shown to be able to metabolize some isomers of polychlorinated biphenyls (PCBs) (Gordon, 2001) . Indeed there is a poplar clone for all purposes and all seasons. POPLARS AS A MODEL SYSTEM FOR WOODY PLANT MODIFICATION The ease with which poplars can be clonally propagated from Cuttings, which is not the case in most woody plants, was a 900d indicator that they might by a likely candidate for e<‘:=‘tse of transformation. That has proven to be the case. Although biolistic projectile bombardment with a gene of interest is possible in many woody plants, transformation L18ing the Agrobacterium tumefaciens system is much preferable. The Agrobacterium mediated gene transfer is the method of choice because of single-copy and single- lOCus insertion compared to other plant transformation teChniques. Although use of Agrobacterium for transformation must be optimized with a number of Va3|:‘iables, the choice of strain, the growth media, inducers and the type of plant material, poplars became the obvious 19 choice for the first woody plant transformation (Hanover, 1987L THE EARLY DAYS OF TRANSGENIC POPLARS The development of a transgenic poplar resistant to the herbicide glyphosate was pioneered in the laboratory of Don Riemenschneider of the Forestry Sciences Laboratory of the USDA in Rhinelander, Wisconsin in collaboration with Calgene, Inc., of Davis, California. By using the Agrobacterium transformation vector, a bacterial aroA gene 1ilhat conferred resistance was expressed in Populus (Fillatti, 1987) . This was a highly desirable modification as plantation losses due to weed competition were enormous. ctJ'RRENT TRENDS FOR THE USE OF TRANSGENIC POPLARS The current research into the use of transformed poplars has focused on two important aspects of poplars, their adaptability to diverse climates, and harsh environments, 20 (often planted on polluted, barren and exposed sites) and their rapid growth for biomass with short rotation. Transformed poplars in bioremediation Genetically modified poplars are being studied in terms of their adaptability to stress situations such as tolerance to heavy metals, ozone, atmospheric H28. (Arisi et al., 1998; Arisi et al., 2000; Herschbach et al., 2000; Koch et a1 - , 2000; Tyystjarvi et al., 1999) Trials are under way with poplar containing the mammalian P450 insert to further understand the mechanisms of detoxification of many harmful Organic pollutants (Ohkawa et al., 1997; Ohkawa et al., 1998L There are many research groups that have sought modifications of poplars for their eventual downstream uses. To achieve this end, there have been studies of E>c>Iblar genetic modifications to render the trees herbicide registant, to improve resistance to insect and fungal pathogens, and to expand their tolerance to environmental ab:iotic stresses. However the largest group of researchers in this field has concentrated their efforts on lignin '“ksdification. 21 Transformed Poplars and Wood Modification As a result of their rapid growth and use for pulpwood, there is great interest in modification of the lignin content in poplars to reduce the cost and environmentally deleterious effects of the pulping process. Any discussion of genetic engineering of poplars must include the current trends in lignin modification. LIGNIN MODIFICATION AND WOODY PLANTS I"1911111 - An Overview Lignin is a complex polymer of phenylpropanoid units mainly d”eposited in plant secondary tissues that contribute to ELIIictions of support and conductivity. It is the second (only to cellulose) most abundant organic polymer on the planet (Zhong et al., 2000) . Lignin is primarily deposited l1'1 secondary cell walls in conductive and support tissues 22 of vascular plants. It is present in some herbaceous plants where its negative effect on the digestive process of ruminants is well characterized. The covalent linkages between lignin and polysaccharides render the cell walls of grasses resistant to digestion (Stone, 1997) . However the high content of lignin in woody tissues used for paper becomes even more undesirable because of the stringent methods used to remove it in the pulping process. Chemical pulping consists of chemical hydrolysis and solubilization Of lignin, by either acid (sulfite) or alkaline (sulfate) pulping while lignin is degraded at very high temperatures and extreme pH. The alkaline pulping or kraft pulping is the most utilized world wide (Baucher et al., 1998) . Genetic manipulation of woody plants for even small amounts of lignin reduction is highly advantageous. Lignin content and composition vary from angiosperm to gymnosperm, from one species to another varying among tree types (ring— p(Drous, and diffuse porous, storied and non-storied earl'lbium, etc.) and even varying seasonally and cieV'elopmentally (Chaffey, 1999b). Characterization of lignin and the process of lignin deposition in woody plants are not well understood. 23 Role of lignin in plant physiology Attempts at lignin modification should include an understanding of the physiological function that lignin has in plants and woody plants in particular. Any modification of lignin must include not only the quantitative results of l ignin produced and pulping characteristics, but also the effects that this modification may have to the plant and any derived wood products. Lignification has played an important role in the adaptation of plants to life on land. Lignin has made possible the development of conductive tissues, E3trengthening them to bear extreme negative pressures while Cc>Ilciucting water. It also assists in maintaining the 1'11’Cirophilic nature of the cell wall. Lignin provides mechanical support strengthening the stem/trunk to uphold the weight of the foliage and canopy and has been suggested to generate an internal strain to reorient the tree to S‘ilz‘ess as a component of compression wood (Timell, 1986) . The lignin polymer provides a barrier of protection against biotic vectors, making the woody stem resistant to decay and can be synthesized de novo in response to wounding or pEathogen infection (Lewis and Yamamoto, 1990). Recent 24 studies confirm that enzymes in the lignin pathway are correlated with biotic and abiotic stresses (Cabane, 1999; Enebak et al., 1997) . These qualities of impermeability and decay resistance are primary factors for the high pollution associated with the pulping process as harsh chemicals are needed to remove the lignin. Lignin analysis - quantity and quality The analytic methods to evaluate lignin composition and cOntent are also problematical (Dean, 1997) . It has been described as a recalcitrant subject with no absolutes for any perspective researcher. Each technique has limitations. Quantitation methods include “Klason lignin” for insoluble (in acid) and Acetyl bromide or Thioglycolic acid for soluble lignins. Characterization of the C20tuposition of lignin includes Nitrobenzene/Cupric oxide degradation and in situ techniques of pyrolysis-GC/MS, I‘l‘L-lc'lear magnetic resonance spectroscopy and others. No Q"~-1:rrent method provides complete or comprehensive lhformation and may be prone to error and false QOnclusions. However improved methods to determine wood 25 quality are being developed as part of the effort to modify lignin content (Tuskan et al., 1999) . L :i. gnin biosynthesis The biosynthesis of lignin is equally not well understood. Generally speaking it begins with the phenylpropanoid pathway, starting with deamination of phenylalanine by phenylalanine ammonia-lyase (PAL) and concluding with the generation of differing amounts of three monomeric S1-112>1;Lnits, hydroxyphenyl (H), guaiacyl (G), and syringyl (S) 1-1ITlit;s differing from each other by their degree of t“etl‘iylation (Whetten et al., 1998). (See Figure 1, page 30 for generally accepted lignin Pathway) . It is stated that the various levels of the three units vary from plant to plant, even within the same plant, from Cell to cell (Chen et al., 1999) . Content and composition of lignin vary developmentally and seasonally as necessity 26 for lignin deposition changes during growth. Storage, transport and synthesis of lignin vary. The biosynthetic pathway itself may vary among different plant families (Baucher et al., 1998) . Attempts at genetic modification of the lignin pathway and the discovery of lignin mutants have resulted in unforeseen diverse and unknown forms of lignin. A loblolly pine ( Pinus taeda) was found to be mutant in the lignin pathway. The xylem is described as red-brown (similar to brown mid— rib mutants) and still maintains vascular function and mechanical support for the tree (Ralph et al., 1997) . However the expression of the gene encoding cinnamyl alcohol dehydrogenase (CAD) is severely reduced and has reduced lignin content (MacKay et al., 1997) . In 1999 the lab of Kazuhiko Fukushima provided evidence of a novel biosynthetic pathway in lignin in differentiating xylem of Magnolia kobus (Chen et al., 1999) . It is not surprising tl'lat a component so essential to plant defense and strength would have alternative and redundant systems to insure the pZ'Z‘voduction of that component. 27 .- I'.‘ .‘ CURRENT TRENDS IN LIGNIN MODIFICATION AND POPLAR Many labs that now study woody plant lignin modification began by looking at herbaceous subjects. The plants of choice have usually been Arabidopsis, Zinnia or Nicotiana (tobacco) (Chaffey, 1999a; Dharmawardhana et al., 1992; Ye et. al., 1994). Arabidopsis as a model for lignification studies continues in the labs of Catherine Lapierre of INRA, Versailles, France (Jounin, 1999). Using Arabidopsis gene arrays and EST collectiong combined with poplar ESTs have become the combined focus in research in Umea, Sweden (Regan, 1999) and in Genome Canada. CLIlrrent research into lignin modification of woody plants, particularly, poplars (and loblolly pine — Sederoff, North Carolina Tree Biotechnology) has concentrated on modification of some of the important and committed steps in the biosynthetic pathway. The review of Wout Boerjan’s g3':‘<>up from Belgium presents a table (17) showing the modification of lignin genes in various plants and results Of those mutations (Baucher et al., 1998) . The article 6‘130 presents the resulting effects in composition and impact on the pulping process. 28 s as; _ ommoosflommflxobm _ \ , \ _ / «We ~§e§u~§ «wage» ~S~§u~§ «we ~E~ou~§ S» ~§-§u~u Kass? 3.3% ssuaxipéum Ibmbssb 8.5.5896. I I I I_ I I I I 4 H200 4 Emu e .5200 e EmU e Sue A See A Sea Ens Emu 933.5 nesgmkaxsgubfim £33m - u~m§ub Al I- stufiasbfi no a 32» 9.33:st is » mamassasuek 29 Figure 1. Legend Figure 1. Monolignol biosynthetic pathway. The enzymes that have been targeted for modification are Phenylalanine ammonia lyase (PAL), Cinnamic Acid 4- Hydroxylase (C4H), Caffeic Acid O—methyltransferase (COMT), Ferulic Acid 5-hydroxylase (FSH), 4- Coumarate:CoA Ligase (4CL), Cinnamyl alcohol dehydrogenase (CAD), and Cinnamoyl-CoA reductase (CCR). The peroxidases and laocases which have been proposed to be the enzymes in the final polymerization step are also being modified. 30 The first modification of the lignin pathway in a woody plant was achieved in 1995 (Vandoorsseleare et al., 1995). Populus tremula x P.alba was modified with a reduction in COMT resulting in a substantial (95%) reduction of enzyme activity. No change was observed in the lignin content, a change was observed in the monomeric composition. The progress in lignin modification till early 1998 was reported in the article from the Boerjan group (Baucher et al., 1998). Since that time numerous groups have studied one or more of the important enzymes in the lignin pathway using transgenic woody plants, in most cases, poplars. 31 CAD: Boudet group, Toulousse, France Bourjan group, Belgium Jouanin group, INRA, Cedex, France COMT Chiang group, Houghton, Michigan Bourjan group, Belgium Lapierre group, Cedex, France Douglas/Ellis group, Vancouver, Canada Ye group, Athens, Georgia FSH Chapple group, West Lafayette, Indiana Jouanin group, INRA, Cedex, France Chiang group, Houghton, Michigan Peroxidases/Laccases Nippon paper industries, Japan Ellis group, Vancouver, Canada Dean group, laccases, Athens, Georgia McDougall group laccases, Dundee, Scotland Table 3. Enzymes in the lignin pathway targeted for genetic modification in woody plants. 32 Global control of lignin biosynthesis - transcription factor, hamsoboxss, and gene silencing cis-acting regulatory elements that are thought to control aspects of lignin formation have been identified in Arabidopsis (Morelli, 1999), poplar (Hertzberg, 1998), loblolly pine (Campbell, 1999), and eucalyptus (Bossinger, 1999). In tobacco an AC-rich motif, PAL box (Ntlml), thought to be important in cis-regulation of phenylalanine biosynthesis was isolated (Kawaoka et al., 2000). Lignin expressing the antisense Ntliml showed a decrease of 70% as compared with control plants. Post transcriptional gene silencing of PAL expression has resulted in striking differences in lignin content and composition (Korth et al., 2001; Reddy et al., 2000). Characterization of these global regulators to modulate downstream expression of lignin genes for the purpose of lignin modification is ongoing. The (at times) heated discussion of the best choice of genes to modify in forest trees to modify lignin composition and content is a long way from reaching resolution. But strides have been made to understand a 33 complex process that is only reaching initial comprehension. Optimism is high that the results so far indicate that the goal is attainable. MODIFIED POPLARS AND THE ENVIRONMENT The potential risks of a long—lived transgenic plantation and its potential impact on the environment is now of primary importance in public perception. Steve Strauss at Oregon State University in Corvallis has ongoing research into prevention of reproduction of such transgenics through the control of flowering. Using the Populus trichocarpa homologue of LEAFY and FLORICAULA from Arabidopsis a gene called PTLF was cloned to examine its expression in a tree species. It would appear that these genes may provide tools for delayed or inhibited flowering in woody plants. (Rottmann et al., 2000) This same group at Oregon State is using population genetics to measure the gene flow from hybrid poplar plantations into surrounding native poplar stands (DiFazio, 34 2001). Hybrid poplars, a result of “classical” breeding programs represent a significant number of short rotation commercial plantations, and have done so for many years. The question is a good parallel to see if a “genetically modified” forest (through selective breeding) can impact a natural population. As ongoing research looks into the effects of transgenic poplars on the environment, it is also beneficial to consider changing poplars to be even more adaptive to adverse environmental factors. Many attempts at poplar modification have directed efforts at resistance to biotic vectors, but little directed at abiotic stresses. Although as a genus they are remarkably versatile in their ability to grow on inhospitable sites, extending their tolerance of poor sites and conditions is equally desirable. The Institute of Plant Sciences and Genetics at The Hebrew University of Jerusalem, Rehovot, Israel has overexpressed a drought/cold/ABA related protein in poplar that shows increased tolerance to salt stress. (Altman, 2001) Open, exposed areas are often available for plantations, poplars are planted, and a situation is prime for failure of the plantation before harvest due to wind stress. 35 WIND STRESS IN POPLARS Why study wind stress in poplars? What does this abiotic stress do to trees, and why is this relevant to the genetics of wood development? And how can studying the genes involved in cambial growth in poplar stems resulting from wind stress contribute to a better understanding of wood formation and tree improvement for field plantations? WIND STRESS AS AN IMPORTANT ABIOTIC ENVIRONMENTAL STRESS IN TREES The effects of wind stress can be influenced by many factors including canopy architecture, planting methods, root structures, soils, morphology and wind velocity (Couts, 1995). The scope of this study refers to the aspect of flexure of the stem of the tree and/or plant. Wind or MP refers to the temporary displacement or sway of a plant stem from the vertical caused by the wind or by other physical or mechanical means. Repeated flexures, either naturally induced by wind or artificially induced by flexing in laboratory experiments characteristically 36 results in a shortening and thickening of the stem, and reduced leaf area. Genetic modification of woody plants and wind stress are topics that are usually not mentioned in the same sentence. In fact wind stress in trees is usually omitted in discussions of abiotic plant stress in general, particularly at the level of gene expression. Chilling Freezing ~\\\‘ ¢ ‘(// Heat Abiotic Stress Flooding T ‘K\\ Drought Salinity Figure 2. Abiotic environmental plant stresses The above diagram is a listing of abiotic stresses on plants (Holmberg and Bulow, 1998). Holmerg does not mention wind stress, nor did Neuman in his description of abiotic stresses on poplars (Neuman, 1996). Wind stress or mechanical perturbation (MP) is frequently overlooked. 37 Plants being stationary objects must provide a defense against wind forces if they are not to blow over or to snap, just as they have developed defenses against other stresses. Although physiological and morphologic effects of the stem flexure (mechanical perturbation - MP) due to wind forces have been well characterized (Telewski, 1995), gene expression has not, with a few exceptions (Janet Braam 1990, l992,l995, 1997, 1998), (Mizoguchi 1996), (Botella 1995, 1996), (Mauch 1997) and (Depege 1997). As wind stress in trees is responsible for the loss of thousands of acres of tree plantations worldwide, poplars, [Harrington, 1993 #268], Pinus sylvestris — in the UK, Pinus radiata in New Zealand (Somerville, 1995), rubber trees in the tropics, it is an abiotic stress worthy of more detailed study (Savill, 1983). Thigmomorphogenesis has been defined (Jaffe, 1973) as a change in growth pattern response or allometry due to touch or flexure. Such flexure will effect transient change in tension and compression of constituent cells within the plane of bending (Biddington, 1986). This is not to be confused with rubbing the plant, vibrating the plant, nor wounding the plant and subsequent breakage of the cell 38 walls. Although these latter stresses may involve some of the same genetic mechanisms of plant defense, we are particularly concerned with those aspects of the wind which result in trees characterized by an increase in stem taper, decreased height and/or increased radial growth and a decrease in leaf area due to flexure of the stem. Staked trees, even with the wind blowing their leaves do not exhibit these physiological changes (Burton, 1973; Holbrook, 1989; Jacobs, 1954), and mechanical bending during dormancy still increases stem diameter (Valinger et al., 1994). A wind stressed tree will be shorter in height, with shorter branches, smaller leaves and increased growth at branch bases, stems and at branch nodes (Telewski, 1995). Table 4 is a proposed model of tree response to wind stress from the time of perception to the time of increased division of the cambium. From initial sensing of the mechanical flexure in the stem a cascade of events is initiated. Within twenty four hours there is increased division of the vascular cambium. In Sweden I observed that the physiological changes in the xylem tissue responding to gravitropic stress in poplar stems were 39 perceptible with microscopic observation three days after induction. WIND OR MECHANICAL PERTURBATION Within first second(E) Within 10-30 min Within 2 hrs (R) Peak with 9 hrs (R) Peak within 15 hrs Within 24 hrs (R) Unknown time (primary stress) FLEXURE OF STEM TISSUE (primary strain) Cytosolic calcium ion accumlutation (H) Decrease in phloem transport (H) Calmodulin transcription (H) Calmodulin synthesis (H) Callose (B—glucan) accumulation (H,C) Ethylene synthesis (H, C) Callose reabsorption (H, C) Increased division of vascular cambium Increased tracheids/Radial file (C, H) Shorter Tracheid length (C, A) Increased Cellulose Microfibril angle in 2ndary cell wall (C) Table 4. Suggested model of tree response, suggested at the cambial level to wind or MP based on woody and herbaceous species (C=conifers, H=herbaceous, A=woody angiosperm) Times are estimates (E), or real (R) recorded periods for responses in woody plants. Telewski, unpublished. 4O Modification of stem taper, additional wood deposition, and changes in lignin content and composition (Pruyn, 1997) (Berlyn, 1979) are direct effects of wind stress. Knowledge of the genetic mechanisms that are involved in changes in stem architecture may be a very important contribution to ongoing research into lignin engineering in woody plants. From the physiological evidence of tree responses to wind as outlined in the proposed model (Table X), gene expression in response to this stress can also be expected. Physiological and biochemical changes that have been experimentally demonstrated will have correlated gene expression. From the table above those genes should be expressed relating to calcium ion channel sensing, calmodulin, changes in hormone levels, and those involved in cambial development and secondary wall deposition, including lignin. Since plant response to wind is an abiotic stress, defense pathways might also be anticipated. Has any such gene expression been found? GENE EXPRESSION IN RESPONSE TO WIND STRESS OR MP 41 Environmental cues are perceived by plant sensory mechanisms, triggering a cascade of internal events to respond to the stress. Wind, which is first perceived as flexure, is known to activate the TOUCH (TCH) genes as characterized by Janet Braam. When she described the touch (TCH) genes that became activated due to touch, wind and water spray, it was also found that TCH gene expression also occurred in response to darkness and temperature shocks (Braam and Davis, 1990). Several studies have discovered genes that might be common to wind stress while studying other stresses such as cold and drought. (Gilmour, 1998; Mizoguchi et al., 1996). Previous molecular studies directed at touch gene expression have been conducted solely on non-woody plants: Arabidopsis thaliana, (Braam and Davis, 1990) (Braam, 1992), mung bean (Vigna radiata)(Botella et al., 1995), wheat (Mauch et al., 1997), and tomato (Depege et al., 1997). In all cases except the mung bean, “touch” was defined as a mechanical flexure (MP) of a selected internode that was moved (or rubbed) back and forth, with hand, glove or glass rod. In the case of wheat, both the flexure and a wind-mimicking fan treatment were used to stress the plants. The mung bean treatment consisted of a 42 torque applied to the leaves by manually bending them downward for a number of repetitions. RELATING GENE EXPRESSION DUE TO WIND STRESS WITH OTHER ABIOTIC STRESSES As cited previously TCH gene expression also occurred in response to darkness and temperature shocks (Braam et al., 1997). Many genes have been shown to be ubiquitous to many abiotic and biotic stress responses as plants arm themselves with defense strategies. Among these are the calcium-channel related genes, implicated as second messengers (Bush, 1995; Haley et al., 1995) and calmodulin genes (Roberts and Harmon, 1992; Sinclair and Trewavas, 1997). Ethylene acts as a signal in many plant processes including transcription of defense genes (Bleecker and Kende, 2000; Ohme-Takagi et al., 2000). Various plant defense genes such as those in the phenylpropanoid pathway, and pathogenesis related (PR) genes (Barin and Zambryski, 1995; Koch et al., 2000; Mauch et al., 1997) also are expressed in response to a variety of stresses. 43 When wind or MP causes a stem flexure, there are changes in the shape and turgor pressure within the cells that is similar to changes in turgor pressure that is observed in dehydration and cold stress. Mechanosensory pathways such as those activated by wind stress have been linked to dehydration and cold stress, (Cowan et al., 1997). Absisic acid (ABA) may regulate gene expression in cold and desiccation tolerance (Chandler and Robertson, 1994) which also relates to turgor sensing mechanisms such as changes in cell shape, dehydrin and dehydration response genes, the late embryogenesis (LEA) protein family. The cold response (COR) genes and in particular a transcription factor CBF controls transcripts that accumulate in response to mechanical stimulation (Gilmour, 1998) Mizoguchi et al. in 1996 report simultaneous induction of three genes in response to cold, wind, and drought stress, two MAPKs (mitogen—activated protein kinases) and an S6 ribosomal protein kinase (Mizoguchi et al., 1996). Recent work of this same lab of Kazuo Shinozaki has monitored expression of 1300 full-length Arabidopsis genes under drought and cold stress using cDNA microarray (Seki et al., 2001). The pattern of gene expression in response to various stresses 44 is complex, has many pathways that are inter-related and involve the same genes or gene families. It is apparent that analysis of wind stress at the molecular level shows some similarity to gene response to other environmental stresses and bears further investigation. The genes described in response to wind or mechanical stress and those mentioned in studies above have been primarily conducted on herbaceous plants. Where are the studies of gene expression of abiotic stresses of woody plants? Again the use of poplar as a model system for such study is advantageous. WIND STRESS AND POPULUS (POPLAR) Poplar is well established as a model angiosperm tree system in molecular studies. The advantages of using poplar as previously described are well—documented, and include small genome size, ease of propagation and transformation and easy extraction of DNA and RNA. For the purposes of studying wind stress in trees as well as cambial development, poplar is a very attractive model. Well-documented field studies have identified wind resistant and wind susceptible clones. This is the same as 45 having Arabidopsis mutants that do not respond to a stress with the same phenotype as the wild type. Mutant Arabidopsis are conserved and valued, mutant trees that are intolerant of certain conditions or with undesirable growth form are usually destroyed. It is fortuitous that these poplar clones have been saved and propagated. A study of the gene expression in the stems of a wind tolerant poplar clone would contribute to the general knowledge of genes involved in modification of stem architecture and growth, give a greater understanding of how the involved genes have a role in stem function, and may provide tools for the better, stronger and more resilient poplar for the future. 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Biomass & Bioenergy, 16, 263- 274. Wood, F.A. and Coppolino, J.B. (1972) The response of 11 hybrid poplar clones to ozone. Phytopathology, 62, 501-502 . Ye, Z., Kneusel, R., Matern, U. and Varner, J. (1994) An Alternative Methylation Pathway in Lignin Biosynthesis in Zinnia. Plant Cell, 6, 1427-1439. Zhong, R., Morrison, W.H., 3rd, Himmelsbach, D.S., Poole, F.L., 2nd and Ye, Z.H. (2000) Essential role of caffeoyl coenzyme A O-methyltransferase in lignin biosynthesis in woody poplar plants [In Process Citation]. Plant Physiol, 124, 563-578. 64 CHAPTER 2 GENE EXPRESSION IN WOOD FORMING TISSUE OF HYBRID POPULUS (POPLAR) ABSTRACT In trees, wood is produced from the vascular cambium. Until recently little was known about the control of cambial differentiation at the molecular level. In this study, hybrid poplar clone 47-174 (Populus deltoides x trichocarpa) was used as a model to examine gene expression in wood forming tissue and as a result of wind stress or mechanical perturbation (MP). Poplar clone 47-174 was stressed with MP to simulate the stem flexure of wind stress. RNA isolated from stressed and non—stressed pOplar stems was examined using differential display of PCR products. One hundred thirty differential display gel bands from stem tissue were cloned and sequenced. Northern analysis and BLAST searches confirmed that more than seventy of these clones represent expressed sequence tags 65 (ESTs) for are poplar genes expressed in wood forming tissue. The evidence to support that this collection of poplar ESTs is derived from genes responding to MP stress in stem tissue is discussed. INTRODUCTION The importance of wood Most woody plants are grown for their stems or trunks, for lumber, for paper pulp and other wood products, while others are grown for fruit or nuts and a few grown for essential oils and secondary metabolites, (citrus, eucalyptus, conifers and the like). Demand for these products continues to grow while land suitable for cultivation of trees shrinks. The acreage dedicated to first growth forests is diminishing and pressures to preserve those remaining are imperative. Demand for forest products has never been greater, thus increasing the need for renewable woody crops. Understanding the mechanisms 66 that contribute to wood formation is necessary as we strive to improve yield and quality of forestry plantations. Wood development and the function of xylem In a review in 1952, Bailey described six stages of wood development. Wood is formed in the stems of plants undergoing secondary growth from the vascular cambial zone during xylogenesis. Cells derived from the cambium initials divide, (zone of division), enlarge, (zone of elongation) and differentiate and mature (zone of maturation). Mature xylem then changes from conductive sapwood, from sapwood to heartwood and to the inner core of heartwood (Bailey, 1952; Larson, 1994; Telewski, 1996). The functions of xylem are diverse. As wood is produced it gives strength and support to the stem that carries a Significant weight of foliage, flowers and eventually fruit. The stem contains conductive tissue and provides 67 strength for the transport of water under extreme negative pressure to the upper most reaches of the plant (Zimmermann et al., 1994). The stem provides protection against a barrage of biotic and abiotic stresses, withstands decay, and reacts and forms wood to reorient the tree with respect to gravity or strengthen it in response to wind flexure. The cell wall of woody plants develops an impervious hydrophobic protective layer, a wall composed of a complex composite of cellulose and lignin. This woody stem that has such an important function for the tree, has also recently become the focus of molecular modifications in attempt to alter its composition. Methods of woody plant modification Traditional breeding programs have either crossed or grafted trees in an effort to increase yield and select for desirable traits, or enhance adaptability to diverse environmental conditions. An example of this is the cultivated tree Gleditsia tricanthos forma inermis, “Sunburst” honey locust. It is a honey locust that has 68 been selected from years of effort to reduce the number of thorns and seed pods normally produced in that species. One can now purchase a grafted cultivar named “Sunburst” locust that has been vegetatively propagated from the original selected tree. It will produce few seed pods and few thorns. As a selection “Sunburst” is commercially very successful. However, the process to develop this tree was time consuming. The advent of molecular technology applied to the modification of woody plants presents an opportunity to shorten the time necessary to introduce or modify desirable traits, increase adaptability and improve wood and fiber quality For example, it is highly beneficial to modify trees to reduce lignin content with the end product being wood fiber for paper. This particular trait has never been a target for traditional breeding methods. However, lower lignin content would facilitate increased efficiency in pulp production, at the same time reducing deleterious pollutants resulting from delignification. Modification of lignin content is one aspect of tree improvement that is desired. Many labs have ongoing research directed toward this end. (Bourjan, Belgium, Jouanin, INRA, Cedex, France, 69 Chiang, Houghton, Michigan Lapierre, Cedex, France, Douglas/Ellis, Vancouver, Canada, Ye, Athens, Georgia and Chapple, West Lafayette, Indiana). Growing plantation trees with increased environmental adaptability, or increased yield without sacrificing tree integrity and wood quality would be equally beneficial. Efforts to modify trees will be aided by a better understanding of gene expression during xylogenesis in the vascular cambial zone of woody plants. We must seek to understand gene function while integrating this with an evaluation of the effects of genetic modification on the whole tree. Gene expression in developing cambium The development of molecular techniques has enabled us to examine wood formation from another perspective. We are :now'able to identify some of the genes that are activated chrring the various phases of xylogenesis. It is possible 70 to discover and characterize those genes and to integrate them with what we know of the anatomical, biochemical and morphological changes during this process. In studies of herbaceous species, Zinnia, Arabidopsis thaliana, and Nicotiana (tobacco) [Dharmawardhana, 1992 #38; Ye, 1994 #169; Chaffey, 1999 #223; Jounin, 1999 #314] some knowledge of xylogenesis has been gained. However in herbaceous plants there is no (or minimal) vascular cambium, and no (or minimal) secondary xylem development. Until recently little has been known about the genetic control of the wood forming process. We may now be able to examine the changes that occur at the most basic cellular levels of xylogenesis in the cambial zone and understand more fully the functions that these genes play in woody plants. Studies are being made world-wide in a number of woody plant species to elucidate the molecular mechanisms of wood formation. One of the most widely studied tree genera is Populus (poplar). Populus (Poplar) as a model system for the application of molecular techniques 71 Poplar has already proven to be a good model system for molecular modification. Poplars are easy to propagate both vegetatively from cuttings, and in tissue culture. They transform easily with Agrobacterium tumefaciens plasmid insertion, their genome size is relatively small (Arumuganathan, 1991), and extraction of DNA is comparatively easy (1997b). In fact, poplar was the first tree to have successfully been genetically modified. A transgenic poplar resistant to the herbicide glyphosate was developed in the laboratory of Don Riemenschneider of the Forestry Sciences Laboratory of the USDA in Rhinelander, Wisconsin in collaboration with Calgene, Inc., of Davis, California. By using the Agrobacterium transformation vector, a bacterial aroA gene that conferred resistance was expressed in Populus (Fillatti, 1987). This was a highly desirable modification as plantation losses due to weed competition were enormous. Since that first engineered poplar, there have been many genetic modifications proposed. Efforts at altering genes in poplars have been attempted for the purposes of increased herbicide tolerance, resistance to fungal and insect vectors, and augmented adaptability to environmental 72 sites which are unsuitable for other purposes, reclaimed from mining, polluted, or in extreme climatic situations. It is the desire to cultivate poplars for plantation harvesting in land that might be unsuited for anything else that has led in part to the examination of gene expression due to wind stress. Wind stress and mechanical flexure Chilling ] Freezing ~\\\‘ l ‘(// Heat Abiotic Stress Flooding )//)' ‘T ‘K\\ Drought Salinity Figure 3. Plant abiotic environmental stresses The above diagram is a listing of abiotic stresses on plants (Holmberg and Bulow, 1998). Holmerg does not mention wind stress, nor did Neuman in his description of abiotic 73 stresses on poplars (Neuman, 1996). Wind stress is at least as prevalent as any of those mentioned in Figure 1. and is frequently overlooked (Stanton, 1997). Plants being stationary must provide a defense against wind forces if they are not to blow over or to snap just as they have developed defenses against other stresses. Although physiological and morphologic effects of the stem flexure (MP) due to wind forces have been well characterized (Pruyn et al., 2000; Telewski, 1995), gene expression has not. There are some studies of gene expression due to touch, or flexure, notably those studies of Janet Braam (Braam, 1992; Braam and Davis, 1990; Braam et al., 1997). However there have been no studies at the molecular level of gene expression due to wind or MP in plants with secondary growth. Wind stress in trees is responsible for the loss of thousands of acres of tree plantations worldwide (poplars, Harrington and DeBell [Harrington, 1993 #268], Pinus sydvestris in the UK (Quine, 1995), Pinus radiata in New Zealand (Somerville, 1995) rubber trees in the tropics (Clement-Demange and Doumbia, 1995). It is an abiotic stress worthy of more detailed study (Couts, 1995). 74 ‘Modification of stem taper and additional wood deposition including lignin (Berlyn, 1979; Pruyn, 1997; Pruyn et al., 2000) are direct effects of wind stress. Knowledge of the genetic mechanisms that are involved in changes in tree stems in response to mechanical flexure may be a very important contribution to ongoing research into modification of woody plants. Gene expression and wood development Since additional wood deposition is one of the morphological responses to mechanical flexure of a woody stem, genes that are specifically expressed in developing cambium and xylem are expected. One source for identifying these genes would be EST databases being generated from poplar cambium and developing xylem tissues (Sterky et al., 1998). Some of these ESTs are now available on the web at PopulusDBase. Several groups are sequencing EST collections from tension wood in poplar. Tension wood is that which is formed on the upper side of stems and branches in angiosperms due to gravitropic displacement. Additional tissue specific libraries from the Swedish 75 consortium will be made available later this summer (Sandberg, 2001). It should be mentioned that databases are being established for gymnosperm wood formation as well. Collections of Pinus taeda (loblolly pine) ESTs are being made at North Carolina under the direction of the Forest Biotechnology Group at the University of North Carolina in the lab of Ron Sederoff. Recently the Canadian government has instituted funding for Genome Canada, the forestry division in collaboration with the University of British Columbia. Genome Canada will be creating EST databases of Pseudotsuga menziesii and Abies grandis. Gene expression and mechanosensory perception Previous molecular studies of touch or movement on non- woody plants are: Arabidopsis thaliana, (Braam 1990, 1992) mung bean (Vigna radiata), (Botella 1996) wheat (Mauch 1997), and tomato (Depege et al., 1997). In all cases except the mung bean, “touch” was defined as a mechanical 76 flexure (MP) of a selected internode that was moved (or rubbed) back and forth, with hand, glove or glass rod. In the case of wheat, both the flexure and a wind-mimicking fan treatment were used to stress the plants. The treatment applied to mung bean consisted of a torque applied to the leaves by manually bending them downward for a number of repetitions. The genes that have been associated with touch (wind and/or MP) are: the Touch (TCH) genes of Janet Braam, calmodulin or calmodulin-like genes (TCH 1,2,3), and xyloglucan endotransglycosylase (XET, TCH 4). XET has been shown to be related to cell wall deposition, which is to be expected in a stem that is depositing new cells at the point of flexure. Botella isolated, l-aminocyclopropane-l- carboxylic acid synthase (ACC) synthase involved in ethylene biosynthesis and a calcium dependent protein kinase due to mechanical strain (Botella et al., 1995). Mizoguchi found MAPKKK (mitogen-activated protein kinase kinase kinase) (Mizoguchi et al., 1996) activated by touch, cold and water stress in Arabidopsis while Mauch described a lipoxygenase (LOX) induced by touch (MP), wind and wounding in wheat (Mauch et al., 1997). LOX is part of a fanuly of enzymes implicated in mobilization of lipid 77 reserves in wound responses. The calcium channel related calmodulins, some protein kinases and the MAPKK from other plants have been implicated as early responses to a number of stresses in plant signal transduction pathways (Gilmour, 1998). The discovery of genes that are expressed due to wind or mechanical flexure has occurred exclusively in herbaceous plants. There have been no molecular studies of MP stress conducted on trees and it is the impact of wind on tree plantations that is increasingly important. Purpose and experimental design for this study The worldwide use of poplar for biomass and its planting on marginal soils in windswept sites (Zsuffa, 1996) creates an imperative for understanding the response of various poplar clones to wind stress. I have investigated gene expression in the stems of hybrid poplar and those expressed in response to mechanical perturbation (MP) during cambial development. Although 78 wind stress is responsible for the loss of thousands of acres of trees worldwide (Quine, 1995), very little is known of this abiotic stress at the molecular level in woody plants. It was anticipated that I would find genes with similarity to some of the above-described genes, as well as some that would be novel. This is the first systematic exploration of this type of stress in stem tissue of a woody plant. Design of experiments The advantages of using poplar as a model system for study of this particular abiotic stress include well-documented field studies which identify wind resistant and wind susceptible clones (Harrington, 1996) and the plantation failures at James River Corporation, Lower Columbia River Fiber Farm (Kaiser, 1997). These clones were available for study in laboratory conditions. To further elucidate molecular mechanisms of plant response to wind or mechanical perturbation (MP), I have chosen hybrid poplar (Populus deltoides x trichocarpa) as a model 79 system. The cross resulting from these two species has produced many clones that have been used extensively in field plantations. Heterosis often results in many of the progeny of the cross possessing hybrid vigor (Stettler, 1996). In this instance the word “clone” pertains to vegetatively reproduced and named cultivated trees that result from selection of the F1 seedlings. In this study I have stress tested a wind resistant clone, poplar hybrid clone 47-174 with mechanical perturbation. My goal was to use the technique of differential display of PCR products to make a comparison of the gene expression of the stressed trees to those of unstressed control trees. To this end I have generated a collection of cDNAs from the mechanically perturbed poplar stems which represent genes expressed in the cambium and developing xylem, including those genes specifically related to the environmental Stress . MATERIALS AND METHODS 80 PLANT MATERIAL AND GROWTH CONDITIONS Poplar hybrid clone 47-174 designated as wind resistant (Harrington, 1996) was received from James River Corporation. Cuttings were rooted in woody plant cutting mix at Beaumont Nursery, MSU and kept under mist until roots were established, then transplanted into one gallon pots. Plants were moved to an environmentally controlled room, as free as possible of biotic and abiotic stresses. Plants were grown under Agro Grow High Sodium Lights (18 inches above the final height of the trees) with 16 hour days and fertilized with Peters 20-20-20 at 100 parts per million. Temperature during the 16 hour days was 26C and 21C during dark hours. One central leader was established, trees were staked and no pruning nor movement of stems or foliage was permitted for one week prior to testing. MECHANICAL PERTURBATION AND HARVESTING One group of 4 trees of clone 47-174 was set to one side of the bench when the plants were moved into the testing room, 81 my: .6. .Y; cc». . ’7“. .-‘v ~u~ G ..‘u V .... u. hr;- .v *r. uh, . 5" In- 6.“ "u u ‘0 ‘30 n 'n. ‘J. 4 . .- ‘M‘. care was taken not to disturb this control set in subsequent watering. An additional group of 16 to 20 trees, specified for flexure tests was also moved into the testing room. The room was maintained as free as possible from biotic or abiotic stress. The trees were never moved, and great care was taken not to move the leaves or stems while watering. One week prior to harvest, the second group of 47-174s were pre-conditioned with 30 flexures of the stem at a selected internode above a mature leaf (Larson, 1994). The stems were displaced 60 degrees from the vertical in both directions. This was done at the same time every day for seven consecutive days. On the eighth day of testing, several control trees were stripped of leaves and stem segments frozen in liquid nitrogen. The stressed trees were given one final treatment and then a time course of stem tissue of all trees was harvested at T = 0, 1, 2, 4, 8, 12 and 24 hours after the final stress. Two control trees, never having received flexures were harvested at time 0 and 24 hours after initial harvest. Stem segments above, below, and including the node of stress were frozen in liquid nitrogen and stored at —80 degrees C. These experiments were replicated eight times and the stem tissues for the same time points were pooled. Different plant tissues were harvested and frozen in liquid 82 nitrogen: stems, leaves, meristems, roots, internode of stress, internode above and below stress. RNA ISOLATION AND DIFFERENTIAL DISPLAY RNA EXTRACTION METHODS The method used to extract RNA was modified a number of times throughout the experimentation. The reason for these changes was the need to extract sufficient quality and quantity RNA for use in northern analysis. Extraction of RNA from leaf tissue of poplars has been demonstrated to be comparatively easy, whereas extraction of RNA from woody stems has not. There is little living tissue, there is an abundance of cellulose, polysaccharides and fibers as well as other metabolites rendering extraction difficult. RNA EXTRACTION METHODS 83 The following is a list of the different methods that were evaluated to extract RNA, either total or messenger RNA, from the poplar stem sections. 1) Current Protocols - Phenol/SDS extraction, Lithium chloride (1997a) 2) Molecular cloning, A Laboratory Manual (1989) 3) Current Protocols — Guanidine thiocyanate (1997a) 4) Hot phenol (1989) as modified by Rujin Chen 11/93 5) RNAgents Total Isolation System, Promega 6) TRIzol Reagent - GIBCO BRL 7) Rneasy Total RNA Extraction kit — Qiagen 8) mRNA Isolation, Dynabeads, Dynal Inc. 9) Boiling phenol (DeVries, 1988) 10) RNA purification from woody branches and needles of spruce (Wang, 2000) 11) RNA purification from mature conifer needles and phloem tissue (Alosi, 2000) 12) RNA purification from Poplar stem tissue, modification of #10 and #11 - aepjones RNA extraction from.pop1ar stem - Protocol #12 84 Five grams of poplar stems were ground in a coffee bean grinder that was RNAse treated, and rinsed with DEPC water. 25 mls of extraction buffer (#10 above) was put into a RNAse treated mortar and pestle. The ground stem powder was added and thoroughly homogenized. This was frozen at —80C in a weigh boat. Removed from the freezer, this weigh boat was floated in a 37°C water bath and when melted transferred to an OakRidge tube to which 5 gm of 8.5M KoAC was added. The protocol continues as in #10 above with the exception that all spins were done in an RCSB Sorvall centrifuge with a 8834 rotor at 10,000 rpms. Lithium chloride precipitation was done overnight, the ethanol precipitation on the second day was also left overnight at -20C. On the third day the resulting pellet was resuspended in 200 pl of DEPC water, the OD read on a spectrophotometer, and then frozen at —80. The modifications combine the triple detergents (cationic, anionic, and neutral) of protocol #10 with the high molarity potassium acetate precipitation of protocol #11. In addition the high speed spins in OakRidge, Saarstedt threaded closure, and Corex tubes done in.a Sorvall RCSB centrifuge were able to guarantee a 85 pellet in each step, while discarding the unwanted cell components. The hot phenol method (#4) was the method used for RNA extraction for the differential display experiments. Differential display is a PCR based method and the RNA extracted with the hot phenol method was of sufficiently good quality to successfully execute the display of PCR products. Northern analysis and hybridization using the hot phenol method proved inadequate. The recently published protocols #10 and #11 and their subsequent modifications resulted in better quality RNA and was used in hybridizations after this time period. After RNA extraction using any of the above mentioned protocols the product was quantified on a spectrophotometer, run on denaturing formaldehyde gels and probed to evaluate quality and quantity. DIFFERENTIAL DISPLAY OF PCR PRODUCTS Tmle technique of differential display of PCR products was developed in 1992 by Liang and Pardee to visually provide a 86 side-by-side display of gene expression of the same organism under two different conditions (Liang et al., 1993; Liang and Pardee, 1992). RNA from stem tissue of mechanically stressed and unstressed hybrid poplar stems was extracted using the hot phenol method. After quantification by spectrophotometer and verification of the quality of RNA by separation on a denaturing formaldehyde gel, the RNA was DNAse I (BMB) treated to remove any residual DNA. The RNA was then reverse transcribed using MMLV Reverse Transcriptase and the cDNA again quantified. The Reverse Transcriptase, the primers and other components of the differential display reaction were obtained from GeneHunter Corporation. 0.2 pg of the DNAse treated total RNA was reverse transcribed using primers with oligo—de with an anchor of G, C, or A to obtain single strand DNA. The products of these three reactions were then used in a second PCR reaction using the same oligo-dT primer in the presence of a second 10mer arbitrary primer. Thirty-two different arbitrary primers (listed in Appendix B) from Gene Hunter Corporation were ‘used to cover an estimated 60 percent of the eukaryotic rmessages (Liang, 1996). The PCR reaction incorporated‘gP CflkTP into the final double stranded cDNAs. These products 87 were visualized on a 6% denaturing polyacrylamide sequencing gel. Control samples which used no reverse transcriptase in the first PCR were also amplified in separate reactions as negative controls; while duplicate samples of the Control unstressed, 2 hour, 4 hour, and 12 hour stressed samples were also amplified and visualized. After gel electrophoresis the polyacrylamide gel was transferred onto Whatman #3 paper, dried for one hour on a gel dryer and the resulting gel and paper were exposed to autoradiograph film overnight. After development of the x- ray film the side-by-side display of control tissue with the stressed samples was possible. Bands that showed a difference in the 2 h, 4h, 8h,and 12h samples from the two control unstressed lanes were marked and then excised through the x-ray film with an Exacto knife cutting the dried acrylamide and filter paper. To extract the cDNAs, the small dried excised slices were resuspended in 100 “L water, soaked for ten minutes and boiled for 15 minutes. After spinning for two minutes to Collect condensation and pellet the gel and paper, the Snipernatant was transferred to a new microfuge tube. cDNA ‘was then precipitated with louL 3M sodium acetate, SuL 88 glycogen (10mg/mL) and 450uL of 100% ethanol overnight and spun in a microfuge for thirty minutes in the cold room. This was resuspended in IOuL of water. CLONING AND SEQUENCING OF DIFFERENTIAL DISPLAY BANDS One hundred forty-five cDNA bands were excised from the differential display gel. 140 were successfully reamplified and were cloned using TA vector cloning. This method takes advantage of the single base A overhang generated by most DNA polymerases in the PCR reaction. By using a vector that has been generated with a T overhang, the PCR product is ligated into the cloning vector (Hadjeb, 1996) and subsequently transformed using Rubidium chloride prepared DHSa E. coli competent cells (Hanahan, 1993). Twenty-four colonies of DH5a E. coli were selected on LB/Ampicillin plates using blue/white screening for selection. These were replicated in microtiter plates and Eitored at —80°C for future screening (over 3500 colonies in Chlplicate). Two clones of each of 140 bands were grown in 89 liquid LB/Amp culture and plasmid DNA was isolated from the bacteria. After verification of the correct size insert with PCR evaluation and BSSHI enzymatic digestion, one copy of each of the 140 remaining cDNAs were prepared for sequencing. Applied Biosystems (ABI) Prism“' Dye Terminator reactions were performed in an MJResearch thermal cycler, cleaned with BIORAD Corporation spin columns and sequenced using an ABI 373 sequencing machine. REVERSE NORTHERNS To elucidate which of the cloned differential display bands was expressed due to MP, the 144 PCR products were dot blotted in three concentrations on duplicate sets of membranes. Restriction digests of the plasmids containing the cloned PCRs were also blotted onto duplicate filters. The RNA from the differential display experiments was reverse transcribed while incorporating a radioactive label. This was used to probe the dot blots and the restriction digest filters. Autoradiography and scanning 'with a Phosphorimager (Molecular Dynamics) were used to evaluate results. 90 NORTHERN ANALYS I S Following RNA extraction (using hot phenol (Method #4) for all gels prior to June 2000, and using the Method #12 protocol subsequently), 20 micrograms of total RNA was run on a denaturing formaldehyde gel. A control sample of RNA from unstressed stem tissue was included with time points as in the differential display, 2, 4, 8 h. The RNA gel was transferred to Millipore ImmobilonW-N transfer membrane using with 10 X SSC (1989). Following overnight transfer, the filter was crosslinked using a Strategene Cross Linker. Probes of 70 cDNAs from PCR products from the cloned differential display bands were made using P32. Prehybridization and hybridization solutions contained Dextran sulfate and Heparin to increase incorporation (Singh, 1984). Hybridized filters were exposed to autoradiograph film (Kodak, Rochester, N.Y.) overnight and subsequently developed. Some filters were re—exposed to x- ray film for one week prior to development. 91 DATABASE SEARCHES Three internet web servers with search engines were used for data analysis: The Arabidopsis Information Resource (TAIR), a publicly available website was the primary search engine used. TAIR BLAST search was done using BLASTx against all protein sequences from the Arabidopsis Genome Initiative (AGI)and total genome protein dataset. TAIR Blastn was used to search again the all higher plant (Viridiplantae) sequence database. The Finch (Geospiza Corp) server is available by assigned password from the Molecular Highthroughput Array Facility at Michigan State University. Using the Finch server, 132 sequences were “batch blasted” periodically using the nucleotide BLAST and protein BLAST default algorithms against various NCBI BLAST databases and dBest. PopulusDB, a Populus tremula x tremuloides genomic sequence database from Sweden was searched one time for all sequence homologies. Last updated in February 1999, the database contains 5,692 EST’s, 4,809 cambium EST’s and 883 xylem EST’s with average lengths of 440 nucleotides. This database has the capability of performing only nucleotide searches. 92 PHYLOGENETIC ANALYSIS AND BOX SHADE ALIGNMENT BioNavigator, a web based bioinformatics program from Entigen, Corporation, California was used for protein sequence analysis. Proteins from database searches were imported into BioNavigator. Protein sets were created and then aligned using ClustalW(Fast) GCG. Pretty Box and Boxshade were used to show protein alignment in specific areas . BioNavigator Macro #26373 was used to create a phylogenetic tree for B-glucosidases. Protein sets were aligned in ClustalW (Fast), then evaluated in ProtDist as a distance measure of relatedness. The Neighbor Joining algorithm then established the phylogeny. Finally DrawGram graphically depicted the relationship of the proteins in a phylogentic tree. 93 RESULTS AND DISCUSSION RESULTS FROM DIFFERENTIAL DISPLAY 141 of 144 excised bands successfully cloned Total RNA isolated from stems subjected to mechanical flexures was compared to unstressed control 47-174 poplar trees using differential display. 144 bands that appeared to be differentially expressed were excised. Based on the autoradiographs only bands that were visually of greater intensity than the two control lanes, or were not present in the control lanes were selected. In addition, these bands were present in two or more of the four time points (2,4,8,12 hrs). 141 of the 144 were successfully reamplified and subsequently cloned into BlueScriptII. One colony of each of the 141 cloned PCR products was selected (after verification for correct size) and sequenced. SEQUENCING RESULTS 94 129 Mechanically perturbed/wind induced (Wimps) sequenced Of 141 cDNA clones that were sequenced, thirteen produced no signal or unreadable sequence. These thirteen were submitted for repeat sequencing. Of the thirteen that were resequenced only the sequence of Wimp 53 produced readable good quality sequence. Eleven other sequences were of poor quality, however were still used for homology searches. One hundred sequences with less than 2% ambiguity, ranging in size from 200 to 450 bases, will be submitted to GenBank. They are named Wimps, an acronym for WInd or Mechanically Perturbed poplar expressed sequence tags (ESTS) . RESULTS FROM DATABASE SEARCHES Putative identity of genes identified by differential display of poplar clone 47-174 'Lable 1 is a presentation of information about 65 ESTs from poplar clone 47-174 which had significant BLASTx or DBest 95 similarities found from database searches. The number of the Wimp EST is in the column that begins with the two Populus Actin clones and is labeled WIMP#, followed by the sequence length in the adjoining column. The BLASTX score from the TAIR search against the Arabidopsis genome initiative (AGI) and Total Genome database from their web site is given in order of probability score. For those twenty-nine with BLASTx hits whose expectation values are <1.00E-4, a putative identity is given. Of these twenty- nine, only 5 had homologies to ESTs from the Populus database. When homologous Populus ESTs and their corresponding WIMPs are compared to the non-redundant protein database using BLASTx, the high hits are a match. This would indicate not only that the Populus ESTs and their corresponding WIMPs are indeed matches, but also that the results are consistent. Putative identity of those Populus ESTs was the same as those of the putative identity for those Wimps. Results of northern hybridization of probes made from those Wimps with high BLASTx scores are listed in the last column. “C” indicates that the hybridization signal was constitutive; “N” indicates there was no apparent hybridization and “2 to 4” represents the up regulation of 96 Wimps #59 B-Glucosidase and #68 Dehydration response protein. If nothing is listed in this column, no northern hybridization was done. Other Wimps, (#31-36 in numerical order) had significant (<5.00E-7) homologies to sequences from the PopulusDBase and the putative identities listed for #3 31-36 are for the PopulusDbase sequence. The PopulusDBase information gives no 3’PolyA terminus for any of their sequences. On pages thirty-seven and thirty-eight are listed those Wimp #s that had high sequence similiarity to sequences from dbEST, a database of expressed sequence tags. These Wimps had no significant hits with BLASTx or other database searches. Database searches reveal some Wimps related to plant defense response Grey-shaded areas indicate database hits that are similar to stress induced or defense genes, or described in MIPS as pertaining to functional categories that relate to plant stress response. For example Wimp68, the putative dehydration response protein showed highest similarity to 97 Arabidopsis thaliana dehydration induced protein RD22. The second grey-shaded group is the WIMPS with strong sequence similarity to ESTs from stress induced plant libraries. 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Many of the Wimps are short sequences, average length of 240 nucleotides, which limits success in homology searches and because of the use of polyA primer for PCR, the sequences are largely derived from the 3’ end of the mRNA. The average length for Wimps that resulted in high BLASTX similarities is 240 bases (80 amino acids in translation). NORTHERN ANALYSIS 65% of Wimp probes hybridize to poplar RNA 103 The most promising cDNAs, those with BLASTs homologies, 3.00e-O4 and lower, or those visualized with reverse northern screening of dot blots of PCR products or of filters with digested plasmids containing the clones, were used to make individual probes for hybridization to blots of stem RNA. PCR products from seventy cloned bands were used to make probes which were used individually to hybridize blots with immobilized RNA from Populus of four to eight different time points and control RNA from unstressed poplar stems. Forty-five of the seventy autoradiographs showed hybridization. Only two of the forty-five had apparent two to four-fold differences in gene expression, Wimps 59 and 68. The other forty-three were apparent constitutive messages in the poplar stems. Hybridization signal varied a great deal from very low to adequate, although incorporation of radioactivity in probe labeling was consistent. Hybridization to a consitituve probe, Actin was also consistent and hybridized strongly to all filters evenly in the control and stressed samples. A number of different strategies was used to increase Northern blot signals. Different pre- and post- hybridization washes were evaluated, varying quantities of 104 RNA on the filters, mRNA on the filters, leaving the film exposed for long periods (one week) and evaluation of results with a PhosphorImager (Molecular Dynamics). Experiments to improve the quality of the RNA continued for three years and eventually led to significant improvement in hybridization results. Putative identity by BLASTx searches of 14 Wimps with hybridization to northerns Putative identity Accession # of Wimp# BLAST 1: score similar protein SARI/GTP binding protein At 4902080 119 6 . 008-34 Aminopeptidase At 1963770 36 1. 003-30 Putative esterase At 2941530 79 1 . 008-16 Hydromethyltransferase At 4913 930 136 2 . 008-11 SINA (seven in absentia) At 5953360 85 4 . DOE-11 (developmental protein) Beta-glucosidase At 3918080 59 5.00-9 S-receptor kinase At 5903700 95 4 . 008-08 Aquaporin At 2616850 128 6.003-08 Ascorbate peroxidase At 1977490 92 7 . 00-08 Selenium binding protein At 4914030 96 3 . 003-06 Kinesin At 3944730 6 1. 003-05 Dehydration induced protein At 5925610 68 8 . 003-05 Thaumatin/Osmotin At 2928790 77 1.008-03 608 Ribosomal protein 6116951 109 4.003-41 Table 6. similarity to proteins in GenBank 105 Wimps that hybridized to poplar RNA with DATABASE SEARCHES SUPPORT HYPOTHESIS THAT WIMPS ARE EXPRESSED DUE TO STRESS RESPONSE Thirty of the one hundred thirty Wimp EST sequences had BLASTx ((probability scores of >3e-O4) similarity to known proteins. However of these thirty, only five were found in the poplar EST collection from Sweden. This poplar EST collection was made from sequences randomly amplified using vector primers. As mentioned previously, Wimps are not random, they were cloned from the 3’ polyA end of each message and they are directional, and cloned from PCRs from stressed tissue. In addition to the five Wimps that amino acid similarity to known proteins and in the PopulusDB, a further five Wimps had high homologies in the PopulusDB, but not in other public databases. An additional release of 4,000 poplar sequences from a leaf library were searched using NCBI dbEST. No further Wimps were found in the new release as of July 1, 2001, so only ten Wimps were homologous to existing poplar sequences. 106 Lack of similarity to the Populus database The two libraries used to construct the poplar database (PopulusDBase) were made from developing cambium and developing xylem. These poplars were not induced by any stress. The poplar library of 5,692 ESTs represents a total of 3,719 unique transcripts (Sterky et al., 1998). The average size of their cloned messages was estimated at 1.2 kb. The readable sequence generated from vector primer (averaging 400 base pairs) from the library was non- directional and occurred randomly throughout the 1.2 kb region. Therefore there exist 800 possible placements for the 400 base pair sequence (within the 1.2 kb clone). 1; the poplar ESTs overlap the Wimps (average 250 bp) by 50 bases at the 3’ end of their 1200 base pair average message then that overlap would give a significant BLAST score. By chance only 200 out of 800 positions (1/4) would result in a significant overlap. Twenty—five percent of the 130 Wimps or 33 Wigps should be found in the PopulusDBase. 107 The fact that only ten Wimps were found to have high homologies in the Populus database would support a hypothesis that these two collections of ESTS, those from Sweden and the Wimps were from RNA populations derived from different methods and from tissue harvested under different plant conditions. Given that conclusion, it is not surprising that only ten of the 132 Wimps were found in the poplar database. The Wimps were cloned from trees that were mechanically stressed. The two Wimps that were found to have two to four-fold up regulation of expression were not found in the Swedish database.. Wimps with similarity to stress induced proteins Although function of the 37 Wimps that had high BLASTX scores has not been verified, the profile of the putative identities suggests some of them are stress related proteins (Kasuga et al., 1999; Seki et al., 2001). Wimp 92, ascorbate peroxidase, Wimp 68, dehydration response protein, Wimp 136, hydroxymethyl-transferase, Wimp 62, subtilisin, Wimp 77, thaumatin/osmotin, and Wimp 59 B- 108 glucosidase represent genes which are stress related. The above-mentioned proteins have been characterized in previous studies (Kasuga, etc. mentioned above) as plant defense, or stress response genes. The dehydration response protein and osmotin are related to changes in cell tension in response to cold acclimation and dehydration. (citations) The changes in cell wall shape during mechanical flexure, alternating between tension and expansion as the stem is flexed, could suggest a similar type of stress response as those of cold, salt and dehydration. All four stresses result in changes in turgor pressure and cell-wall changes. Over twenty Wimps (in addition to the 37 mentioned above with BLASTX protein similarity) had high hits in dbEST datasets from libraries created from stress induced plants: drought, bacterial pathogen, cold or salt stress (see Table 1). Although this is not conclusive evidence that the Wimps are expressed due to MP, it would support the hypothesis that genes expressed due to mechanical perturbation could be similar to those genes found in response to cold, drought, or salt stress where there is also a change in turgor pressure within the plant cells. It also supports the hypothesis that mechanical 109 perturbation is an abiotic stress of plants that induces a number of stress response genes. Although the data from the northerns would indicate that the Wimp ESTs are largely constitutive, the Wimps were cloned from PCR products from poplars under stress conditions. From the database searches there is an abundance of representation of genes whose function is stress related. Wimps not found in the developing cambium.library Further evidence that the Wimps represent a collection of stress induced CDNAs comes from results of screening of the Swedish library. A library screen of six different Wimps using the Swedish developing cambium library (from Magnus Herzberg, Department of Forest Genetics and Plant Physiology, Umea, Sweden) failed to hybridize to plaques, while Wimp 128, putative aquaporin, a highly expressed constitutive gene did yield colonies from the Swedish library. 110 In summary, of 130 Wimps, greater than half are putatively expressed in developing xylem and phloem and putative identity of these cDNAs suggests that a number are implicated in plant stress response pathways. WIMPS 0F PARTICULAR INTEREST Several Wimps had similarity to proteins that could be of particular interest. Two of these had two- to four-fold upregulation in repeated Northern analysis, Wimp 59, B- glucosidase, and Wimp 68, dehydration response protein, (Figures 4 and 5). Figure 4. Wimp 59 B—glucosidase: The photo shows northern hybridization of Wimp 59 to Poplar mRNA. Lanes are control, 0, 2,4,8, and 12 hours after MP. 111 ,m Ethidium bromide- Normalized signals for Wirrp 68 Relative Intensity Time Figure 5. Wimp 68 dehydration response protein: The top photo shows northern hybridization of Wimp 68 to total Poplar total RNA. Lanes are control, 4, 8 and 12 hours after MP. The bottom photo shows the Poplar total RNA gel (used to transfer) stained with ethidium bromide. The graph shows relative intensity of Wimp 68 hybridization after normalization of the ethidium bromide stained gel. 112 LIGNIN BIOSYNTHESIS AND WIMPS 59 AND 62 Two Wimps that could be involved in lignin precursor storage and polymerization are Wimp 59 — B-glucosidase and Wimp 62, Subtilisin. Figure 6 indicates proposed pathways for the possible transfer, storage and polymerization of the monolignol alcohols (Whetten et al., 1998). These compounds are volatile, they degrade quickly and are toxic to the plant. They must be polymerized rapidly, in other words there must be de novo synthesis as required or they must be converted to glucosides and stored. Although this has been proven in the case of conifers (Dharmawardhana et al., 1995) there is little evidence to support this pathway in angiosperms. Noritsugu Terashima has studied the behaviour of monolignol glucosides in Magnolia and Ginkgo lignin, but no B-glucosidase has been cloned specific to this pathway (Matsui et al., 1994). There is also a lack of knowledge of the polymerization process in angiosperms or gymnosperms. The research of Gordon McDougall at the University of Dundee has focused on the enzymes involved in lignin polymerization, particularly 113 laccases (Richardson, 2000; Richardson et al., 1997). An oxidase found in his lab contained two polypeptides responsible for the oxidase activity, the larger of the two was homologous to a number of plant subtilisin-like serine proteinases. Wimp 62 shares homology with this group. . glucosyl transferases Symplastic ,' Storage cinnamyl ‘e (vacuole?) alcohols B glucosidase Transport? Transport? L . Apoplastic p glucosidase Apoplastic cinnamyl .{. cinnamyl alcohols glucosides 1accase(s)? peroxidase(s)? V’ Lignin polymerization Figure 6. Different storage, transport and deglycosylation pathways for monolignol alcohols (Whetton and Sederoff, 1995) 114 WIMP 59 - POSSIBLE ROLE IN LIGNIN PATHWAY After the two- to four-fold up regulation seen on the autoradiograph of Wimp 59, numerous attempts were made to obtain a full length clone. They were not successful. However ClustalW alignment, BoxShade, and phylogenetic analysis of plant B-glucosidases reveal that Wimp 59 is different from other Populus B-glucosidases found at present, either in Sweden or in British Columbia in the lab of Brian Ellis (Figures 7 and 8). It is more closely related to those cloned from HOrdeum vulgare (barley) and Oryza sativa (rice). B—glucosidases are a large family of enzymes that catalyze the hydrolysis of glycosidic linkages and are found in plants, fungi, animals, and bacteria (Esen, 1993). Swiss—Prot lists one hundred eighty two 8- glucosidases, while a BLINKS (Blast Links) search revealed 32 bacterial, 63 metazoan, 5 fungal and 100 plant 8- glucosidases. Plant B—glucosidases have been studied in many metabolic events, particularly in defense against pathogens. However the function of the many plant 8- glucosidases has not been well studied. Wimp 59 is certainly a member of this large family, it is eXpressed due to mechanical flexure in wood forming tissue, and it does not have homology to any poplar B-glucosidases 115 It is a good candidate for further found previously. seen mucwmmummn HUmD HmHQOm ob poms .mmspflmmu pm>ummcoo >H£mfln mo mmmhm monocmp mono pmpmnmuxmnm mnfi .Hmoz um wmmnmnmo Ohm IHCI ucmpadpmhncos one Eon“ mums mmocmdqmm CHmDOHQ Hmnuo Had .Iumbv mHHHm cmflum mo and mnu Eonu mocmsqmm mmmpflmoodam-m memHHQSQCS Cm .mscflEHmu wxonnmo msu Mo ucmficmfiam one mumumcmm mm: IUUUIIumMmV gamumSHU .mmmmpflmooSHmum Mo ucmEsmHH¢ .6 msdmflm uamrzmmAAxxmzwfimZmemzdIZHND>W>HUmmHmHm uuuuuuuuuuu mHmmWMMJhMMhBEfimZMmmquwzzwfl>whAwwm>UNO¢ZZmUZQAAm3 In:uIIMAEQMmBN4m¢MQZKMQBuZmQ>N>H0hMwBVUmazmmZQaqu ummfixmmdzzxm3fldmflxmwm¥qhuZmQ>M>meméhwwamgmmZqumz mUO mz¢ uQmOAmomm30dmAMmWMMODmHm0>mwdwmmHfiwwm<3mmzmqqm3 mOQUAmmBmzmmeMMHmBmZmBmZ>WQAOmDHBWGQHmeZQdmmZ study. mm mass m mamflamnu .d m>eumm .O mummas> .m muscucoo .m Homo umaoom 116 At 3660120 At 3918080 Wimp 59 Prun us serotina P. contorta Populus UBC 2 Populus UBC 3 Populus UBC 1 At 67312 At QQLV33 Populus SWE 1 H. vulgare ~|—- O. sativa Figure 8. Plant B-glucosidase protein family. This figure shows a phylogenetic tree created using BioNavigator (see methods). Populus UBCs are unpublished fi-glucosidase sequences from the lab of Brian Ellis (UBC). The Populus SWE 1 sequence is from PopulusDBase. All other protein sequences were from the non-redundant (nr) protein database at NCBI. 117 Wimp 62 - Subtilisin, another possible candidate in the lignin pathway Wimp 62, with homology to subtilisin is also interesting. Subtilases have been demonstrated to be serine proteases. Gordon McDougall’s lab has postulated that such proteinases could have a role in wall modification events in enlarging cambial cells or in maturing xylem elements. The following is the protein alignment of Wimp 62 with an Arabidopsis thaliana subtilisin that is the highest hit for the Wimp 62 translation. 3 ' AYQVNTEYS FGSLTWTDGVHIVRSPLSVRTEFLQPYI (Wimp62) VNT-H- FGSL WTDGVH V P+SVRT+F++ Y+ 3' SHRVNTDFYFGSLCWTDGVHNVTIPVSVRTKFMRNYV (At Subtilisin) Figure 9. Protein alignment of Wimp 62 Subtilisin The laccase purified from developing xylem tissue of Picea sitchensis (Sitka spruce) is differentially regulated in compression and non-compression wood branches (McDougall, 2000). One domain of this laccase in the N-terminus has protein homology to the subtilisin family and is implicated in.lignin polymerization. The research group in Dundee, Scotland has also purified an angiosperm laccase from Poplar from lignifying tissue (Ranocha et al., 1999). Wimp 118 62, which has protein similarity to this family is another candidate for further characterization. Wimp 68 - Dehydration induced protein A class of proteins first described in 1998 shares a conserved domain at the C-terminus with Wimp 68. Called the BURP domain for the four members of the group in which they were first identified: §NM2 from Brassica napus microspore derived embryo, ESPs, abundant non-storage seed proteins, 3D 22, a drought induced protein from Arabidopsis, and gGlB, the B-subunit of polygalacturonase isozymes (Hattori et al., 1998). The group shares some structural features, but no patterns of tissue specificity nor functional similarity. Wimp 68 has the highest similarity to RD—22 which is expressed due to drought, and as characterized by Shinozaki, is mediated by abscisic acid (ABA) (Yamaguchishinozaki and Shinozaki, 1993). 3' AYQVXNVKPGTVPVCHFXLQDHVVW (Wimp 68) A++V VKPGTVPVCHF + vaw 3' AFKVLKVKPGTVPVCHFLPETHVVW (At RD22) Figure 10. Protein alignment of Wimp 68 Dehydration response protein. 119 The above alignment of Wimp 68 and Arabidopsis thaliana RD22 shows grey-shading for 100% amino acid similarity to the conserved amino acids of the BURP domain. It can be concluded that Wimp 68 is expressed in developing cambium, shows two to four-fold up regulation on a northern and is part of a family of plant genes characterized by a BURP domain. It shares protein similarity to a gene from Arabidopsis that is induced under stress conditions. Wimp 68 should be further characterized. DISCUSSION OF NORTHERNS Radiolabelled probes were prepared from seventy different Wimp cDNAs and used to probe northern blots. There are three categories of results: twenty-five which had no hybridization (after being left on film for a week), forty- five with hybridization (in some cases, weak), and of these forty-five, three indicated a slight difference in gene regulation (two to three fold difference from the control lane). Two of these Wimps, numbers 59 and 68 were examined repeatedly, up to ten times each in an effort to maximize 120 hybridization. These two showed the greatest difference in hybridization intensity; as well as being putatively linked to stress proteins and/or the lignin pathway. The remaining forty-two appeared to be constitutive, showing equal hybridization of control lanes to those in the stressed lanes. It should be mentioned that Northern analysis was ongoing over three years as different protocols were used for RNA extraction in an attempt to test for up regulation. CLONED BANDS HAVING NO HYBRIDIZATION TO NORTHERN BLOTS Of the seventy cloned Wimps that were chosen for hybridization to Northern blots, twenty-five yielded no signal after exposure to autoradiograph film for one week (Wimps 2,3,4,8,10,11,12,13,15,20,25,28,58A,62,66,101, 106,115,130). Appendix A presents a detailed history of these bands including sequence data, primers used, database similarity results and northern hybridizations. These bands likely are expressed in poplar stems, however the methods used for verification of gene expression are not 121 sensitive enough to detect messages with very low expression. NORTHERNS, GENE EXPRESSION, AND UP-REGULATION OF MP GENES Although there were apparent differences (two to four-fold) in gene expression on autoradiographs for two Wimps, the majority of the forty-five hybridizing Wimps appeared to be consitutively expressed genes. If these are truly constitutive, then the technique of differential display and the experimental design failed to discriminate among genes that were expressed due to the stress and constitutively expressed genes. Alternatively, a greater number of the 141 cloned cDNAs may represent MP induced genes but the methods used to detect differential expression were not adequate. This could be due to a number of reasons: a) the quantity of message in the harvested stems, b) rapid growth of both stressed and unstressed plant material, and c) quality of the RNA transferred to the nylon membrane. These factors could cast doubt on the results of the northerns. 122 RNA EXTRACTION Some discussion of the difficulty that I encountered in the extraction of the poplar RNA may provide guidance for future workers. From the number of articles published dealing with poplars, it would now seem that this should not have been such a problem. However there is a variability in the type of poplar clone, or species being used for extraction. In Sweden, I observed the extraction of RNA from a hybrid cross of Populus tremula x P. tremuloides. The stem sections frozen in liquid nitrogen were then slit vertically through the bark which was then peeled back. A scalpel was used to scrape the bark section (cambial), and in a second scraping of the middle core (xylem). This is the method described in creation of the Poplar cDNA library (Sterky et al., 1998). RNA extraction was then done using Dynal magnetic polyT beads. The stems of the Swedish hybrids were the same age and height of the 47—174 clones that were used in the experiments at Michigan State University, however they were almost an inch or more in diameter making cambial scrapings an option. The 47-174 Stems were often only 1/4 inch in diameter and could not be 123 used for cambial scrapings except in field grown poplars of this clone. Field grown poplars could not be used in these experiments because a variety of biotic and abiotic stresses exist under field conditions. Wind stress comprises elements other than stem flexure such as canopy size and shape, wind direction and speed. These compounded factors could not be segregated in field grown poplars from the exclusive aspect of stem flexure. Because of the selection of poplar clones, for purposes stated above, it was necessary to develop methods to successfully use these clones for the MP experiments. Over a three year period I continually tested different methods to optimize results of extractions. After the modification I made to protocols #11 and #12, the RNA was of good quality. However, time constraints became a limiting factor. It took six months from rooting of cuttings to harvesting stressed tissue and sufficient plant material was not available. Since the development of my extraction modifications, my protocol has been requested by a USDA Forest Service laboratory performing RNA extraction on elms and 124 experiencing problems. (Jennifer Koch, USDA Forest Service, personal communication) All of the factors mentioned above may have contributed to the lack of conclusive evidence of up-regulation for most Wimp genes. It is my opinion that the combination of the above limitations coupled with the difficulty of extracting high quality RNA that led to the difficult demonstration of upregulation. An additional factor is the rapid growth of the poplar clones during the experiments. The stems of poplar clone 47-174 (control and stressed) grew almost a foot in height from the time of pre-stressing to one week later at harvest. Cambial development would be similar in both control and stressed stems, the differences in the two samples could be difficult to detect. The stressed stems should be depositing additional xylem at the internode of stress. Although in one set of experiments RNA was extracted from the specific internode of stress (and the internodes above and below the stressed internode), there was no apparent difference in gene expression. The gene expression may be at constitutive or basal levels in all samples, the differences being undetectable with current 125 It is a matter of allocation of resources, a technology. change in the deposition of material that is almost certainly being continually deposited. LIMITS OF DIFFERENTIAL DISPLAY The differential display procedure as applied here will in tiheory screen sixty percent of the Populus genome (product Iliterature of GeneHunter Corporation; Liang and Pardee 1.963). Because of cost limitations, each set of primers ‘Mnas used in only one differential display reaction. However as there were two control lanes and four time Ipcsints represented in the use of each set of primers used, t:11is was considered sufficient to act as a control to limit Ifatlse positive reactions. If a band was observed in two or ITKDzre of the time points, and not in the controls, it was c2C>risidered to be significant and selected for further a i3r1€ilysis. Despite measures to decrease false positives, w“Gill-documented pitfall (Bauer, 1993; Callard, 1994) in differential display, is the PCR re-amplification at low Stringencies (42°C) that can result in non-specific 126 ‘ amplification. Those bands that did not re-amplify, or produced poor quality unreadable sequence could be the result of nonspecific amplification. In the population of bands that were sequenced it is unknown the number which truly are not poplar bands, nor specifically expressed to the stress conditions. Only fifty of the seventy cloned bands that were used for radiolabeled probes showed hybridization to poplar RNA. An additional twelve (which were never made into probes) had homology to sequenced ESTs from the Populus database, confirming that sixty-two were indeed poplar genes. However a lack of hybridization in the twenty Wimps that did not hybridize to the RNA filters could represent the difference between a PCR based technique (differential display) and northern hybridization. It cannot be ruled out that these differential display bands were the result of false positive selection. Until the emergence of microarray technology to monitor global gene expression, differential display was widely used by researchers in human medicine and in other fields to analyze differences in gene expression in two different tissue types, or the same tissue type under different 127 conditions (Liang, 1996). It is still a powerful and valuable tool in organisms that do not have, nor will have in the near future a sequenced genome or collection of expressed sequence tags (ESTs). For certain physiological conditions it remains a useful technique (Liang, 2000). Microarrays made from cDNA libraries that are not normalized to guard against the bias of abundant transcripts may not detect many low abundance messages. Microarrays in general lack sensitivity for low abundance transcripts (Goldberg, 1986). Differential display may sample rarer products more efficiently by reducing the ‘Ca: effect' (MathieuDaude et al., 1996). Two sequences revealed high homology to Pseudomonas putida flagellin, Wimp 121 (BlastX 7e-30) and Wimp 124 (9e—30). Interestingly a cDNA probe of me 121 revealed hybridization to all lanes of poplar total RNA. No probe was made of me 124. These high homologies to a bacterial protein sequence could indicate the presence of bacterial contamination from the surface of the poplar tissues used for RNA extraction. This could only be possible if bacterial RNA was extracted along with the plant RNA for both the differential display and in the RNA that was blotted onto a filter for the Northern hybridization. 128 Although it is unlikely that bacterial RNA could be reverse transcribed in the first strand synthesis since oligo-dT primers were used in these reactions, in rare cases some bacterial RNA does have polyA 3’ termini. (Marie Edmunds citation) However an alternative explanation is that bacterial RNA survived the isolation and was PCR amplified from poly A rich regions. It can therefore be concluded that at least half of the Wimps (sixty-two) that were sequenced from isolated differential display bands are ESTs from poplar stems in developing xylem and cambium. This is supported by Northern hybridization (fifty) and by significant BLAST scores within published Poplar EST sequences (twelve). An additional thirty-five sequenced Wimps had significant hits to plant ESTs in the NCBI database. This is strong indirect evidence that these Wimps are indeed poplar genes from the developing cambium/xylem region. Twenty-seven Wimps for which Northern analysis was not done revealed either “no hits”, nor significant hits with TAIR, PopulusDB or Finch searches. 129 SUMMARY It can be concluded from database searches and from northern hybridizations that the majority of Wimps are derived from the developing cambium and xylem of poplar. The Wimps were generated from poplar tissue that was mechanically stressed using PCR techniques. The differential display which uses PCR to amplify and display tissue under different conditions may represent a level of sensitivity in gene expression that cannot be verified with northerns. The majority of the northerns that were done showed constitutive levels of expression, yet sequence similarity to genes of known functional categories reveals a contrasting picture. It can be concluded from database searches that there is an abundance of representation of stress related genes that bear sequence or protein similarity to a majority (65) of the Wimps. 130 FUTURE DIRECTIONS Given time and increased funds a more complete coverage of the RNA population could be accomplished with increased efforts to reduce the incidence of false positives. Using a complete set of primer combinations, replication of the differential displays and using a gel apparatus to obtain longer sequences would all improve the significance of the results. Using hybrid poplar clones that were more amenable to cambial scraping and RNA isolation would be desireable, but at the expense of being able to use field evaluated wind tolerant poplars. The technology in the laboratory of Bjorn Sundberg permits sampling of RNAs from a cell specific gradient across the poplar stem. These improvements would optimize results. Having full length clones or obtaining sequences of greater length would also be an improvement. A differential display of cDNAs from proven wind tolerant versus wind intolerant poplar clones would be highly desirable. These Wimps are a contribution to the public domain databases. It has been shown that a majority of the sequenced Wimps are from plants, and most are from hybrid 131 poplar stem tissue. Some show evidence that they are representative of genes that would be expressed under wind stress conditions. This collection should be expanded. All of the poplar ESTS, including those from the Swedish database, which is being expanded, the French tension wood database, and hopefully the newly funded Canadian EST collections should be combined on a comprehensive poplar microarray chip. An EST collection of cDNAs from mechanically perturbed poplar stems, akin to the ones that I have cloned, should be included. 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(ed.), Biology of Populus and its implications fer.management and conservation. NRC Research Press, Ottawa. 143 CHAPTER 3 CULTIVARS OF QUERCUS CERRIS x QUERCUS SUBER: Q. x HISPANICA, THE LUCOMBE OAK AND INTER-SIMPLE SEQUENCE REPEATS (ISSRS) ABSTRACT Morphological and chromosomal evidence for the identification of cultivars of Quercus x hispanica, the Lucombe oak has often been inconclusive. The use of DNA fingerprinting techniques has proved helpful for such identifications. Using inter-simple sequence repeat (ISSRs) primers, 66 Q. x hispanica (and related) samples were analyzed and compared for band pattern differences to establish identities. DNA evidence revealed that some named cultivars had identical banding patterns, which then aided in identification of other named or unnamed cultivars with the same DNA banding patterns. The cultivar named 'Lucombeana' with nine different grafted samples showed no 144 two had identical banding patterns, while two grafted trees on the grounds of the University of Exeter, and dating in age approximately to the time of William Lucombe, did have the same banding pattern and may indeed be of the original clonally propagated ‘Lucombeana'. INTRODUCTION From the time that William Lucombe of Exeter discovered a different looking oak seedling in his Quercus cerris L. seedbed (probably from a Q.cerris pollinated by Q. suber) in 1763, (Elwes & Henry 1906) there has been confusion about the name and identity of that oak. Called Q. lucombeana by Sweet, in 1826, it has subsequently been called the Lucumbe oak, the Lucombe oak, Quercus x hispanica Lam.var. lucombeana, and the Exeter oak among other names. Within a short time Lucombe clonally propagated thousands of ramets of this oak by grafting onto Q. cerris rootstock and distributed them, while the original may have been cut down for Lucombe’s coffin boards when the tree was about twenty years old.(Elwes & Henry 1906). Lucombe’s son named a group 145 of seedlings from the original tree, which are now regarded as cultivars: ‘Crispa’, ‘Suberosa’, ‘Incisa', ‘Heterophylla’, and ‘Dentata’. Subsequent cultivars originating from other crosses of Q. x cerris and Q. x suber (Q. x hispanica) include ‘Cana major’ of 1849 from the Hammersmith Nursery, ‘Diversifolia’ from the Smith Nursery) and perhaps the most famous other than that of Lucombe, ‘Fulhamensis’ Loudon 1838,from the Whitley and Osborne Nursery at Fulham, England. (Bean 1976) Morphological characteristics, such as corky bark formation, leaf form, leaf retention, height at maturity, acorn size, etc., are quite variable. Chromosome studies of the number and morphology of the chromosomes showed no variation among the forms examined (Caldwell 1953). Because of these difficulties in identification, it is more than inappropriately named. It was decided to use DNA fingerprinting on a group of Q. x hispanica cultivars in an attempt to shed light upon the confusion surrounding the Lucombe oak. Are the many cultivars named ‘Lucombeana’ vegetatively identical, and is it possible to establish their lineage as the same as that original cloned (grafted) material of Lucombe? .Although 146 many fingerprinting techniques have been successfully used on plant material (Weising 1995) and on oaks specifically (Dow 1995 and 1996), it was decided to use ISSRs that had been used successfully in population studies of oaks (Marquardt personal communication). ISSR markers are generated from single-primer PCR reactions where the primer is designed from di— or trinucleotide repeat motifs with anchoring sequences of one to three nucleotides. (Wolfe 1998) These occur in all eukaryotes' Using polymerase chain reaction (PCR) primers created to bind to these repeated regions, the intervening areas are amplified allowing variation to be detected between samples. Plant cultivars that are vegetatively propagated should all have an identical banding pattern. All of the clonally propagated oaks from Lucombe’s nursery and all those that were grafted later from this first cross will exhibit an identical, unique banding pattern for each primer used. It should be noted that seed propagated cultivars (e.g. Oilseed Rape), will have a wide range of band patterns, as can individuals within the same species. 147 MATERIALS AND METHODS Sixty-four different plant samples of Q. x hispanica and related materials were collected, see Table 1. Other genera from the Fagaceae Dumortier (Castanea and Fagus) and one outgroup (Rhododendron) were used to evaluate various extraction methods and choice of primers (data not shown) Samples from various sources in the United Kingdom (UK) (see Table 7 for specific locations and accession numbers) were mailed to Michigan State University, Michigan, USA in plastic bags through regular postal channels. One sample, Q.hinckleyi, was obtained from a herbarium voucher at MSC Herbarium (Plovanich-Jones 002). DNA was extracted using the method of Scott et a1. 1996 that was developed for use on ‘rain forest’ plant species but which has often proved quite useful for samples high in polysaccharides and secondary metabolites. The posted samples proved difficult to extract with other methods because of long transit storage conditions. 148 Name Accession # Name Accession # Castanea dentata (MSU) Q. x hispanica 'Ambrozyana' 80.0280A Castanea sativa (Hillier’s) Q. J: hispanica ‘Ambrozyana' 82.0296A Q.cerris 'Laciniata ’ 44.0004 Q. x hispam'ca ‘Ambrozyana' 77.4402 Q.cerris L. 14.0079 Q. x hispanica 'Ambrozyana' 77.2213 Q.cerris L. 75.0105 Q. x hispanica ‘Cana major' 27.0057 Q.cerris 'Wodan' 89.2595 Q. x hispam'ca ‘Cana major 27.0056 Exeter lower Q. x hispam’ca ‘Cana major' 27.0055 Exeter graft union ( Q. robur L.) Q. x hispam‘ca ‘Cana major’ 43.0457 Exeter middle Q. x hispanica ‘Cana major’ 43.0455 Fagus sylvatica L.(MSU) Q. x hispanica ‘Cana major' 27.0058 Kensington “A” West Ham Park Q. x hispanica ‘Crispa ' 43.0463 Kensington “B” West Ham Park Q. x Iu'spanica 'Crispa ’ 43.0464 Kensington “C" Kensington Gardens Q. x hispanica ‘Crispa’ 43.0465 Kensington “D” Chiswick House Q. J: hispanica 'diversg'folia' 87.2194 Q x hispanica Lam. 95 .0694B Q. x hispanica ‘diversifolia' 08.0320 Q x hispanica Lam. 95.0694A Q. x hispanica ‘Fthamensis' 86.2228 Q x hispanica Lam. 04.0434 Q. x hispam'ca ‘Fulhamensis ’ 82.0142 Q x hispanica Lam. 43.0472 Q. x hispanica ‘F ulhamensis' 78.1942 Q x hispanica Lam 77.5535 Q. x hispanica 'Hemelryik’ 93.0030 Qx hispanica Lam. 44.0403 Q. J: hispanica ‘Hemelryjk’ 93.00308 Qx hispanica Lam. 77.1306 Q. J: hispanica ‘heterophylla’ 95.0454 Q x hispanica Lam 09.0047 Q. x hispanica 'Lucombeana' 77.5412 Qx hispanica Lam 95.606958 Q. x hispanica 'Lucombeana’ 14.0133 Q x hispanica Lam 77.1306 Q. x hispanica ‘Lucombeana' 56.0039 Q. Iaceyi Sma11(Texas) Q. x hispam’ca ‘Lucombeana' 43.0384 Q. hinckleyi C.H. Muell.(Texas) Q. x hispanica 'Lucombeana' 77.7380 Quercus robur L. (MSU) Q. x hispanica ‘Lucombeana ' 88.0329 Rhododendron species Q. x hispanica 'Lucombeana’ 44.0037 Q.suber L. 79.9326 Q. x hispanica ‘Lucombeana' 17.0048 Q.suber L. 77.4783 Q. x hispanica ‘Lucombeana’ 41.0425 Q.suber L. 43.174 Q. x hispanica ‘suberosa' 95.1629 Q.suber L. 78.3131 Q. x hispanica 'Waasland' 97.0075 Q.suber L. 76.9326 Q. x hispam'ca ‘Wagem'ngen' 89.2597 Q. x hispanica ‘Wageningen' 89.2596 Table 7: Plant samples used for DNA microsatellite analysis. the Arboretum furnishing the plant material. PCR conditions: final concentrations of IX PCR buffer, cresol red, 200uM each dNTP, 0.07U/ul Amplitaq, 30 ng total DNA , 1.8uM UBC primer. An MJ Research, 149 Inc. Accession numbers for some samples are those of 30ul PCR reactions were performed with 12% sucrose, PTC 100 thermocycler was used with the following conditions: denaturation at 94°C for 2 min. (using a hot start, 3.5 mM MgClzwes added when temperature exceeded 80°C) 29 cycles at 92°C, 30 sec., 52°C for 45 sec., and 72°C for 2 min. A 2% agarose gel was used for resolution of the PCR products. 25 pl was loaded into each well (the addition of the Cresol red dye to the PCR reaction obviated the need for any further loading or running dye) and run at 50 V for 16 hours using a cooling, re—circulating tank maintained at 12°C. Bands were visualized by staining gels with Ethidium bromide for thirty minutes and destaining in 0.5x TAE for an additional thirty minutes. DNA Proscan, (Nashville, Tennessee) was used for scoring of bands and evaluation of results. All samples were evaluated with a minimum of two gels for each UBC primer. Four UBC primers (UBC 807, 811, 834 and 841) were used for each sample. Although UBC primer 807 can produce a number of bands that will also appear using primer 834 likewise for 811 and 841, it will also produce many unique bands. The four primers will produce four different banding patterns for each clonally unique cultivar. Samples of ‘Lucombeana' from twenty different PCR reactions and the four primers were repeated as the results 150 were somewhat startling and this was the focal point of the study. Results for all gels were consistent and repeatable. RESULTS The ISSR banding patterns of 'Ambrozyana', and 'Fulhamensis' and the two trees from the University of Exeter were identical within each sample group. (Figure 11 Panels a and b) Banding patterns are identical if the same banding phenotype is obtained with all primers used. Figure la shows four samples of ‘Ambrozyana’ using UBC 834, the bands are identical for all four samples. ‘Ambrozyana’ band patterns for the other UBC primers were consistent and identical within the four. This was also the case with the two samples from the University of Exeter shown in Figure 1b. Again using UBC primer 834 in Figure 11c , three samples of ‘Fulhamensis’ Lanes A, B, and C, match one of the unnamed trees from Kensington, London, Lane D. 151 Exeter M ‘NMWVIV‘ (\w' mu“ m .1....) a) WI Gamma) “(1* humu- ‘w l K i .111". wt. C) , m m m w m Mi .1) .... ' ‘” Mun V . m Figure 11: Banding pattern UBC 834: a) ‘Ambrozyana' Lanes A-D, M = Gibco 100 bp molecular weight marker b)E = Exeter oaks, c) ‘Fulhamensis' Lanes A, B, C, and unnamed oak from Chiswick House, London, Lane D, Exeter, Lane E 152 The cultivars labeled ‘Crispa‘ and 'Cana Major' were different from each other and furthermore no 'Crispa' was the same as the other two Crispas, (Figure 12) and no 'Cana Major' identical to the other five Cana Majors. (Figure 13) a) 5 mm Figure 12. Banding pattern a) UBC 811 and b) UBC 841 of three cultivars of ‘Crispa’ 153 MABC DE FExeter :mfiflmwm :uuuumfiflfl .... wu- ”We“ m ‘M Mum MN W m . “Una“uu My ‘mn mlw ‘“ W m m w u .” "J“ ”W‘““mw M ,. WM “ h W)“ (“W Vin“) Figure 13: Banding pattern UBC 834 for six samples of var. ‘Cana major’ 'Lucombeana' showed that none of the nine different samples all named ‘Lucombeana’ had identical banding patterns (Figure 14). It should be noted that a different banding pattern always had more than two bands different from another sample being considered with the same name. There 154 were no instances of samples being different by only one band. MABCDEF GHM (I'VMIH ‘in 1‘ .. “M‘ mm M NW“ N nmmlm m..." m mum-u. ., w... Wm) (WM) WNW) ”.11va n m Mum. iii-‘1 ‘ M11111“) MN m M m H‘" IWNH‘W MM ‘ l. 111“) .v. H in. (NW “mm M“ Will *1. (new MM!) ' m Figure 14. Banding pattern UBC 834 for nine samples of ‘Lucombeana’. One lane is unmarked due to confusion on the label of the posted sample. 155 The banding pattern from the three samples of 'Fulhamensis' matched those of three of the samples from the London area, Kensington 1, 2, and 4 and that of one sample named 'Lucombeana' #88.0329 (Figure 15). ML1K1K2K4F1F2F3M Figure 15: Lanes marked M are molecular weight standard, all other lanes are banding patterns from UBC 841. Lane L1 ‘Lucombeana’ 88.0329, Lanes K1, K2, and K4, unnamed oaks from the London area, Lanes F1, F2, and F3 ‘Fulhamensis’ 156 The two accessions of 'Diversifolia' were identical to each other, as were those of 'Hemelrijk' and the 'Wageningen'. These latter two cultivars, often referred to as Q. x hispanica differed from each other as well as the third Dutch cultivar, 'Waasland'. (These data are not shown). These cultivated varieties have been named more recently and are still being propagated and sold in the nurseries in which they originated. The ten Q. x hispanica with unspecified cultivar name showed no similarities to each other nor to any other named cultivars. These data are also not shown. It is more than likely that these were propagated from acorns from any of the Lucombe oaks across the United Kingdom. DISCUSSION It can be concluded from gel evidence that in the case where banding patterns are identical in multiple different samples of the same named cultivar (four or five minimum) that the given taxonomic designation is correct. Other unnamed samples with the same ISSR profile as the accepted cultivar can subsequently be named. In the case of 157 Ambrozyana numbers 80.0280A, 82.0296A, 77.4402 and 77.2213, the different samples are clonally identical and this cultivar is almost certainly a valid one. The three ‘Fulhamensis’ samples, 86.2228, 82.0142 and 78.1942 are identical and the unnamed samples from West Ham Park and Chiswick House (K1, K2, and K4) that match them can be considered the same clone. And one clone from Hillier’s Garden and Arboretum, Accession #88.0329, is incorrectly named as ‘Lucombeana', it is most definitely ‘Fulhamensis’. Certain cultivars remain problematical, ‘Cana Major’, ‘Crispa’ and ‘Lucombeana’ revealed no banding pattern that was identical within each sample group of the same name. Cultivars that had no more than one or two samples could be said to be not identical to other cultivars if their banding patterns using the same primer (and others) did not match banding patterns of any other cultivar. These include ‘Laciniata’ ‘Suberosa’, ‘Diversifolia and ‘Wodan' which are different from each other and other named cultivars. The ten unnamed Q. x hispanica samples did not match any named samples and could possibly have grown from acorns (seed propagated), rather than have been vegetatively propagated. 158 The case of the Lucombe oak is particularly interesting. As no consensus was found from the samples received as ‘Lucombeana’, we would offer the following suggestion. A much larger sample set of ‘Lucombeana’should be collected including those from historical collections (such as Kew, Cambridge, etc.) which date from the time of Lucombe and are vegetatively propagated, a graft union being visibly obvious. The remaining tree from Exeter should be evaluated and included in this collection. Additional primers (7 - 10) should be used to evaluate the larger ‘Lucombeana’ sample set. A minimum number of samples with identical banding patterns for all primers used should be established as a criterion for the identification of a cultivar. When such a number is determined, then one could establish what is validly a ‘Lucombe’ oak. Such a proposal is not of course our decision and until the time that determination has been made, we will still be in search of the Lucombe oak. ACKNOWLEDGMENTS 159 The following persons aided in the collection of samples, photographs, DNA extractions or PCR ISSR evaluations. Frank W. Telewski, Beal Botanical Garden, Michigan State University, USA; Hugh C. Angus, Westonbirt Arboretum, UK; Steven Scarr - University of Exeter, UK; Elinor Wiltshire - Carroll House, London, UK; Glen T. Jones and Jan Rademaker. Michigan State University, USA; Paula Marquardt — USDA, University of Wisconsin, Rhinelander,USA; Bryan Epperson — Department of Forestry, Michigan State University, USA; M.V. Ashley - University of Illinois at Chicago, USA, MSU US DOE Sequencing Facility — Michigan State University, USA. REFERENCES 160 Bean, W.J. (1976). Quercus in Trees and shrubs hardy in the British Isles. Vol III, pp. 456-521. John Murray, London. Caldwell, J. & Wilkinson, J. (1953). The Exeter Oak — Quercus lucumbeana,. Transactions of the Devonshire Association: 35—40. DNA Proscan. (1997), DNA Pro Scan, Inc., Nashville, TN 372l2 Dow, B.D., & Ashley, M.V., & Howe, H.F. (1995). Characterization of highly variable (GA/CT)n microsatellites in the bur oak, Quercus macrocarpa. Theor. Appl. Genet. 91: 137-141. Dow, B.D. & Ashley, M.V. (1996). Microsatellite analysis of seed dispersal and parentage of saplings in bur oak, Quercus macrocarpa. Mol. Ecol. 5: 615-627. Elwes, H.J. & Henry, A. (1906). Quercus lucombeana, the Lucombe oak. In The trees of Great Britain and Ireland, Vol 5. Pp. 1259-1267. Privately printed, Edinburgh. lflitchell, A., (1994) The Lucombe Oaks. The Plantsman, 15(4): 216-224. Scott, K. D. & Playford, J. (1996). DNA extraction technique for PCR in rain forest plant species. _iotechniques 20: 974-78. 161 Wang, Z., Weber, J.L., Zhong, G., & Tanksley, S.D. (1994). Survey of plant short tandem DNA repeats. Theor. Appl. Genet. 88: 1-6. Weising, K., Nybom, H., Wolff, K. & Meter, W. (1995). DNA fingerprinting in plants and fungi. 332 p. London: CRC. Wolfe, A., Qiu-Yun, X., & Kephardt, S. (1998). Assessing hybridization in natural populations of Penstemon (Scrophulariaceae) using hypervariable intersimple sequence repeat (ISSR) bands. Mol.Ecol. 7: 1107-1125. Zietkiewicz, E., Rafalski, A., & Labuda,D. (1994). Genome fingerprinting by simple sequence repeat (SSR) - anchored polymerase chain reaction amplification. Genomics 20: 176-183. 162 CHAPTER 4 A PHYLOGENY OF THE INTERNAL TRANSCRIBED SPACER (ITS) AND EXTERNAL TRANSCRIBED SPACER (ETS) REGIONS FOR.MONTANOA (ASTERACEAE: HELIANTHEAE). ABSTRACT A phylogeny of the genus Mbntanoa based on the Internal Transcribed Spacer (ITS) and the External Transcribed Spacer (ETS) is presented. The combined dataset supports the monophyly of the genus and an early evolutionary split that coincides with geographic distribution. The two clades revealed by parsimony analysis have each approximately half of the number of species in the genus. One lineage is composed mostly of central and southern Mexican species whereas the other lineage contains those species endemic to Mesoamerica and South America. The molecular phylogeny is compared to previous phylogenetic 163 hypotheses based on morphological characters. Key features in the structure of the capitulum of Mbntanoa, such as pale morphology, heavily used in the past to construct hypotheses of relationship within the genus, are viewed as of minimal value to circumscribe natural groups. The relationships of Mbntanoa to other genera in the Heliantheae are briefly discussed. Introduction The genus represents one of the most conspicuous genera of Asteraceae of mountainous Mesoamerica and northern South America. Some species are ruderal and abundant in recently disturbed areas in tropical deciduous forests whereas others such as M} revealii are large buttressed trees up to 20 m in the cloud forests of western Mexico. The shrubby to arborescent habit of most species of Montanoa along with its dichasial capitulescences combine to create a spectacular display of white flowers along roads and areas with secondary vegetation. Mbntanoa is a member of the mostly Neotropical tribe Heliantheae which is characterized by heads with receptacular bracts or pales and trinerved 164 leaves. Most of the 25 species of Mbntanoa are found in Mexico and Central America with five species endemic to northern South America. The genus is characterized by its sterile white ligules, white to yellow, rarely black disc corollas, a chromosome number of x = 19, and pales that continue to grow and expand after anthesis (acrescent pales) turning the flowering head into a spiny fruiting ball. The phylogenetic position of.MOntanoa within Heliantheae has always been enigmatic and controversial. Funk (Funk, 1982) revised the genus and clarified the taxonomy by recognizing 25 species from 125 available names but was unable to identify the sister taxon of Mbntanoa nor clarify the relationships of Mbntanoa within Heliantheae. Robinson(Robinson, 1981) believed the genus to have an isolated position in the Heliantheae and placed it in its own monotypic subtribe Montanoinae in his tribal classification scheme. Both authors relied on morphological characters traditionally used in Asteraceae classification to identify potential sister taxa. According to Robinson(Robinson, 1981) the sterile ray flowers of MOntanoa tend to group the genus with members of subtribe Helianthinae and Ecliptinae whereas its cypselae 165 characteristics point to a close relationship to Melampodium and allies. Funk (Funk, 1982) hypothesized that given that Mbntanoa has an unusual chromosome number in the Heliantheae of x = 19, genera sharing this chromosome number could be close relatives of the genus. She compared salient morphological features of Mbntanoa to genera such as Jaumea, Venegasia, Villanova, Synedrella, Actinospermum, Amblyolepis, Gaillaria, Calea, and Podachaenium concluding that these genera did not form a monophyletic group nor any of them was the sister taxon to MOntanoa. Rojasianthe superba was believed to be distantly related to.Montanoa in spite of shared features such as acrescent pales, an x = 19 chromosome number, white ligules, and opposite leaves. Given that no outgroup could be identified, Funk (1982) polarized characters by using functional groups within Mbntanoa based on pale morphology. This exercise produced three groups or lineages, which were in turn used as reciprocal outgroups to each other. Molecular studies based on approximately 20,500 bp of the chloroplast genome for 124 genera of Heliantheae have been recently completed (Panero et al. unpublished). Results from these studies indicate that Mbntanoa is the basalmost lineage of tribe Ecliptinae and sister to Rojasianthe. 166 Based on these results we have chosen Rojasianthe and Idiopappus (subtribe Ecliptinae) as outgroups. We initiated this molecular study of the genus Montanoa with the primary interest of testing the phylogeny based on morphological characters advanced by Funk (1982) in her revision of the genus. Species relationships and other infrageneric groupings previously recognized are based on cladistic analyses of morphological features with a special emphasis on character state series describing various pale characteristics. We wanted to ascertain if pale morphology is an important predictor of relationships in the genus. Secondly we were interested in understanding the origin and evolution of Montanoa. To test these hypotheses we built a phylogeny based on the Internal Transcribed Spacer region (ITS) and the External Transcribed Spacer region (ETS). Several studies have shown the ETS to be useful in increasing the phylogenetic signal and support for monophyletic groups in phylogenies based on the ITS region (Baldwin and Markos, 1998; Clevinger and Panero, 2000; Markos and Baldwin, 2001). 167 MATERIALS AND METHODS Plant Samples Thirty-one samples corresponding to 22 species of Mbntanoa, Rojasianthe superba, and Idiopappus quitensis were collected in the field. Herbarium specimens were used for DNA extraction of M1 atriplicifolia, M1 pteropoda, and M3 echinacea. Table 8 lists the samples used, and GenBank accession numbers. 168 GENBANK TAXON ITS 1 ITS 2 ETS Idioggpus quitensis AY0381 16 AY038149 AY038083 [Montanoa angulata AY0381 17 AY038150 AY038084 Efiontanoa atriplicifolia AY038118 AY038150 AY038085 [Montanoa atriplicifolia AY0381 19 AY038152 AY038086 Mntanoa bipinnatiflda AY038120 AY0381 53 AY038087 [Montanoa echinacea AY038121 AY038154 AY038088 lMontanoa fragrans AY038122 AY038155 AY038089 [Montanoa frutescens AY038123 AY038156 AY038090 IMontanoa frutescens AY038124 AY038157 AY038091 lMontanoa qrandiflora AY0381 25 AY0381 58 AY038092 [Montanoaguatemalensis AY038126 AY038159 AY038093 [Montanoa heggona AY038127 AY038160 AY038094 [Montanoa hibiscifolia AY038128 AY038161 AY038095 [Montanoa imbricata AY038129 AY038162 AY038096 IMontanoa karwinski AY038130 AY038163 AY038097 [Montanoa karwinski AY038131 AY038164 AY038098 [Montanoa laskowski AY0381 32 AY038165 AY038099 [Montanoa leucantha subsp arborescens AY0381 33 AY038166 AY038100 IMontanoa leucantha subsp leucantha AY038134 AY038167 AY038101 Wontanoa liebmannii AY038135 AY038168 AY038102 [Montanoa liebmannii AY0381 36 AY038169 AY038103 [flantanoa mollissima AY038137 AY0381 70 AY038104 Wontanoa ovalifolia AY038138 AY038171 AY038105 [Montanoa pteropoda AY0381 39 AY0381 72 AY038106 Mntanoa pteropoda AY038140 AY0381 173 AY038107 [Montanoa quadrmaris AY038141 None AY038108 IMontanoa revealii AY038142 AY038174 AY038109 [Montanoa revealii AY038143 AY038175 AY038110 Montanoa speciosa AY038144 AY038176 AY0381 1 1 [Montanoa standleflL AY038145 AY038177 AY038112 [Montanoa tomentosa AY038146 AY0381 78 AY0381 13 [Montanoa tomentosa subsp microcephalal AY038147 AY038179 AY038114 Montanoa tomentosa subsp. tomentosa AY038148 AY038180 AY0381 15 Rojasianthe superba AF171947 AF171986 AF 172025 TABLE 8 . List of taxa used in phylogenetic studies. specimens collected by J. Panero and deposited at TEX except when noted. 169 DNA isolation Total genomic DNA was isolated from fresh leaf tissue collected in the field, stored in liquid nitrogen or silica. The method outlined by (Saghai et al., 1984) and modified by Doyle & Doyle (1987) was used to isolate DNA from fresh and silica dried material. Herbarium samples were extracted using either the method of (Paabo, 1993) and gel purified or the rainforest method (Kirsten and Playford, 1996). Amplification of the ITS and ETS regions The ITS and ETS regions were amplified using Polymerase chain reaction (PCR) in either 50 or 100 pl reactions. ITS 5 and ITS 4 primers of White et al. (1990) were used to amplify a region of approximately 700 bp. A primer was developed upstream of the 5’ end of the ITS region to improve amplification efficiency of M; echinacea and M. bipinna ti fi da (Primer 7 . 5 : 5 ’ GAGTCATCAGCTCGCGTTGACTA 3 ’ ) . 170 ETS primers las—E and ETS-Hel-l developed by Baldwin & Markos ((Baldwin and Markos, 1998) were used to amplify a region of approximately 400 bp. Amplification of the ITS and ETS region was performed under the following conditions: one cycle of 4 min denaturation at 959C, primer annealing at 48°C for 45 sec, primer extension at 72°C for 1 minute followed by 32 cycles with similar conditions to initial cycle except for 1 minute denaturation and an additional 2 seconds for every successive extension. This was followed by a final extension of 10 min at 72 °C. PCR products were cleaned and concentrated with Ultrafree-MC filters (Millipore Corporation) prior to sequencing. Dye terminator sequencing was done at the Michigan State University DNA sequencing facility and the University of Texas sequencing facility following manufacturers instructions and protocols. Internal primers 2 and 3 of the ITS (White et al., 1990) and IBS-E for the ETS (Baldwin and Markos, 1998; Markos and Baldwin, 2001) were used in sequencing reactions. 171 Sequence alignment and phylogenetic analysis Sequences were assembled into contig files and aligned manually using Sequencher (Gencodes). Parsimony analyses were performed using PAUP 4.0b6b (Swofford, 2001). Heuristic searches were performed with 100 random entries for the combined ITS-ETS data matrix using ACCTRAN, MULPARS and TBR options. Support for monophyletic groups was assessed using 100 bootstrap replicates (Felsenstein, 1985). Tree statistics such as the consistency index (Kluge and Farris, 1969) and the retention index (Farris, 1989) were calculated by PAUP. RESULTS Maximum Parsimony analysis of the ITS data matrix produced 136 equally parsimonious trees, tree length 262, CI excluding uninformative characters of 0.85 and a RI of 0.89. Similar analyses of the ETS data matrix produced seven trees of 146 steps, CI excluding uninformative 172 characters 0.85 and a RI of 0.92. The combined data matrix produced twelve trees of 410 steps, CI excluding uninformative characters of 0.85 and a RI of 0.90. The strict consensus tree from the combined data matrix is shown in Figure 16. Sequence length is comparable to that of other Asteraceae. The combined ITSl and ITSZ region of Mbntanoa varied in length from 480 to 483 bp. There was little variation in length in the ETS region among the species of Mbntanoa but the outgroup genera have smaller ETS regions. The ETS of Mbntanoa ranged in length from 413 to 415 bp whereas the ETS of Rojasianthe is 384 bp and that of Idiopappus is 339 bp long. 173 M. gaidiflora 95 ’12—: Mhbn'mta .64.. —— M.speciosa M. bipinnatifida 84 70 J— M. leucanmaarborescens L—— M. leucantha Iamntha 88 MW M. mollissima M. standleyi 96 M. Iiebmam11 M. fiebmarviiz M.hexagona — _— M.Mentosaxmfliifofia 75 100 M. tomentosamiaocephala M.tomentosatomettosa M.Meswm M.MmZ M. aigulata M. quadrangularis M. fragrans Mackinaw M. atriplidfofm MatriplidfoliaZ Mgiatemlensis M.ptempoda1 M.pteropodaz M11bisdiolia M. ovalifolia 'M.kaMiski1 M.kamins|d2 M. revealii1 M.avaaiiz ldiopapptsquitensis Rojasianthe superba 1 .83 97 , 100 52 71 HT 11111 NW 100 1; l | Figure 16. Phylogeny of ITS and ETS of Genus Montanoa . Mexican species (I) MesoAmerican and Northern South American species 174 The monophyly of the genus Mbntanoa is well supported with a bootstrap value of 100. The Mbntanoa species are distributed in essentially equal proportions between two main clades. The first clade contains only Mexican species and has moderate support with a bootstrap value of 66. This clade contains two clades that have moderate to strong support. The first clade contains most species from central Mexico that have large heads and showy white ligules and has a bootstrap support of 88. The cloud forest species M. standleyi is the basalmost lineage. There is no resolution between four lineages represented by the two samples of M3 leucantha, ML mollissima, M2 laskowski, and the clade composed of the species M1 bipinnatifida, ML speciosa, M; imbricata and M1 grandiflora. The other clade contains three lineages in a trichotomy and has a bootstrap support of 75. The two samples of M1 frutescens are in the same clade with a bootstrap support of 97, the three samples of M1 tomentosa, cluster together in a trichotomy with bootstrap support of 100, and the third lineage contains M1 hexagona as sister to the two samples of M1 liebmannii. The bootstrap support for this clade is 99. 175 The second main clade includes Mexican, Central and South American species. Relationships among the different species of this clade are not supported in the bootstrap analyses. The Mexican species ML karwinskii and.M. revealii are sister taxa and this relationships is supported by a bootstrap value of 100. This clade is sister to a clade that has weak support with a bootstrap value of 52. This clade contains four lineages. The western Andean species Montanoa ovalifolia represents a single lineage. The second lineage is represented by two samples of ML atriplicifolia that are sister to M} guatemalensis; this clade has weak support with a bootstrap value of 53. The Mesoamerican species M1 pteropoda and M3 hibiscifolia are sister with a bootstrap value of 71. The Mesoamerican species M; echinacea is sister to the Venezuelan species ML angulata, M3 fragrans and M} quadrangularis, however, the relationships shown in this clade have no bootstrap support. DISCUSSION 176 The genus Mbntanoa has always been regarded as one of the most distinctive genera in the Heliantheae and its monophyly has never been questioned. The molecular data presented supports this View. Mbntanoa and Rojasianthe are the only genera in the Heliantheae that have pales that grow after anthesis. Pales have been used to circumscribe species and infrageneric groups in Mbntanoa (Funk, 1982). Mbntanoa has two types of pales characterized by the shape of their apical halves. Some species have tapered or pointed pales, whereas others have cuneate or truncate pales. Truncate pales have small pointed or tapered tips and only differ from tapered pales by differential growth and vascularization of the pale body. The distinctive morphology of the pales was used by Funk (Funk, 1982) to place species in two subgenera. Subgenus Mbntanoa has all the species with tapered pales whereas subgenus Acanthocarpae has all the species with truncate pales. Results from our study show that pale morphology is not a good indicator of relationships as each pale type has arisen multiple times in the evolution of the genus. We believe this structure is extremely useful in the identification of species and in the construction of phenetic treatments, that is to say those based on morphological features, phenotypic treatments. 177 Our studies reveal that there was an early split in the evolution of the genus with two lineages containing each approximately half the species in the genus. One clade contains species from central Mexico (Mexican clade) whereas the other clade contains Mesoamerican or southern Mexican species and the South American taxa (Mesoamerican/South American clade). The species relationships revealed by our molecular study do not agree with relationships suggested by cladistic analysis of morphological features in Funk (1982). The Mexican clade is characterized by two main lineages. All the species in the first clade, with the exception of M} hexagona, have tapered pales. The four taxa in this clade are represented by multiple samples of species from different areas of Mexico. The three samples of M} tomentosa are sister to M3 hexagona and M} liebmanii. Mbntanoa hexagona was placed by Funk (Funk, 1982) in series Hibiscifoliae of subgenus Acanthocarpae because it shares with M; hibiscifolia a distinctive cypselae wall ornamentation. In our study M; hexagona is sister to the diploid species ML liebmannii, a weak shrub or perennial herb from central Oaxaca. MOHtanoa hexagona is an 178 octaploid species and therefore may include multiple genome combinations. Mbntanoa tomentosa was placed by Funk (Funk, 1982) in its own series within subgenus Mbntanoa as it is the only species in the genus with densely pubescent pales. In addition, the head of M. tomentosa contains only one fertile, central flower whose cypselae is shed as a unit along with all the extended pales of the senescing head. Mbntanoa tomentosa is a variable and abundant species of central Mexico. Funk (1982) placed multiple taxa in the synonymy of M; tomentosa that appear to be variations of leaf forms from specific regions of Mexico. She did not provide a hypothesis of relationship for this taxon except for the fact that it represented a distinctive member of subgenus Mbntanoa. We sampled three of the four varieties of the species and they cluster together with high bootstrap support. Mbntanoa frutescens from central Mexico is characterized by its greenish disk corollas and reflexed pales. Mbntanoa frutescens, like M3 liebmannii and M; tomentosa, has tapered pales. Funk (Funk, 1982) allies this taxon to M. guatemalensis and M; mollissima. 179 The second lineage of the Mexican clade is composed of species that have large heads with ligules ranging from creamy white to bright white and orange disc corollas. Mbntanoa standleyi is the basalmost lineage of this clade. This species, like M; andersonii inhabits cloud forests and is endemic to the states of Chiapas and Oaxaca. It can be easily distinguished by its distinctive palmate leaves and recurved pales. Mbntanoa standleyi shares several features with M; andersonii including similar pale and head morphologies. Mbntanoa mollissima, M} laskowski, and.M. leucantha each represent single lineages in a polychotmy that also includes the clade of large headed species of central Mexico including M1 bipinnatifida, M3 grandiflora, M; imbricata, and M3 speciosa. Mbntanoa mollissima, except for its tapered pales, has a similar habit and overall morphology to M; leucantha. This species is a small shrub of xeric areas immediately north and south of the neovolcanic range of central Mexico. Mbntanoa mollissima is further distinguished by its ovate ligules that like those of M3 leucantha, can vary from creamy to bright white. Mbntanoa laskowski is unusual in Mbntanoa in that it grows in the scrub forest of the Pacific coast of the states of Colima and Jalisco. Like M3 mollissima it has 180 bright white oval ligules. .Mbntanoa leucantha is one of the most abundant species of Mbntanoa, variety arborescens being a common treelet of disturbed, montane areas of central and southern Mexico. The nominal variety differs from variety arborescens by its bright white ligules and «- Tr smaller shrubby habit. The two varieties of M3 leucantha were sampled and both taxa are in a sister relationship with a moderate bootstrap support of 70. , The large headed species of Mbntanoa are clustered together in a clade that has a moderate bootstrap support of 64. Mbntanoa bipinnatifida is the basalmost lineage, with M1 speciosa, M3 imbricata, and M1 grandiflora grouped in a terminal clade with a strong bootstrap support of 95. Montanoa imbricata and M1 grandiflora are sister and characterized by having large white ligules and capitulescences. They grow in ruderal areas, especially along roads and abandoned agricultural fields. The second clade contains species from western Mexico, Mesoamerica and South America and is characterized by two main lineages with species north and south of the Isthmus of Tehuantepec. The first clade contains two distinctive species from the Pacific coast of central and southern 181 Mexico north of the Isthmus. .Montanoa revealii is one of the most spectacular species in the genus. It is a large tree of the cloud forest of the states of Guerrero and Oaxaca. This is a very distinctive taxon that can hardly be confused with any other species in the genus. .Mbntanoa revealii is a hexaploid species with tapered pales. Funk (1982) placed this taxon in series Apertae Funk as the only member of this group. .Mbntanoa karwinskii is another distinctive species in the genus that can be easily recognized by its leaf morphology in which the blade starts at the point of divergence of the three main veins of the leaf. This condition is sometimes observed in M1 revealii. Mbntanoa karwinskii has truncate pales and therefore, it is a member of subgenus Acanthocarpae. It has a large geographic range along the coast and intermountain valleys of western Mexico. The strong relationship shown in our analyses between M3 karwinskii and M3 revealii is puzzling given the morphology of key taxonomic features that will support relationships to other species in the genus. It is possible that M1 revealii represents an allopolyploid of complex origin in which one or two additional species of Montanoa may have contributed to its genetic makeup. 182 The other clade contains species that grow exclusively south of the Isthmus of Tehuantepec. Most of these species are endemic to the montane regions of Central and South America. Four lineages, most of these with weak bootstrap support were revealed by our analysis. The first clade has the Mesoamerican species M} echinacea as the basalmost lineage of a clade containing three of the four South American species sampled. None of the relationships shown E in the strict consensus tree have bootstrap support. Mbntanoa ovalifolia subsp. australis a native of southern Ecuador and northern Peru did not cluster with the other South American species in our analysis. In some of the twelve equally parsimonious trees this species is basal to the clade containing M; guatemalensis and M3 atriplicifolia. Montanoa guatemalensis is a dodecaploid tree from Central America. This taxon is sister to the diploid species M} atriplicifolia, a variable taxon from Mesoamerica. The relationship between these two species is weak as the clade has a low bootstrap support of 53. Funk (Funk, 1982) placed these two species in different subgenera and consequently her discussion of their potential relationships is compromised by pale morphology 183 considerations. The two species are parapatric in some areas of their respective geographic ranges. Funk (1982) considered M1 atriplicifolia to be sister to M3 pteropoda. She based this assumption in the fact that both species share a scandent habit, rather small disk flowers, and a distinctive pale morphology. In our studies M1 pteropoda is sister to M3 hibiscifolia. All these species, with the exception of M; guatemalensis, were considered by Funk (1982) to be closely related. The results obtained in this study show that the geographical distribution of species should be regarded as an important consideration in the circumscription of infrageneric taxa above the species level. We believe MOntanoa to be of Mesoamerican origin and that most of the species we observe today are the result of subsequent radiations into the southern and northern areas of the montane Neotropical region. The Isthmus of Tehuantepec appears to have been an effective barrier to the movement of certain groups of taxa of the Mexican flora and apparently impacted the course of evolution in the genus MOntanoa. The role of the Isthmus of Tehuantepec as an effective barrier or filter for certain groups of taxa has 184 been much discussed by several students of the Mexican flora (Miranda, 1952; Rzedowski, 1983). Finally, the significant incongruences in the phylogenetic relationships depicted by the molecular and morphological studies may be the result of emphasizing pale morphological features in the classification of the genus. Pales are structures that apparently play a role in the protection of 1 developing cypselae (Stuessy and Spooner, 1988) and therefore, may have a limited use in reconstructing phylogenies in Mbntanoa and possibly other composites. ACKNOWLEDGMENTS I wish to thank Jose Panero for the opportunity to study such an amazing group of woody plants. Through Jose's love of the Composites of the New World, I gained an appreciation, wonder and respect for this flora through his enthusiastic descriptions. His unbounded excitement about these incredible plants is contagious. It is due to Jose’s confidence in me and support that I was able to undertake this project. I also want to thank Sue Stoltzfus for her 185 help and encouragement with our sequencing effort and Anna Weise for sequences as well. I also wish to thank the support and assistance of Dr. Alan Prather in preparation of this chapter. His continued encouragement and support with this and in revisions of the oak project has been generous and without personal benefit. I owe a great deal of my knowledge of systematics to Alan Prather and his patient teaching. The study and aepj salary was supported by NSF grants DEB 94-96174 and 99-093800 to Jose Luis Panero. REFERENCES Baldwin, B.G. and Markos, S. (1998) Phylogenetic utility of the external transcribed spacer (ETS) of 188-268 rDNA: congruence of ETS and ITS trees of Calycadenia (Compositae). Mblecular Phylogenetics & Evolution, 10, 449-463. Clevinger, J.A. and Panero, J.L. (2000) Phylogenetic analysis of Silphium and subtribe Engelmanniinae 186 (Asteraceae: Heliantheae) based on ITS and ETS sequence data. American Journal of Botany, 87, 565- 572. Farris, J.S. (1989) The retention index and homoplasy excess. Systematic Zoology, 38, 406-407. Felsenstein, J. (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39, 783-791. Funk, V.A. (1982) The systematics of.Montanoa (Asteraceae, Heliantheae). New York Botanical Garden, New York. Kirsten, S. and Playford, J. (1996) DNA extraction technique for PCR in rain forest plant species. Biotechniques, 20, 974, 977, 979. Kluge, A.G. and Farris, J.S. (1969) Quantitative phyletics and the evolution of anurans. Systematic Zoology, 18, 1-32 . Markos, S. and Baldwin, B.G. (2001) Higher-level relationships and major lineages of Lessingia (Compositae, Astereae) based on nuclear rDNA internal and external transcribed spacer (ITS and ETS) sequences. Systematic Botany, 26, 168-183. Miranda, F. (1952) La vegetacién de Chiapas. Ediciones del Gobierno del Estado. Tuxtla Gutierrez, 2 vols. Paabo, S. (1993) Ancient DNA. Scientific American, 269, 86— 92. 187 Robinson, H. (1981) A revision of the tribal and subtribal limits of the Heliantheae (Asteraceae). Rzedowski, J. (1983) Vegetacién de México. Editorial Limusa, Mexico D. F., 1-432. Saghai, M.M.A., M, S.K., A, J.R. and W, A.R. (1984) E Ribosomal DNA Spacer-Length Polymorphisms in Barley ' Mendelian Inheritance Chromosomal Location and i Population Dynamics. Proceedings of the National W Academy of Sciences of the Uhited States of America, 81, 8014-8018. Stuessy, T.F. and Spooner, D.M. (1988) The adaptive and phylogenetic significance of receptacular bracts in the Compositae. Taxon, 37, 114-126. Swofford, D.L. (2001) PAUP: Phylogenetic analysis using parsimony (and other methods) ver. 4.0b8. Sinauer Associates, Inc., Sunderland, MA. White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M: D. Innis, D. Gelfand, J. Sninsky, and T. White [eds], PCR protocols: A guide to methods and applications. Academic Press, San Diego, CA, 315-322. 188 CONCLUSION DO WOODY PLANTS AND MOLECULAR TOOLS HAVE A FUTURE TOGETHER? Problems associated with woody plants and traditional (non molecular) experiments In the introduction I stated that there were difficulties involved with experimentation on woody plants because of their very nature. Traditional studies are made more difficult due to the slow growth habit, their size and space requirements, dormancy, the difficulty of propagation and length of time for flowering. In addition, the woody stems are a significant barrier to some experiments and the presence of many secondary metabolites are also problematical. 189 I have worked on three woody plant genera, oaks,.Mbntanoas, and poplars and to a great degree I have had difficulties due to some of the above-mentioned problems. The oaks were dormant during five months of the year, and to receive the leaf samples from the UK sometimes took waiting an additional six months for some leaf samples. Although none of the other problems were an issue for the oaks, the high phenolic content was a problem for shipping of the samples. The.Mbntanoas proved a difficult woody subject for another reason; not having to do with their woodiness. I was determined to include all twenty-five species of the genus in the study. One species, Montanoa joseii is only found in a remote mountainous area in northern Colombia. I have wanted to go and get a speciment of this plant, however the presence of armed conflict due to the drug wars has made this a challenge. It has only been collected twice, by Jose Cuatrocasas (for whom it is named) and by Vicki Funk. DNA extraction from a herbarium specimen resulted in truncated segments. The poplars did present a number of traditional difficulties. It took two years to develop a successful method of propagation. At one time we had to wait until 190 the dormant season in Washington State to obtain specimens. At that time, rooting cutting from green tissue was thought to be undesirable. Tissue culture, mist propagation, different media and different greenhouses were all tried. At the conclusion of these studies, a success rate of 90% of cuttings rooted. Indeed the poplars did occasionally go dormant in the greenhouse at the onset of short days. However under the Agro-Gro lights it was possible to stimulate them to break dormancy. The idea of slow growth is almost a contradiction in terms referring to poplars. While growing, they can elongate up to a foot per week. However from the time of cuttings till the time of stress initiation, it was six months. Space was equally a problem. Only a limited number of trees could be stress tested during one set of experiments. The maximum was twenty trees in one gallon pots. Length of time for flowering in woody plants was not an issue for these studies, since no flowers needed to be collected, no crosses needed to be done. All material was vegetatively propagated, or collected from stems or leaves. 191 Problems associated with woody plants and molecular experimentation Were these molecular studies made more difficult by the fact that I used woody plants? The problems associated with woody plants and molecular tools often refer to the size of their genomes, the difficulty of transformability and problems associated with introduction of transgenes into a long—lived species. Extraction of DNA and RNA for some woody plants is often mentioned as an additional problem. Since my studies did not involve transformation and introduction of a transgene, many of the issues raised about experimentation on woody plants were not involved. Only the issue of extraction proved problematical. With the oaks and the Mbntanoas, the molecular studies used DNA extracted from leaf tissue. This was no further problem of extraction than many other plant tissue types. In the years of working with Jose Panero on DNA studies from many exotic and unusual plant specimens, the oak and Mbntanoa DNA extractions were simple by comparison. Occasionally the oak DNA was brownish in color due to the 192 115‘ presence of phenolic residues, but did not present difficulty for further enzymatic manipulation. The poplar RNA proved to be a real problem. I routinely isolated poplar RNA from leaf samples with ease, but the stem sections proved a formidable barrier. It is with some satisfaction that I have received requests for my modified protocol for RNA extraction from poplar stem tissue. If" The methods that are being used around the world in poplar studies on stem sections either are not available for use here, or because of the selection of poplar clones could not be applied. Despite difficulties, I have been able to reach satisfactory answers to two of my questions, and a partial answer for the third. I have been able to identify cloned cultivars of the “Lucombe” oak, and shown conclusively that the eleven samples received from the UK which are called “Lucombe” oak are not identical cultivars. I have answered the question of the relatedness of the genus Mbntanoa to other new world composites. And I have been able to show that its evolution has developed 193 geographically as it has radiated from a MesoAmerican center both north from Central America to Mexico and radiated south from Central America to northern South America. I have been able to hypothesize that wind stress on poplar trees does induce gene expression; and the genes that E appear induced indicates a relation to other stress induced genes. I have been able to identify a set of cDNAs that are expressed in the lignifying tissue of poplar stem tissue. Answer: Woody plants and molecular tools g9 have a future together Despite the difficulties involved working with woody plants, the problems can be surmounted. Each challenge that is unique to using molecular tools on trees can be met with patience, effort, and resources. There must be a commitment to each of these. Working with woody plants is not easy, however, as more questions are answered, it becomes easier. 194 The need for the preservation, conservation, yet continued harvesting of this very precious resource has never been greater. So go out, and hug a tree! But only pick up your pippettor and think about using your molecular tool kit if you have real dedication! 195 APPENDICES 196 ‘: an“! ..r..- APPENDIX A COMPLETE WIMP RESULTS: PRIMERS USED IN DIFFERENTIAL DISPLAY, SEQENCES, DATABASE SEQUENCE SIMILARITIES, NORTHERN DATA 197 WIMP 1 - PUTATIVE IDENTITY - UNKNOWN PRIMERS 5’ -AAGCTnG-3’ and 5’-AAGCTTTGGTCAG-3’ >wimp1 TTTTTTGCGTTGGAGAGAAAATGAGTTTCCTTAAATNAAAATTAACTTGGCATTACAAT ACATATATTAAATAATNGCAATCANNAATAANAAAAGAAAANNGGATGGCCTTATTTTT GNCTCAGCAAATTGCTTTTGAGGCCTTGTTTTATTCTTCTCTTCTGCTGGAATATACAT TCCAGCANGGATTCNCCATGGGCATGGCATTNGNAGTNNAGTNTCNTGGCNATCCAGCT T C 1 2 4 8 12h 198 WIMP 2 - SEQUENCED TWICE - POOR SEQUENCE PRIMERS 5 ’ -AAGCT11C—3 ’ and 5 ’ -AAGCTTTGGTCAG- 3 ' WIMP 3 - PUTATIVE IDENTITY - UNKNOWN PRIMERS 5’ -AAGCTnC—3’ and S’-AAGCTTTGGTCAG-3’ >Wimp3 CAAAAGACAAAAATATCTCACAGCCATTAAGTTTATATGGTATCGACATA CAAGCTGAACTGAAATACGTTGGACAGCATGTTTTACATATAACTCGATA ACATATTTACAAGGATAAGGAAACTACTATAGTACATCCTTTGTATTTGA TCCTTTCGGCAATCAAGCTTAATCAAGCTT WIMP 4 - PUTATIVE IDENTITY - UNKNOWN PRIMERS 5’ -AAGCTnC93’ and 5’ -AAGCTTTGGTCAG- 3’ >Wimp 4 AAGCTTTGGTCAGGAAACCCCATATCAAGTCGTGGCATCTATGAAAGCTC TTGGGGCATCGTACATGTGCTTTAACATTCTTCATAATAAGTGCGGTCCG ATTATACTGCCTGGTTCGCTTCACAACAAAATTAGGCGATGTAGNTTTGT ATATTTTCATAGCTTTGAGTAGTTGTCAAAATTGATCCAATTTGACTCCA AATCATTTGAGGCTGTCCTGTTACATTTGTTGCTATATCAATCAATTGGT ACTCAGAGTCGGTACTCGTTCTCGAAAAAAAAAAAG 199 WIMP 5 - PUTATIVE IDENTITY CYTOCHROME f PRIMERS 5’ -AAGCT11C-3' and 5’ -AAGCTTTGGTCAG- 3’ >Wimp 5 AAGCTTTGGTCAGGGAGACGCANAAATAGTACTTCAAGACCCATTACGTG TCCAAGGCCTTTTGTTCTTCTTGGCATCCGTTATTTNGGCACAAATCTTT TTGGTTCTTAAAAAGAAACAGTTTGAGAAGGTTCAATTGTCCGAAATGAA TTTCTAGATTGCGAATTTATCAACATCAAGTTCTTATCTTTTCTTAATTT CTTAAAAAGAACAAAATTTTTGTTGGAAATTCTGTATGAAAAAAAAAAA BLASTX homology >gi|6723761IemblCAB67170.1| (AJ271079) cytochrome f [Oenothera elata subsp. hookeri] Score = 94.0 bits (230), Expect = 3e-19 Query: > 5 FGQGDAXIVLQDPLRVQGLLFFLASVIXAQIFLVLKKKQFEKVQLSEMNF 154 FGQGDA +VLQDPLRVQGLLFFLASVI AQIFLVLKKKQFEKVQLSEMNF Sbjct: 269 FGQGDAEVVLQDPLRVQGLLFFLASVILAQIFLVLKKKQFEKVQLSEMNF 318 200 WIMP 6 - PUTATIVE IDENTITY KINESIN LIKE PROTEIN PRIMERS 5’ —AAGCTnC-3’ and 5’ —AAGCTTTGGTCAG— 3’ >Winq96 AAGCTTTGGTCAGCAAGGTGGTTAAGGGATTTCTCAGCATTGCTTGTTTC TCAGGGCACACAGCTTGGACTGCTCCTGAAGAAAATTTTGAAGGGCGATA TTGGTTCCCTATCAAAGACTGAATTCATAGAAGCCATTTCCCAATATCTT AGACAAAGAACTANTTTGGCTTCAAGTGATTTCTCCAAGTTTTGTGTCTG TGGTGGGAAAAAAAAAAA BLASTX homology >gil2392771|gb|AAB70034.1|AAB70034 (AC002534) putative kinesin—like protein [Arabidopsis thaliana] Score = 61.3 bits (146), Expect = 1e-09 Query: > 64 LGLLLKKILKGDIGSLSKTEFIEAISQYLRQRTXLASSDFSKFCVCGGK 210 LG L+KILK D G LS++EF+ A+ +YL+ R L S +FSKFC CGGK Sbjct: 186 LGSFLRKILKCDNGDLSRSEFLAAVFRYLQHRKDLVSKEFSKFCKCGGK 234 C 2 4 8 12 h Band 6 201 .5 WIMP 7 PUTATIVE IDENTITY - UNKNOWN PRIMERS 5’ -AAGCTnC- 3’ and 5’ -AAGCTTCGACTGT- 3’ >Wimp? TTTTTTTTTTTCAAGAAAGAACAAATTAGACATCCATAAAGCAAGATTCA GGTTTCCATAAATAAATTACTCTCTTCTTCTACTACTACTACGACTACTA CAATATTATCTATGGATGACAGTAAGAAAACAAAGCCCTTACATAGGAAA TATCTTTTGCAGATTCTACCTAATTGAGAATTACAACTGACACATTGAAA GTCCTGAATGAGGGATGAAAACAAAACGTAGCTGCCACCTCCTGTTGCTT ATTCTGAACTCATCAAGCACTCTGACCAAAGCTATCAAGCTT WIMP 8 PUTATIVE IDENTITY — UNKNOWN PRIMERS 5’ -AAGCTnC- 3’ and 5’ -AAGCTTTGGTCAG- 3’ >Wimp8 AAGCTTTGGTCAGCAGTCCATGTGTGCGTGTTTGACATTACAATTGAAAT CGATGTTGATTGAAAAAGCATCCTCTTGATGCATTTCACATTTCCAATCT CAGTCCCAAATGGGTTTAGAGCCTGTTTGATTTTTAATAGTATTTTTGAA AAAAAAAAA WIMP 9 - PUTATIVE IDENTITY - UNKNOWN PRIMERS S’ -AAGCTnAr 3’ and 5’ -AAGCTTCGACTGT- 3’ >Wimp9 CTTTTTTTTTTTAANANCATNANACTCTTTGATTTGGTCATTAATTTTAT TTTTTCTCGTCTTTATNTATTAATCTCTACAACCTTCTCTCATAATTTGT GCTACNTACCTTTATATCCATGGACAATACACATACCTTCAAGCCACTAN AGCTATGCTCTCTCCCAATGAAATGTATAACGGTGTATGGAGAAGCCTTA CTACANTCGAAGCTT 202 “.n- u-r-n l_' WIMP 10 - PUTATIVE IDENTITY - UNKNOWN PRIMERS 5' -AAGCTnAr 3’ and 5’ -AAGCTTCGACTGT- 3' >Wimp10 TTTTTTTTTTTATAAGAACTACGCAATCACATCATTGTAATTATATATTA ATGCGGGAATTACATTAGTTGTCTATGATAGCATGACATTACACAAACAT GATCGCTCTATTTTATATGACTAAATTTCTGTCAAGTGTCTGTAATCTCT GCATTGAACAAGATATGCATAAAATATACAGTCGAAGCTTAATCAAGCTT WIMP ll - PUTATIVE IDENTITY - UNKNOWN PRIMERS 5' -AAGCTnAr 3' and 5' -AAGCTTCGACTGT- 3' >Wimp11 TTTTTTTTTTTATAGCAAAATCATCATTTTCATCCATTCAGCTCACATAC GATCATCCCATATAATCACTGTTCTCTTTTACGTCTACATGCATAGAATT ATGTGCATAATTTTAAACATATAACAGTCGAAGCTT WIMP 12 — SEQUENCED TWICE - POOR SEQUENCE PRIMERS 5’ -AAGCTuG- 3’ and 5’ -AAGCTTCTCAACG- 3' 203 WIMP 13 - PUTATIVE IDENTITY — UNKNOWN PRIMERS 5’ -AAGCTnG- 3’ and 5’ -AAGCTTCTCAACG >Wimp13 AAGCTTCTCAACGAGGACAGGTATGTTTTGCTATTTGTGAACCTGTATGC CCAGACTTTTTGATAAGAACTTCCCTCCCATAACTACTGAGTTTGAATAT CTAAGCAGCGATGAAGAAGACACAGCTTCTGCATCCCATACCCAAAAAAA AAAA WIMP 14 — PUTATIVE IDENTITY - UNKNOWN PRIMERS 5’ -AAGCTnG- 3’ and 5’ -AAGCTTAGTAGGC- 3’ >Wimp14 AAGCTTAGTAGGCAGATTATGAGCACTGCAAGTTTGGCTTATGTAACTCT AGCGTATTGTTGTCTCGGCATAGGCATTGGTATTGCCGCTTTTATGATTT CTGCCATTGGATGCTTCATGTTAGGATTTTGATTGTCTAATGTGTAGCCT TTCATAGAGCTAAATCATATGGAAGACCGCATTACCATTTTTTAATAAAG ACATTATCGTTTGAATTAATGCAAGTTTTGATGGTTATTTTGGCTTCAAA AAAAAAAA WIMP 15 - PUTATIVE IDENTITY — UNKNOWN PRIMERS 5’ -AAGCTnG- 3’ and 5’ -AAGCTTAGTAGGC- 3’ >Wimp15 TTTTTTTTTTTGGCCCAGCAAAGCTACTTATATTTGTTCCACAGATATAT TCACAGACAAAATAATGACAGGGAACATTTATTAGTTAAAACATTATCAG AAGTGGTCATAAACCACTCAAATAACCACCAATAGCCTACTAAGCTT 204 WIMP 16 - PUTATIVE IDENTITY - UNKNOWN PRIMERS 5’ -AAGCTufiF 3’ and 5’ -AAGCTTCTCAACG- 3' >Wimp16 TTTTTTTTTTTCAACTGCTTGCAAGAAAAGATCATTGAACTATTAATTAT TAAAAGAGAAGCCTTTAAAGCACTGTGCATGCATGATACCCTCAAGACTG CATGTTTACTTTTCCAGGCAAGAACTGAAGGTGAGTTGGGGTTTTGACTA CAAGCTGAAATATCAAAGTAAAGCGCTAATTCTAGGCTAACATGATTCAT GGCGAAGAAAATCCAGTGCTT WIMP 17 - PUTATIVE IDENTITY - UNKNOWN PRIMERS 5’ -AAGCTHCF 3’ and 5’ -AAGCTTCTCAACG- 3’ >Wimp17 ANAATAGTCTCCATGGCTATAAATCATTTAACACGTTGAGAAGCTTAATC NAGCTTATCNATACCGTCGACCTCTAGGGGGGGCCCNGTNNCNAATTCTC CCTANAATGANTCCTANTANCCCCCCTCNCTGGNCTTCCTCTTNCTANAT CGTGACTGGNAANACNCTGCCTTTACCCANCTTNATCCNACCTCCTAGGG GNGGCCCTNTCNCCAC 205 WIMP 18 - PUTATIVE IDENTITY - HYPOTHETICAL PROTEIN PRIMERS 5’ -AAGCTnCF 3’ and 5’ -AAGCTTGCACCAT- 3’ >Wimp18 TTTTTTTTTTTCGAGAAAGACAAACCATGAAACAACCATTTTGCTTAAGG TACAATGTACTGATTTGTTCGTGATTGATTTTGGATTATATGCCATCTGT GTTTCCAACGATAAGAGCACACTGAACGATCCCAGCAAGGATAAACATTC ACACTATACTAGGCGGATCCCCCACCCAATTGCAAACTGCACGAGACCAG CGAGTTCTAATACAGTCCACTAATGGTGCAAGCTT BLASTX homology >gi|7523680lgblAAF63119.1IAC009526_4 (AC009526) Hypothetical protein [Arabidopsis thaliana] Score = 56.6 bits (134), Expect = 5e-08 Query: < 229 APLVDCIRTRWSRAVCNWVGDPPSIV 152 APLVDCIRTRWSRA C+W GDPPSIV Sbth: 369 APLVDCIRTRWSRAACSWSGDPPSIV 394 206 WIMP l9 - PUTATIVE IDENTITY - UNKNOWN PRIMERS 5’ -AAGCTn£F 3’ and 5’ -AAGCTTGCACCAT- 3' >Wimp19 AANCTTGCACCATGATCATAATAAAACTGCCTAAATAGGTGTTCGCCTCT ATTGATGGGTCTCCACTANGGCGAGTCCCCCATTCAGTCTTGTGTCGTTG TCTTTTTTTTGTTTTTTTAATGAGTANCATGTGTCGTTATCTATGTATCC TTTGGCGGTATGAAATATGTAATGCAAATAAAATTCTACTATTTTGTTGA AAAAAAAAAAG WIMP 20 — PUTATIVE IDENTITY — UNKNOWN PRIMERS 5' -AAGCTn£F 3’ and 5’ —AAGCTTGCACCAT >Wimp20 AAGCTTGCACCATGTACTGTAGTTGAAGAGTCTAAAGTAGCTTTAGCAGT GATAATGATGATGGTTATGTATGCCATCGATTGTGATGCCCTTCTCGGAC CCTCCTTTGGAATCATTACCACTTTTGGTCTTCTGTTTTTTTGACATGCA ATCGATTGCCACTCCCTTTTCCTTTCCGAAAAAAAAAAA WIMP 21 - PUTATIVE IDENTITY UNKNOWN PRIMERS 5’ -AAGCTnAr 3'and 5’ -AAGCTTCTCAACG- 3’ >Wimp2l ATAAGAAGGGTCACCTCAATAATGCTCGACAATCAAGTCCGATCTACAAA TCAGGGCAAAAAGTCTAACAAAAACATCACTGCAAGTACACTCGTATCCG GTCAGATTTTCTCTCCATAAGTTGTCGTTGAGAAGCTTAATCAAGCTTAT CGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGT GAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTG GGAAAACCCTGGCGTTACCCA 207 WIMP 22 — PUTATIVE IDENTITY — Ribosomal protein 188 PRIMERS 5’ -AAGCTuG- 3’ and 5’ -AAGCTTAACGAGG- 3’ >Wimp22 TTTTTTTTTTTNATTAATNAAAACNTCCTTGGCAANTGCTTTCNCANTTN TNCNTCTTTCATAANTCCAAAAATTTCACCTCTGACTATNAANTACAAAT GCCCCCNACTGTCCCTGTTAATCATTACNCCNATCCCNAAGGCCAACACA ATAGGATCNAANTCCTATNATTTTNTCCCATGCTAATNTNTCCAAAGCNT AGGCTTGCTTTNANCACTCTANTTTCTTCAAATTAACAGC WIMP 23 - PUTATIVE IDENTITY - HYPOTHETICAL PROTEIN PRIMERS 5’ -AAGCTn£%’3’ and 5’ -AAGCTTAACGAGG- 3’ >Wimp23 TTTTTTTTTTTGGTTACTAAAATNTTTCANTTCGCCAGGTTGTCTCTTGC CTGCCCATGGATTCGGCAGCANTTTGAAAGGTTAACCTATTCGGGAATCT CCGGATCTACNCTTATTTTCAACTCCCCNAAGCATTTCGTCGCTTACTAC GCCCTTCCTCGTCTCTGGGTGCCTAGGTATCCACCGTAAGCCTTTCCTCG TTAAGCT BLASTX homology >gi|6723805|emb|CAB67216.1| (AJ271079) hypothetical protein [Oenothera elata subsp. hookeri] Score = 46.9 bits (109), Expect = 3e-05 Query: > 5 FFWLLKXFXSPGCLLPAHGFGSXLKG 82 F WLL+ F SPGCLLPAHGF S KG Sbjct: 29 FLWLLRCFSSPGCLLPAHGFSSSSKG 54 208 WIMP 24 — PUTATIVE IDENTITY - ENOLASE PHOSPHATASE PRIMERS 5' -JMAGCHH;G- 23’ano155’ -PU¥3CIWTVLACCIK:- 3' >Wimp24 TTTTTTTTTTTGCAAAGGAAACAGCCGCATTTTCTTTTAAAAGCGACATT TTTATTTATGTGTGCGTTGTGAAAAGCATTTCATACATTTGCAATAACAC AAGTACATATCTGCTTTCAGATTTCCAAGCCAAACTTAGCGTGATAAAAA TTGCATACATATCTGCCATCAGATTTCAGCGAAGGAGGTGATTGTCTTGA AACCGTGATTCTCTGGAAGAGGTGCATTTCCTGGCCGGATAGAAATCATC ACATCCAAACCTGCTCCTTTTGCGGTAAAAGCTT BLASTX homology - Wimp 24 >gi|10177247|dbj|BAB10715.1| (ABOO7644) contains similarity to enolase- phosphatase~gene_id:K19P17.1 [Arabidopsis thaliana] Score = 64.0 bits (153), Expect = 4e-10 Query: < 2 82 AFTAKGAGLDVMISIRPGNAPLPENHGFKTITSFAEI 172 A AK AGL+ +ISIRPGNAPLPENHGFKT+TSF++I Sbj ct : 471 AVAAKAAGLEAIISIRPGNAPLPENHGFKTVTSFSQI 507 WIMP 25 NO SIGNAL ON SEQUENCE PRIMERS 5’ -AAGCTnG- 3’and 5’ -AAGCTTTTACCGC- 3’ WIMP 26 SEQUENCED TWICE - POOR SEQUENCE PRIMERS 5' -AAGCTnG— 3’and 5- -AAGCTTCATTCCG- 3’ .209 WIMP 27 - PUTATIVE IDENTITY - UNKNOWN PRIMERS 5’ -AAGCTnG- 3’and 5- -AAGCTTCATTCCG- 3’ >Wimp27 AAGCTTCATTCCGTACTTACCAAAGAGATGGCACTTCTTCAGGGCCATTT GTTGAACTGAGCCTTTTTTCACCGAGTCCAGATGCCACTCCGCCAGAGTC ACCTCCGCCTCTAGGCTGGCCTATGCGCCCGCTTGTGGACCCGAAAACTG GGCAAGGAACTGATTTCTCGCAGTGAATATCAATTTTGTGAAAAAGAAAA ACTGATTGTAAAGGGTTAGTCTCCATAAGGATTTTTATTACTGGTTTGTT TTCCAAAAAAAAAAA WIMP 28 - PUTATIVE IDENTITY - UNKNOWN PRIMERS 5’ -AAGCTuG- 3’and 5- -AAGCTTCATTCCG- 3' >Wimp28 TAAGCTTCATTCCGCTACTGGGTTAAGTACTTACGTTTCTGGGAGCAGTC TCTGGTGTGGCCTTTCTNAGGCCTNGGTCTTTGTNCATTTTCTCTTTATC ACCNAAAGAGTTATCATGTGAAACGTTTGATCTTTAGTCAAAATATTCTG NCATGCTTCTTACAAAAAAAAAAA WIMP 29 - NEVER REAMPLIFIED NOR CLONED 210 WIMP 3O PUTATIVE IDENTITY - UNKNOWN PROTEIN PRIMERS 5’ -AAGCTHG- 3’and 5’ -AAGCTTCCACGTA- 3' >Wimp30 TAAGCTTCCACGTATAATATGAATGGGATCAGACGGAAGTTCTTGGTTCA GTGGGTTTGGAACCATTTCGTTAGTTACTGTACTGGCCCTAATGCCCTTG ATCAGTAGTGGTATTTTCCTATTTTGACCTTGTTTAATTTCTGTAATTTG GGGTTTTATCGCTCTTTTTTTTGTTGGGTTTTGGATTTTGGGGTTATTAA TGCATTAAGGTAGTTAAGGTTGTTAAATCTGTAATAATCTCTATTATACT ATTTTTGTACACCACTTTGCTGTCTTCTCCCAAAAAAAAAAA BLASTX homology >gi|9758197|dbj|BAB08671.1| (A8018109) gblAAD25141.1~gene_id:K17N15.10~similar to unknown protein [Arabidopsis thaliana] Score = 65.2 bits (156), Expect = 2e-10 Query: > 7 STYNMNGIRRKFLVQWVWNHFVSYCTGPNALD 102 +TYN+NGIRR++L+QW+W+H VSYCTGPNALD Sbjct: 306 ATYNVNGIRRRYLIQWLWSHVVSYCTGPNALD 337 WIMP 31 - PUTATIVE IDENTITY - UNKNOWN PRIMERS 5’ -AAGCTnG~ 3’and 5’ -AAGCTTCCACGTA- 3’ >Wimp3l TTTTTTGGAAATATCAAGGGTGGTCCTTCATTTTCTTTTATGANTAAGAT TNTGCTGGAAAAAACTCAAACCAATTGTCTAACCATAGTCATCTTCTTCN TACTTTCAAATAATTTAAGACTANAATAATTTCAATATCTAATACGTANA TAATCNTACTGGAAACTANAATCTTATTTACGTTAATTATAGTACTCAAA GCCCATACACTGGGTAATTCANTCCANTAATCAATCTCAATGACTACCAT ACACCTCACAATGCCTTNCGTGTGAAGCTTT 211 WIMP 32 — PUTATIVE IDENTITY - UNKNOWN PRIMERS 5’ -AAGCTnG~ 3’and 5’ -AAGCTTCCACGTA- 3' >Wimp32 TCCACGTATGTTATTGAAGAATATAAAATCTACTGCATTCTAGTTTGTAT TGCGGGTAAATGAACTTCACTAGGATATCNACAAAAGTTCCTCTTTACTC TCATCTNGTTAAAAAGTACGAATATTGTTGATGAACGGGGAAAGCTNGCT AGTANTTAAAATCTGTTCTGTTCTGGGTTTGTTTNCATTACGTNGTANNN NCCACTAATTATTGTTGCTTGCATGTTTCAGTTATTTTTTCCAAAAAAAA A WIMP 33 - PUTATIVE IDENTITY - UNKNOWN PRIMERS 5’ -AAGCTnG~ 3’and 5’ -AAGCTTCGGGTAA- 3’ >Wimp33 AAGCTTCGGGTAATGATGTAATCTTATGTAGACAATTCAAGCTGCTGACA TTTTTCAAGCAGCCTCCTACAACTGGAAAGTTTTAGATATAAATAGCCAT TCTATCAAGAGGAAGCTTGTGGTTAAATCAATTTTCAGTGCTGCTACTGT CTTGAAATCAAATGATATATCACGTTGAGTCTGCAGATG WIMP 34 - PUTATIVE IDENTITY - UNKNOWN PRIMERS 5’ -AAGCTHG- 3’and 5’ -AAGCTTCGGGTAA- 3’ >Wimp34 TNCCTCCCTCAGTTCTAGAACGATCNATNACCTTCGGGTAAATGAACTTC ACTAGGATATCAAGAAAAGTTCCTCTTTTCTCTCATCTAGTTNAAAAGTT CGAATATTGTNGATGAAGGGGGAAAGCTTGCTACTAGTTAAAATCTGTTC TGTTCTGGGTTTGTTTTCATTTTGTTGTAATATCCANTAATTATTGTTGC TTGCAAAAAAAAAAA 212 WIMP 35 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTuG- 3’and 5’ —AAGCTTCGGGTAA- 3' >Wimp35 AAGCTTCGGGTAATGCAGTGAAGATTTGAAAGAGGATTAGTTTAATCTTC TTACCTCTTTGCTTTTGGCTGCTAATGCTTTACACCTGTTTCATGCCTTC TCTTGTTACTTAAAATCTTGCTCTTGTTTCCTTTATGACTTTGATTTGGT CATTTACTCAAAAAAAAAAA WIMP 36 - PUTATIVE IDENTITY AMINO PEPTIDASE PRIMER COMBINATION 5' -AAGCTnC- 3’ and 5- -AAGCTTCATTCCG- 3’ >Wimp36 CTTTTTTTTTTTCCAGAATGGAGATTATCATTTGGAATCACGCCATTTAA CAATCCTATCACCACCTTCTGGTAAATTTACGTTGGAAATTGTTACTGAG ATATATCCCCAGAAGAACACATCATTGGAGGGACTTTACAAGTCATCTGG GAATTTCTGCACTCAATGTGAAGCAGAGGGTTTCCGCAAAATTACATATT ATCAGGATCGCCCTGATATAATGGCAAAATACACTGTCCGGAATGAAGCT TAATCAAGCTTATGGAAAAAAAAAGGCNGGCGGTTTTTTCCCCTTCCNCC CCC WIMP 37 - PUTATIVE IDENTITY — UNKNOWN PRIMER COMBINATION 5' -AAGCTnC- 3’ and 5— -AAGCTTCATTCCG- 3’ >Wimp37 CTTTTTTTTTTTCCATTACAAAAACGTTTTGCCTCGTTGAACATATCTAG CCACTTGTTACAGGAAACAACATTTTGCCAATATTTCACCAAATTTGGGG CACCAAAGACCACATCATCTTGAACACAAGAGTACCAGAGTACCAAATAA ATGACATATAGTGTTTCCTTTTCCCCGGAATGAAGCTTAATCAAGCTT 213 WIMP 38 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTNCF 3’ and 5- —AAGCTTCATTCCG— 3’ >Wimp38 TTTTTTTTTTTCCATCACATTCAAACATCCATTAAAGTTGGGATTTGGCA AACACAAGTGACAAAGATAATCCCATCTCCTATCAAAATTCTAAACAAAA TTAAGGGGAAAAAACCCAACACACCACACATTTTACGGAATGAAGCTTAA TCAAGCTT WIMP 39 - NEVER EXISTED PRIMER COMBINATION NONE WIMP 40 - NEVER REAMPLIFIED PRIMER COMBINATION 5’ -AAGCTM£F 3’ and 5- -AAGCTTCATTCCG- 3’ WIMP 41 — PUTATIVE IDENTITY — UNKNOWN PRIMER COMBINATION 5’ -AAGCTULF 3’ and 5’ —AAGCTTCCACGTA- 3’ >Wimp41 TTTTTTTTTTTCGATACCCAAGAGGCAAATAAAAATGCCTTCATAAACTA TAGAACACTATCACACACATACATATATATATAAATATTTAAGGAAAAAA 214 GGTCCAAAAGCTACATCACTCAAAATTCTTTATGATACATAAACAGTAAA ACCAAGGCTCAACAAGAGGTAACCCAGATCGTTTTCAAGAAGCTTGGCCA AGAAAAAGGACTCCAATGGCCAGTAGCCGCAGCAGCTACTTAATTAGTAC GTGGAAGCTTAATCAAGCTT WIMP 42 — PUTATIVE IDENTITY — UNKNOWN PRIMER COMBINATION 5' -AAGCTu£F 3’ and 5' -AAGCTTCGGGTAA- 3’ >Wimp42 CCACACAGTAACAATTAAATATATACCATTCAACTAAAGCATAGAGTATT TGCTAACTTTAGACAGAACGCAAACCTAGAACTTGGGCTGTGATCATGTC ACTACTGTCAGAAGAGGTCTAATTTTTCATTGTTTCTCATCATTGTTGGC GTCCTCATCAAAAGCACCCAGCAGCTCCTGCACATCTCTCTCCTTTATAC TTGATTTCTTCACATTGCATCTCTCTCCTTCGCATTTATGAACAAGCCAC AACATCTCTCTTACCCGAAGCTT WIMP 43 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTnC- 3’ and 5’ -AAGCTTCGGGTAA- 3’ >Wimp43 TTTTTTTTTTTCGACCAAACCAATTCGAATTTTCCAACTTCTGTAGCCCA AATCCCTATAATTCTGTTCTCGTTTTTACAGTGATCCGCTATGCCAAAAC TATTTTATAGAAAAAACTCAACAAGAAAACCCAGTCAATGGCAAACCTTC CAATGTCATCAGCACTGAGACCCCTGTTTACCACCTTTGTCCAGTTTCAG AGACTTAGGATCAAGTGGGGATAGTACCCACTGTTAGATTACCCGAAGCT TAATCAAAGCTT 215 WIMP 44 — PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTn£F 3’ and 5' -AAGCTTCGGGTAA- 3’ >Wimp44 CTGCAGGATTCGATAGCTTCGGGTAAAATGAAATATGTCGCCTTTGCTTC ATGCTATGTGACAATTGCTACCATAAAGTTGCTGCATCTTTGTGATAAAC AGCTGGAATTCTCTTCTGTTTCTCCTTTTCTTCTTGTGTAATTGTGAACA AAATTTGGTTGAAAAAAAAAAAG WIMP 45 - PUTATIVE IDENTITY — UNKNOWN PRIMER COMBINATION 5’ -AAGCTnAr 3’ and 5- -AAGCTTCATTCCG- 3’ >Wimp45 AAATTGAGCAGCTGGCATGCATGGCAGGAAGGAGGTTCCTTTTCTAATGA CACAGATCACCATATATTCATTTTTGTTTTTTGATTCGTGATTTTTTACC CAATGATGAAAGTTGTAGTTCCTGTGACGTAGATTTGTTTTGCAACAAAC GAATTGAAGATTCAATTCTAAAGCCATTATTTTATAAAAAAAAAAA WIMP 46 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ ‘AAGCTnAr 3’ and 5- -AAGCTTCATTCCG- 3’ >Wimp46 AAGCTTCATTCCGGGGGAAGCTATGTTAAAGTGTACATATTATTTGTGCA GTTCATTTGTAGTTTATTATTATTATTAGGGTGTACCTTGCTGTATTTGA TAAAAAGAATTCCAATGCTCTGCATTCAATAATTCAGGTTGCATGTGTAA AAAAAAAAA 216 WIMP 47 — PUTATIVE IDENTITY — UNKNOWN PRIMER COMBINATION 5’ -AAGCTnAr 3’ and 5’ -AAGCTTCCACGTA- 3’ >Wimp47 TTTTTTTTTTTAGGGATTGATAATGTTGATGTCAAATTTACTATTCGAAT GTGACATTGTTTGAAGACATTGGGAGATAAAACGACACATGGACATGCCT ATCAACTTTCATGCATTCGTTGAAATTTAGATCTGCGTGGTATACATGAC TTCCGTTGGAGCATCGTGACCATGTACGTGGAAGCTTAATCAAGCTT WIMP 47A - PUTATIVE IDENTITY — UNKNOWN PRIMER COMBINATION 5' -AAGCTnAr 3' and 5' —AAGCTTCGGGTAA- 3' >Wimp47A CTTTTTTTTTTTAAGAAAAATTGGATATTCATTACAGGTTACAATTACAT TATTCTCAAAGCATAAACATGGGATGACAATACTACTGCAACAAAGTAAA AAATTGTAGAACCTGGTACCACTTTTGGATGTTACCCGAAGCTTAATCAA GCTT 217 it '“"““— WIMP 48 — PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTMG~ 3’ and 5’ -AAGCTTCGGCATA- 3’ >Wimp48 . TTTTTTTTTTTGGAAATTATGAGTGCAGGCCTACAACTTGGAATGCTGTA ACATCAAAAACAAAACAATGTGAAGAAAAATCAAAACAACATCCTCCCCC CTATAAAATTCTCTAGGCGAACGTTCTTATGTTGGTGCCATTTGAACACC CCTCCCCCCTTTCTCTCTCTCAGGGATAAAGGTGAAACTTCTATTGGCCT ACTTTTTAACGTCCAAGACCTTGCCCAAGTTCCTGTCATCTGAAGGGAGC AGGGAGCANGATTGGACGCAACAATAGTTAAATCAAACTAGATTACACTT AGCTTGCAGACACCTTCTTCCGAACAACCCTCTTTCTACGAACCTTTGGT GCTGGTGATTCCCCATTAATGGAATCAGTATTGCCTTGTTTTATGCCGAA AGCTT WIMP 49 - PUTATIVE IDENTITY - RIBOSOMAL PROTEIN PRIMER COMBINATION 5’ -AAGCTnG- 3’ and 5’ -AAGCTTCGGCATA- 3' >Wimp49 TTTTTTTTTTTGGAAAACGGATACTGAAATTAGAAACAAAGTCCGTCCAT CATTCTCAGCTCATACGATATTTAAACTTAGCTTATAAAAAGTAAAACGA CAATCCCTACAAGACATCGAATGGACCCAAGCTGCTACCTGTAAAGAGCC TGAGGAACTCAAATGTTGCTGGGATACATGAAAACTCTTACACGACATCC CATGGACTTTGGAGGCAAGTTGGACTTGAACTTAGCTCTGACAACACCAC TGTTTCCATGAGGCCTTGTGACCTTGCCCCAAATGCAGCGATAGTGGGTC CCATCCCTCTTCACCTTGGCCTTGTATATGTATGCCGAAGCTTATCAAGC TT BLASTX homology >gilB954039|gb|AAF82213.1|AC067971_21 (AC067971) Strong similarity to a ribosomal protein from Arabidopsis thaliana gb|AL161667. Score = 116 bits (288), Expect = 9e-26 Que ry: < 3 3 5 AYIYKAKVKRDGTHYRCIWGKVTRPHGNSGWRAKFKSNLPPKSMGCRVRVFMYPSNI 1 6 2 AYIYKAK K++G+HYRCIWGKVTRPHGNSGWRAKF SNLPPKSMG RVRVFMYPSNI Sbj C t : 5 5 AYIYKAKTKKNGSHYRCIWGKVTRPHGNSGWRAKFTSNLPPKSMGSRVRVFNYPSNI 1 l 2 218 WIMP SO - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTnG~ 3’ and 5’ -AAGCTTCGGCATA- 3’ >Wimp50 CTTTTTTTTTTTGGCGAGACAAGCTACCTTTAGCTAGGAGCCTCTTTTTC CTTCTGCCCGAAAACAACGTTCGACTTGCATGTGTTAAGCATATAGCTAG CCTTCCTTCTGAGCCAGGATCAAACTCGTCTTTTGAGCATGATCACGCCC TGCAGTGGTAGAACCTAGTGAACCAGACGTACGACTTCTGAACCCGAAGC TCTTCTCTTATGCCGAAGCTTATCAAGCTTA WIMP 51 - PUTATIVE IDENTITY - UNKNOWN PRIMERS 5’ -AAGCTNCF 3’ and 5’ -AAGCTTCGGCATA- 3’ >Wimp51 AAGCTTCGGCATAGAAGAAAGTTGGAATGAACAGAGTAATACAGTGAGTT AGAATTGACTAGGCTGAGCTACTCTTTTGAAGTGAAATTGTGACCTCAAC AGTGTGTTGTATATCGCGAGCAAGATCATGTATATATGAAAATCTCTTTC TTTCGATGCTGCAAAAAAAAAAA 219 WIMP 52 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTu£% 3’ and 5’ -AAGCTTGAGTGCT- 3’ >Wimp52 GCTTGAGTGCTGTGCATTGGTTATGTGGTGAGTTGATTGACTGCTACGTA GTTTATCGATGCAAATATGGTATGAGCCATTTTTATTGTTATTTGTAGAG TGTGAGATTATTAGTCGTAGTGGTGTTAATTCCCTAGAGTCGTTTCTGCA ATCATGTCCAATGAGGTTTATAATAAGGAAATTTCAATTACTTGATGTAG TTGAGAAGCCATCTCAGACAAGTTGATATATTTTGTGTTGGTCAAGTTTA GGATTCATGTACAGAAACCGTATTATGTTGCAGGTAATGAATTCATATTT CCTTGAAAAAAAAAAA WIMP 53 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5' -AAGCTHCF 3’ and 5' -AAGCTTGAGTGCT- 3’ >Wimp53 CTTTTTTTTTTTCGAGGTAATATGTTGATTTAATTTAGCCATTTAAAAAA GGATAGAAAATANTTAATTGCATCCATTTTACCTGCAAGAAATAATTATT AGCATGTTTTTCAAATTCCAATGCCAAACTAATCATCTAACTCAATCATC ATGACCAACTCCATCCTCTTCCTCTTTCTCTTCCCACCCTTTATTTCGGG TCCAGTTTCTACAAGTGCTGGTTGGAACGGAGCAACCAGTGCTTGAACCC TCAGCTACTTGAGATACCTCTAATTGAGCACTCAAGCTTAATCAAGCTT WIMP 54 — PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTnfiF 3’ and 5’ -AAGCTTGAGTGCT- 3’ 220 >Wimp54 TTTTTTTTTTTCCAGAGAATAGATTGGTCATGCAACTTCAGTTGGGAAAT CAAATATTTGTATGTAAATATTTAGAGTTTCTTTTACTAAAATAAAAAAC CCCCCCTTCTTGGGAATGAAACAATATTTTGAAGTCTATCCTTTCCATTT ATGTAGTTCCTCAGCACTCAAGCTTAATCAAGCTT WIMP 55 — PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTnC- 3’ and 5’ -AAGCTTCGGCATA- 3' >Wimp55 TTTTTTTTTTTCCCAAAACGAAAACCGTGATCATTATCTGAAATATATCA AATCAGTTGCACCCAACATGCATATTCCCAGCAAGTCGATGCTATATACC AGAATGACTAGTTTAGCAAACTTGGAAGATTTGTTTCCTCGAGTGGCTAG TTAGTACAGTTACTTCATCCTAAAACAAGTACTAAATTCTTGCCAGTGTA TGCCGAAGCTTAATCAAGCTT WIMP 56/57 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTNLF 3’ and 5’ -AAGCTTCGGCATA- 3’ >Wimp56/57 CTTTTTTTTTTTCATACAACTCCACGAAAAGCCAAGTTCAAAACCATATA TAGATATCCATACTGATAGCTATCACCCCAAGATATGCACAGAAGGTGCA GGTTACAACGAATTCTAAATGGTTCACTGAAGGGTCTGATGGCCACCAGT TCTTCCCTTGATTAAGATATGCCGAAGCTTAATCAAGCTTATCCCTCNNG GCTTTGGGNTTTCGGAATTNCGNACCTTTCC 221 WIMP 58 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5' -AAGCTHC- 3' and 5' -AAGCTTCGGCATA- 3’ >Wimp58 AAGCTTCGGCATACAGATGATGAACTGTAAGATCCAAAACGGCGTCATTT GGCATGGAAAATGACAAATTGTAGTTTGGTCCCTGATGTTTTGAAATGTA TCAGTTGGGTCCCTTAATCAATAAATTTTAATTTTGCCCCTTGAAAAAAA AAAAG WIMP 58A - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTnA: 3’ and 5’ -AAGCTTCGGCATA- 3’ >Wimp58A AAGCTTCGGCATAGTATATGTAGTTTCTTCTCCAGTGTAGATTTCATGGA TTTACAATTTGGGGCACTTGTCTATTTTATATCAAAGTAGAGGCATTGGT CGAAAAAAAAAAA 222 WIMP 59 — PUTATIVE IDENTITY - B-GLUCOSIDASE PRIMER COMBINATION 5’ -AAGCTn£# 3’ and 5’ -AAGCTTCGGCATA- 3' >Wimp59 AAGCTTCGGCATAGTATATGTTGATTACACCAACCTAAAGAGGTACCCCA AAATGTCTGCCTATTGGTTCAAGAAACTACTGGAGCGGAACAAACACTAA ACATAGCAGCCAGGCTTTCCTTTCGATTTAGTTGTCTGGAAATTAATATT ATTACAGTAAAGCTATCAAGAACATTGTGTTAAAAAAAAAAA BLASTX homology >gi|9294063|dbj|BAB02020.1| (AB020749) beta-glucosidase [Arabidopsis thaliana] Score = 55.4 bits (131), Expect = 8e-08 Query: > 5 FGIVYVDYTNLKRYPKMSAYWFKKLLERN 91 FGIVYVDY LKRYPKMSA WFK+LL+RN Sbj ct : 4 82 FGIVYVDYKTLKRYPKMSAQWFKQLLKRN 510 Wimp 59 Probe 2/12/00 223 WIMP 59A - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5' -AAGCTHC- 3' and 5' -AAGCTTCGGCATA- 3’ >Wimp59A AAGCTTCGGCATAATGATCGCAAGTTTAAATAGCTACATTCTTTCAATCC ATTTTTCCTTGTAAGTTATGTCAATTTTATGCATGTAAATTTCGTTTTTA TACATGTTTTTCCCCTCCACAATCAATTACCGTTATAATTCCCCTTTGAA AAAAAAAAA WIMP 59B - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTnAr 3’and 5’ -AAGCTTCGGCATA- 3’ >Wimp59B TTTTTTTTTTTAGCAATTGATGACATGCATTAGAAGTAAATCATTTAGTT TCCTCAGAATCTAACAGGCGCTACTTGATCAAAACAATACATATTATTCA TAATTGTACACTATGTATGCCGAAGCTTAATCAAGCTT WIMP 60 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTHA- 3’and 5’ -AAGCTTTAGAGCG- 3' >Wimp60 .AAGCTTTAGAGCGGCAGTACTAGAAATCGAACGTTGGAGATGTAAAACGA AGCAACATTTTTGGTTTCTGTCCCATTTCGATCATGGATTGGTTGCCATT CATGGATAACGAACTCTCCCAATAATTCTTTTCATATGTGATACTGTGAG TTGCCATCAACAAGATGTTTATTTATTTATTTTTACAGAAATGGATGCAT GGATAGGCTAATAAAAAAAAAAA 224 WIMP 61 - PUTATIVE IDENTITY — GLYCINE/PROLINE RICH PROTEIN PRIMER COMBINATION 5’ -AAGCTn£# 3’ and 5’ -AAGCTTACCAGGT- 3’ >Wimp61 TTTTTTTTTTTGGAAGCAGAAATCAGACATATATATATTATGTTTATGTT GGACCAGCAATGTAATCATCAAAGTCAGCTAAAAAGGTTAGCTGGAGATG GTAAAAAGTAAGATTTATTCAAAGAAACCAGAATCTGCTAATGCGTAGCC AATCAAGACTAAATCCAGCTGACATGCAGGATGATCACTTCCACTTCTTA AATTTCCCTCCTCCGTGCTTCTTCCCAAACTTCCCACGCTTGAACTTTCC ACCGCCGTGCTTGCCGCCGTGCTTGAATTTTCCATGACCACCACCATAGA ATCAAGAAAGAGCTCTCAGTCTGTCAATCCTTACTATGTCTGGACCTGGT AAGCTTAATCAAGCTT BLASTX homology >gi|2129603|pir||865780 glycine/proline-rich protein GPRP - Arabidopsis thaliana>gi|1465364|emb|CAA59059.1| Score = 54.3 bits (128), Expect = 6e-07 Query : < 2 9 8 YGGGHGKFKHGGKHGGGKFKRGKFGKKHGGGKFKKWK 1 8 8 YG GHGKFKH GKH GKFK GK G GGGKFKKWK Sbj ct : 14 5 YGHGHGKFKH-GKH- -GKFKHGKHG-MFGGGKFKKWK 177 NORTHERN - NO HYBRIDIZATION 225 WIMP 62 - PUTATIVE IDENTITY - A. THALIANA SUBTILISIN-LIKE PROTEIN, P. ABIES ANTIFREEZE-LIKE PROTEIN PRIMER COMBINATION 5’ -AAGCTnG- 3’ and 5’ -AAGCTTACCAGGT- 3' >Wimp62 TTTTTTTTTTTGGAAACATAGAATGAATAGATACATATGAAAGAAATTAC TATTTAAATTGAATTTGTCTATGTGAAATAATGATCCTCGTGTGGAAGCA AGTGTTTCTTAGTTTTCATCAAATATAAGGCTGTAAGAATTCTGTCCTGA CAGACAAGGGACTTCTCACAATATGCACTCCATCAGTCCATGTAAGGCTC CCAAAAGAGTACTCCGTGTTCACCTGGTAAGCTTAATCAAGCTT BLASTX homology >gi|12323571IgbIAAG51764.1IAC066691_4 (AC066691) subtilisin-like protein; 10849-13974 [Arabidopsis thaliana] Score = 52.3 bits (123), Expect = 1e-06 Query: < 232 AYQVNTEYSFGSLTWTDGVHIVRSPLSVRTEFLQPYI 122 +++VNT++ FGSL WTDGVH V P+SVRT+F++ Y+ Sbjct: 717 SHRVNTDFYFGSLCWTDGVHNVTIPVSVRTKFMRNYV 753 NORTHERN HYBRIDIZATION - NO SIGNAL 226 WIMP 63 — PUTATIVE IDENTITY — UNKNOWN PRIMER COMBINATION 5’ -AAGCT11G- 3' and 5’ -AAGCTTACCAGGT- 3’ >Wimp63 AAGCTTACCAGGTGAAAGGTAGAGTGCTAGATACATAGTCAGTGTAAATA TGTACTGACATGTTTTGTGCTGTGTGAAACTAGCTGTATTTGCAATATAA TTATTATTGCTTTCTTTTACTTGCAGCCAAAAAAAAAAA WIMP 64 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTHG- 3’ and 5’ -AAGCTTAGAGGCA- 3’ >Wimp64 CTTTTTTTTTTTGGGAGAGTAAGATCACCGTATTATTAGCTTAAAACATA TGTACAAAAGCATGCAATTGAACTAAAAAAACATCCAGCAAAATAGAAAG GGCATCACAAACAACATAGTTGTAAGTAATGTTATTAACTACAAAATACA TTTGTGAATAATAAAGTTCTAGGCATAATTCATTCAGGAATAACTAAACT TATTGCTACTTCACCAAGTGACATCCGAACAGCTCTAGTTGATCAAATTG AGATTCAGTTGGTGTTTCGCTTGTTTCGCTTTTCTTGATTCTACATGAAA TTCACATGTTTGGATAAGCCCCGATGCTAATTTTCTAAGGAATATGACCT ATCCTTCCTCATCCTCAGATTGAGACGTGTTACTTGTGGTAAGTTATCTG CCTCTAACTTAATCAAGCTTA‘ 227 WIMP 65 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTUEF 3’ and 5' -AAGCTTAGAGGCA- 3’ >Wimp65 TTTTTTTTTTTGAAACTCAATGCAGATTAGATTAACCATATACAACGTAG CGAGAAGACAAACAGCAATAAAATCAACAAAGATGACTCCGATGCCCTCT CATTTGCCCAAATGAGTGAACCCATGTTCAGATTAAGATTACAATATTAT AGCCAGCAATGAAGTCGACAAATTTTCTCCATTAGAGTGATGCCAACAGA GGCTTCACCATAGGAACTTAATTTGTGTGATCTCCAAGAGACGAAAATTC ATCCACTTCACCACTTGAGGCAATCCATTTATTACATTGCAAATTGTCTA AAGTTATTATAATGCTCATCGCCCAGCAAGTGCTCTGTAGCAAACCGCTC CCTTGTGTGCCTCTAAGCTTAATCAAGCTTA WIMP 66 — PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTHG- 3’ and 5’ -AAGCTTAGAGGCA- 3’ >Wimp66 AAGCTTAGAGGCAGAAAGGGAATTCCAGGCTCTGGTCAGTTTAGAGGGTG ATTATCTTGGACCGGTGTCTTAAAGTTCTACCTTGCACTCAGCTGGAGTA TCATATCTTCTGAAGTTTTCTAATCAAGTTAGTTTAACTGCCAGCTTGTT GGTTGTTTCTGTAGTATTGTATATCCTTGTGCCACTCTACTCCTTTTTGT GACTGTTGTCTTGGAGTTCTTGCAACTTCAGTGATTGCTAGACTATATGT TTAGCAACTTGATGTTTCCTTCTGTATGCAGATGTTGACAAATAGTATAA TTGATTCGAGGACATCTCATCTTTTGTCAAAAAAAAAAAG WIMP 67 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTHCF 3’ and 5’ -AAGCTTACCAGGT- 3’ 228 >Wimp67 TCAACCTTATTGATAGCGGTCAAANTCAANGGGGGGGCNCGGNTTCCAAT TCCCCNTATTNGNTTTTTTTTCNNNNCTCTCGGGGGGCNTTTANAACNTT TTANTGGGGAAACCGGNGNTCCAATTTTTAGNCTNGGGGTTCCCNTTTCN CTNGGTTTTTNAAAAGNCNCATTTNCTTCCAAATTGGCGCCGATGGGAAG GAATNACCTAATTNTNTAANCNNTATTTGNTTGNCTTTTANNAGNGAANG AACCTTNACAAAAAAAAAA 229 WIMP 68 — PUTATIVE IDENTITY —DEHYDRATION RESPONSE PROTEIN PRIMER COMBINATION 5’ -AAGCTuC- 3’ and 5’ -AAGCTTACCAGGT- 3’ >Wimp68 TTTTTTTTTTTCGATTAACAAAATGCTCAAGTAGGCCCTTTGTGCTGGTT TACTCTTCTCATGGAGGTCTCTCTTTTCACATTACATGCTCATGTGGATA GGAAAATAGCAACAGAAGGAGATGTTATCTAATAATAAACACCGTGATTC ACTGAATCTATAGCAAGCTTTATATTATACATAACCTTTGTAGTGGGTTT TAGATGTATTATTATGAAAACCGAGTAGATATATAAGCATACAACATTCC AATTTCAGCTACCGTGGACCGAAGAGAAACGAAATGACTGGGATCCANAC TCCTAGTTGGGAAGCCACACAACATGATCCTGAAGAANGAAATGGCAGAC TGGTACGGTTCCTGGCTTAACATTGANCACCTGGTAAGCTT BLASTX homology >gi|1172874|sp|QOBZ98|RD22_ARATH DEHYDRATION-RESPONSIVE PROTEIN RD22 PRECURSOR >gi|479589|pir||S34823 dehydration-induced protein RD22 — Score = 43.4 bits (100), Expect = 0.001 Query: < 389 AYQVXNVKPGTVPVCHFXLQDHVVW 315 A++V VKPGTVPVCHF + HVVW Sbjct: 365 AFKVLKVKPGTVPVCHFLPETHVVW 389 NORTHERN HYBRIDIZATION 4 8 230 WIMP 69 — PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTnC- 3’ and 5’ -AAGCTTACCAGGT- 3’ >Wimp69 AAGCTTACCAGGTTAAATAAAGGTGTTCTCAACTAGCCTACCTGGTTAAA TAAAGGTGTTCTCAACTGGCCTACCTGGTTAAATAAAGGTGTTCTCAACT GGCCTACCTGGTTAAATAAAGGTGTTCTCAACTGGCCTACCTGGTTAAAT AAAGGTGTTCTCAACTAGCCTACCTGGTTAAATAAAGGTGTTCTCAACTG GCCTACCTGGTTAAATAAANGTGTTCTCAATTAGCCTACCTGNTTAAATA AAGGTGTTCTCATCTANCCTACCTGNTTNAAATAAAAGGTGTTCTCAACT . AACCCTACCTGNTTTAAATAAAGGTGTTCTCCAACTAACCTACCT 1R WIMP 7O - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTMCF 3’ and 5’ -AAGCTTACCAGGT- 3' >Wimp70 AAGCTTACCAGGTCATGCCATCATTTATTTGTACAAAGATGCACGCACAA GTGCGCTTCCTGTGAATTTGACTCCTTCAAAAAGACAGGATAGCAATAAG GTCAAACATTGTTATTGATATTGAGTTTGAAACTGATCTTGCAGTTTCCT GTAGAAGCCAACAGTACCCGATGAAATATGCTGTTGTTTCATGTAAAATT GTTATTATCATAACTAATAAGGCATTTTTCTTCTCCGAAAAAAAAAAAA WIMP 71 — PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5' -AAGCTHLF 3’ and 5' -AAGCTTACCAGGT- 3’ >Wimp7l TACCAGGTCCAGACATAGTAAGGATTGACAGACTGGGAGCATGTCGGTTG TGCTTAGATTACTGGGAAACTACATTCCAGCTGTAGTGTGAACCAACACC 231 AGATATTTAAATAAAATGCCCGTCTTTCTTGTGTTGGCAGAACTTCTTTC AGGGTTGTTTGGATATAGCATGTCAAGAAAATTTTAGTATTTTATCTTTT AATAAGACAATATAGTGTGAAAAAAAAAAA WIMP 72 — PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ —AAGCTnC— 3’ and 5’ -AAGCTTACCAGGT- 3' >Wimp72 TTTTTTTTTTTCCATCCAAAAGATAGGCATCGAATCATCCCAAGTAGTTC AAGCTTACAAAAGGATCAAAAAGGCAAATTATAAACTGGCAAAGACAACA ACCAAGTTTCACAAGACAAATTAAAGGAAGAGTGAACATTCCAACAAAAC CACCTGGAAAAGCCACCAACACCACCTGGTAAGCTTAATCAAGCTT WIMP 73 — PUTATIVE IDENTITY — UNKNOWN PRIMER COMBINATION 5’ -AAGCTnC- 3’ and 5’ -AAGCTTACCAGGT- 3’ >Wimp73 AAGCTTACCAGGTGCCAGATCAATGCTTATTATGAAGGGCATTTAGCTTT GTTTGAGTTCATATTTCTCCTCTTATTGTTTTGCAGAGTTTTTGTTTCAG TCTCTGTGGTTAAATAGACTGATGACTTTTGGGAAAAAAAAAAAG WIMP 74 — PUTATIVE IDENTITY — UNKNOWN PRIMER COMBINATION 5’ -AAGCTnAr 3’ and 5' -AAGCTTACCAGGT- 3’ >Wimp 74 ACAACAATCATTTANCATTTCTATANCCAAAATTCACATNAATNCCAGTC CAANAAGGTTTCTGAAGATAGATTAAAGTGTATAAAGGGGAANAANACNC 232 ATTTCAGAGCTCGAGGGACANGTCNANCTTGTNAATNCGACCATCTTNAT CNGTCNNCNNGTGATCNTTGCCTATCNCCCTACCNTNTCCACNGTCTGCC GGAGACACTNTCTNCTNNTCCCCCCCTTNTTNACCCTTNCNACACTGGCC A WIMP 75 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTnAr 3' and 5' -AAGCTTACCAGGT- 3' >Wimp75 TTTTTTTTTTTACGACAAGGAAAGGGGGAGCAGAAGACCTCGAATTACTT l TATTTACCGTTTAAAAAAGCATTGCAGATGATAATTGGTAGAGACATGGC I ATCGTTATAGTTTCTGTGAAGTCGGCATTGCTAGAGAAACTCCACCACCA CCTGGTAAGCTTAATCAAGCTT WIMP 76 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTuAr 3’ and 5’ -AAGCTTAGAGGCA- 3’ >Wimp76 TTTTTTTTTTTAACTCGCAACATCATATATTAAACTAAAATGCCAGTGCT TGAGGACTTAAAGCACCTCATGGATAATACATAGTAAACATAGATAGATT CAATCTGAAAATATAATTAAACATTACTGCGTATCTTGCTTATCCTGCCT CTAAGCTTAATCAAGCTT 233 WIMP 77 - PUTATIVE IDENTITY — OSMOTIN LIKE PROTEIN PRIMER COMBINATION 5’ -AAGCTnA- 3’ and 5’ -AAGCTTATCGCTC- 3’ >Wimp77 CTTTTTTTTTTTAGCAAGACGAAAGCATCTAAAATTAAGATTTGATTAAA CAGAAACCCCAAACATGACATTCCCATACACACAACATAAAGACCAGAAG ATTATGACATTATCAATCTTTCCTAATATTCCAAGCTGGGCCAGAGAGAG AATTAGGATAGATCATACAAAAGACCAAAACATAGATACGATATTCATTC TTCGACTTTGCTACTTACAGACTCTTTTAGTGACAAAAGATAACTTTAAG TTCACGTGGCGAAGAACACTCATGCGTGAGCGATAAGCTTAATCAAGCTT BLASTX homology >gi|2501182I8plQ41350|OLP1_LYCES OSMOTIN-LIKE PROTEIN PRECURSOR >gi|2129934|pir||JC5237 Score = 41.4 bits (95), Expect = 0.003 Query: < 289 SLSLTHECSSPRELKVIFCH 230 S SL HECSSPRELKVIFCH Sbjct: 233 SPSLMHECSSPRELKVIFCH 252 NORTHERN HYBRIDIZATION 234 WIMP 78 - SEQUENCED TWICE POOR SEQUENCE PRIMER COMBINATION 5’ -AAGCT11A- 3’ and 5’ -AAGCTTATCGCTC- 3’ 235 WIMP 79 — PUTATIVE IDENTITY - ESTERASE PRIMER COMBINATION 5’ -AAGCTME# 3’ and 5’ -AAGCTTTCTCTGG- 3’ >Wimp79 TTTTTTTTTTTGGAGGGTGCCGTTGGGGACGGGAAGAGGAAGTACAAAAG TTACTCCAGATCACCATCACGACATAGTTACCACATTATGTATGGATTAC TGCAGACTTAAAGTTTAAGAGCATGAGCATGGTGGTGAATGTGGTCATCG ATGAAAGTGGCAATGAAAAAGTAGGAGTGATCATAACCAGGTTGTAATCG CATCAGAACTGAAACATTTGCACTCCTGCATGCCTCTTCAAACTTGTTTG GCAGCAGCTGATCATGCAAGAATTTATCCTCATCTCCCTGATCAATTAAA ATGGTAGCAGATACATCATGGACCTTTGACACCAGAGAAAGCTTAATCAA GCTT BLASTX homology >gi|6984138|gb|AAF34769.1|AF227624_1 (AF227624) putative esterase D [Euphorbia esula] Score = 129 bits (320), Expect = 2e-29 Query: < 338 SLVSKVHDVSATILIDQGDEDKFLHDQLLPNKFEEACRSANVSVLMRLQPGYDHSYFFIA 159 SLVSK HDVSATILIDQG +DKFLH+QL+P KFEEACR ANV +L+R PGYDHSYFFI+ SLVSKFHDVSATILIDQGGDDKFLHEQLMPGKFEEACRLANVPLLLRTHPGYDHSYFFIS 115 Sbjct: 56 Query: < 158 TFIDDHIHHHAHALKL 111 TFIDDHI HH AL L Sbjct: 116 TFIDDHIRHHVQALNL 131 NORTHERN HYBRIDIZATION C 1 2 4 8 1 2 Band 79 236 WIMP 80 PUTATIVE IDENTITY — UNKNOWN PRIMER COMBINATION 5’ -AAGCTnAr 3' and 5’ -AAGCTTATCGCTC- 3’ >Wimp80 AAGCTTTCTCTGGGGATCAACTGACTTCTGCTAAAGAAAAAGCGAGCAAC TGGTTGCATATGAAATGTTACCATAGAAAGGAGTTATGCATCTGGCCACA CTTTCAGTGCGACAAGGGTCCGTTTGTTAACAAACGCCACTTAGTGAGAA GTTCTATATCGGGCCACGCCTGTTGGACTGATCGTTTTGTGCCTATATTT AATTGATGAGGATTTCATTTTGTTACTCTAATCTTCACGCTATTTTGTTA CCCTTATTCTTGTTACTTGTGATGCCCTGTCCCAAAAAAAAAAA WIMP 81 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTuG- 3' and 5’ -AAGCTTGTTGTGC- 3’ >Wimp81 TCGCAGGAATCGATTAAGCTTGTTGTGCAGTTAAATATCATGTACAATGT AAATTAAATAATAATAATAATAACAGCACTCTATCTCCTTTATTGTTTAT CCAAAAAAAAAAAG WIMP 82 — PUTATIVE IDENTITY — UNKNOWN PRIMER COMBINATION 5’ -AAGCTuG- 3’ and 5’ -AAGCTTTTGATCC- 3’ >Wimp82 AAGCTTTTGATCCTGTAGTAGCGTGTAAGTCCTTTCAAATTGTTCTTTTT TCTACTCACAGTGGGGAATAAGTCAGGCTTCAATGGCTAAGTTATCACTT TAGAGTATGTTTTATTTGTTTTTTCATGTCTTATTGTTTTAGCCTGTGTT ATTTCTTTCTTTCTTGCCTTCTTGTATACGGTTTGTGATCTGTATTTTGT GGTANATACTGTAGATCCATGGAAAACGTTGTTATTGTCATCGACCAGAA GATTTTGGCCTANTAAAATAGAATTCTTGTGTGCAAAAAAAAAAAG 237 WIMP 83 - PUTATIVE IDENTITY — UNKNOWN PRIMER COMBINATION 5’ -AAGCTuG- 3’ and 5' -AAGCTTTTGATCC- 3' >Wimp83 CTTTTTTTTTTTGGGACGATAAAGCAAGGGTATTTGTGCCATACAAGTTT AAGAAATTATCTCATACAACATGGAGGAGATGAACAGTATCATTTCTAGC GGCCAAATACTTTGGGATTGGGATCAAAAGCTTAATCAAGCTT WIMP 84 — PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTnfiF 3'and 5’ -AAGCTTTCTCTGG- 3’ >Wimp84 CACGTATGTAAATTTACACAGAACAACTCTGAAAAGATACAACATAGATG AGCATCGACCGAAGAGAAAGGTCTGCAACATTTGGAAAAGAAATGAGGAA AAAATTACAACATACAAGGAAACCAAAACTCTACAAACCATCTAAAGCAC AATTAGAAGATGTTGTTATACTGACTGGTGAGAGAATCCCTGANAATTGA ATAAAGACGTAAAGCGAGTACTGTCNAAAGTTTTCCCCTCCTACCNTGAA TTTAAAACNCAAAAATTNTTGTCNNNTGCCAAAAAAACC 238 WIMP 85 - PUTATIVE IDENTITY - SINA PROTEIN PRIMER COMBINATION 5’ -AAGCTnCF 3’and 5’ -AAGCTTTCTCTGG- 3’ >Wimp85 AAGCTTTCTCTGGTGGGGATAGGAANGAGCTGAAGCTAANAGTTACCGGG AGGATATGGAAGGAACAACAAAATCCANAAACTGGAGTGTGTATACCCAA CCTTTGTAGCTAAGGCAAAGTGATTTTGAAGACATTTACACTATTTGTGT GTTTGGGACTGTTGCGTGTTGTCCCAACCTCTTCCACCCTTCTCTCACAT GCTGTAATGGATTGAATTAATGAAATTCCCCATTGTTTTTTTTTTAAATA AAATTGTATAATAAACAGCCGTTCTTCCCGAAAAAAAAAAAA BLASTX homology >gi|9759183|dbj|BABO9798.1| (AB013388) developmental protein SINA (seven in absentia) [Arabidopsis thaliana] Score = 63.6 bits (152), Expect = 6e—10 Que ry : > 6 FSGGDRXELKLXVTGR IWKEQQNPXTGVC I PNLCS l 1 0 FSGGD+ ELKL VTGRIWKEQQNP +GVCI ++CS Sbj Ct : 2 2 8 FSGGDKKELKLRVTGRIWKEQQNPDSGVCITSMCS 2 62 NORTHERN HYBRIDIZATION C C 2 4 8 11211 Band 85 239 WIMP 86 - PUTATIVE IDENTITY -UNKNOWN PRIMER COMBINATION 5’ -AAGCTnC- 3’and 5' -AAGCTTTCTCTGG- 3' >Wimp86 CTTTTTTTTTTTAGAAACACGGAATGTACTAGTATTATTCTAATTTGTAT TTGTGGGGAATGGCAATTCAACACTATCAATCAAAAACGAAAATGAAAAG CAACATGAGCCTTCCAGCTGCAAATTGGGCCTACATGCGAAATGATTTTA CCAGAGAAAGCTTAATCAAGCTT WIMP 87 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTnAr 3'and 5’ -AAGCTTTTGATCC- 3’ >Wimp87 AAGCTTGTTGTGCGAAAGTAATGGAAGGATGAAAGAGAGTGCCATTGAAT AATATCATCCAGAAAAATGTTTAATGGCCTGTTTACAAGGCCAAAATGCT ATCAATCAGTATCTGTCTTGTTTAAAAACGTGTATATGCATTATATTGTG ACCAGTTCAAAGAGAGTAGCTGGATAATTAAATCTGCTGGCAAAACATGA GCGCGGATGCCCTCGCCCCCTCCCTGTATTTTGTACTTTGTTTAAATTTA AACACATTTTAATTTTAAATTGTTTCCTCCTAAAAAAAAAAA WIMP 88 NO REAMPLIFICATION - NOT CLONED 240 WIMP 89 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5' -AAGCTnAr 3’ and 5’ -AAGCTTTCCTGGA- 3’ >Wimp89 TTTTTTTTTTTACAAGGAGAATGAGAATTATTATAGAAATGCAGACCTTA ACTGTACAGAACATTAATTCAAGCTGTCCAGAGAAAGTATAAATTGTATT ATACAATTACAACCAAGGAATGGCTATAATGAAACATCAATTTTCTTTAT TCTTTGAGGGGCAACTTTTTTATTTTGGGTTGTCGGTAAATATCATTAAT CCAGAGAAAGCTTAATCAAGCTT WIMP 90 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ 'AAGCTnAr 3’ and 5’ -AAGCTTTCCTGGA- 3' >Wimp90 CTTTTTTTTTTTAGAAACACGGAATGTACTAGTATTATTCTAATTTGTAT TTGTGGGGAATGGCAATTCAACACTATCAATCAAAAACGAAAATGAAAAG CAACATGAGCCTTCCAGCTGCAAATTGGGCCTACATGCGAAATGATTTTA CCAGAGAAAGCTTAATCAAGCTTAANTCCCGCCCGTNANAAAAATTGGGC NATTATGGCCCCCC WIMP 91 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTnAr 3’and 5’ -AAGCTTTTGATCC- 3’ 241 WIMP 92 - PUTATIVE IDENTITY - ASCORBATE PEROXIDASE PRIMER COMBINATION 5’ -AAGCTuG- 3’ and 5’ -AAGCTTGGCTATG- 3’ >Wimp92 AAGCTTGGCTATGAAACAGAAGATACGAGCAGAGTATAAAGCAATTGGTG GAAGCCCAGATAAGCCTCTCCAGTCTTAACTATTTTCTAAATATCATGAT CACGATTGCTGTTCTTGCATTCTTAACATATCTTCTTGGAAATTATGTTT GTTTTTGAGGATTGTTATAAAATAGTGTTGTTTCTACCCAAAAAAAAAAA BLASTX homology >gi|7484621|pir||T12282 L-ascorbate peroxidase (EC 1.11.1.11) precursor — common ice plant Score = 35.6 bits (80), Expect(2) = 1e-08 Query: > 8 AMKQKIRAEYKAIGGSPDKPLQS*LF 85 +M+QKIRAEY+ GGSP+ PL + F Sbjct: 385 SMRQKIRAEYEGFGGSPNNPLPTNYF 410 Score = 42.6 bits (98), Expect(2) = 1e-08 Query: > 78 NYFLNIMITIAVLAFLTYLLGNY 146 NYFLNIMI +AVLA LTYL GNY Sbjct: 408 NYFLNIMIVVAVLAVLTYLTGNY 430 NORTHERN HYBRIDIZATION C 2 4 £3 12Ih Band 92 242 WIMP 93 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTHG- 3' and 5’ -AAGCTTGGCTATG- 3' >Wimp93 TTTTTTTTTTTGGAAACAAAGGACCAAACAACGTGGAAATACCTTCCAAC CATGATTATACAAAGACGATGTCTGACCAATACAGTATGCAACATCATTG AGCTTAAGAGCTAAACAGATCTTCATTTCCTATAGCTCACTACTTGATAT ACTGAGAAAGCCACCTAAAACCCTCTCCATAGCCAAGCTTAATCAAGCTT WIMP 94 - SEQUENCED TWICE - POOR SEQUENCE 243 WIMP 95 - PUTATIVE IDENTITY - RECEPTOR KINASE PRIMER COMBINATION 5' -AAGCTuG- 3’ and 5’ -AAGCTTGGCTATG- 3’ >Wimp95 TTTTTTTTTTTGGTAAGTTGCTACTAAAACATCTCGATAGACTTGAAACT AGCTGACCCGAGATTTTTATACGGGCCGGGTGATACCTCAGTCTTCTCCT CCAAGACCCGTTTAGGCCCTCTTCTCCTCCTATTCCAGATCTTATAACCT CCATAGCCAAGCTTAATCAAGCTT BLASTX homology >gi|11358818|pir||T48397 S-receptor kinase-like protein - Arabidopsis thaliana Score = 52.3 bits (123), Expect = 7e-07 Query: < 170 MIKLGYGGYKIWNRRRRGPKRVLEEKTEVSPGPYKNLGSASFKSIEM 30 M+ + Y G++ W R KRVLEE +SPGPYKNLGS SF S+EM Sbjct: 437 MVAMVYVGFRNWRRE----KRVLEEDNGLSPGPYKNLGSDSFNSVEM 479 NORTHERN HYBRIDIZATION 244 WIMP 96 - PUTATIVE IDENTITY - SELENIUM BINDING PROTEIN PRIMER COMBINATION 5’ -AAGCTuG- 3’and 5' -AAGCTTCACTAGC- 3’ >Wimp96 AAGCTTCACTAGCCCATGAGATGAGATACCCAGGAGGTGACTGCACATCA GATATATGGATCTAAGCATCCTGGATGGTTTGGCAGTGAGGGGTTACCTG AGAACAGACCTTAGGAAGCAATAATATCAATGTGTAAATTGCAATGGCAT GTTGTGGCACTTACTCTTTGATGGATGAGTAACTTCGTTCCTTTTAATGC TTTAATCCATCGTTTAATATCAAATGATAATTGCAGTTTCATAATCAACA TAAACTTATGCCAAAAAAAAAAA BLASTX homology >gi|6094242|sp|023264|SBP_ARATH PUTATIVE SELENIUM-BINDING PROTEIN >gi|7488183|pir||D71401 Score = 47.3 bits (110), Expect = 4e-05 Query: > 6 SLAHEMRYPGGDCTSDIWI 62 SLAHEMRYPGGDCTSDIWI Sbj ct: 472 SLAHEMRYPGGDCTSDIWI 490 NORTHERN HYBRIDIZATION <3 1 2 4 23 12 h Band 96 245 WIMP 97 - SEQUENCED TWICE - POOR SEQUENCE WIMP 98 - PUTATIVE IDENTITY - UNKNOWN WIMP 99 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTuG- 3’and 5’ -AAGCTTCACTAGC- 3’ >Wimp99 CTNGCNTNCCTNAGTTCCTGAGTNCTAGAGCGANCGANNAANCTTTTGGG NTCTGGACTAACNTACCTCGTAATAATATACGCTTATCCATATGTCACNN NANNTNTGCTNATGANNAGNTACACATANCTTATTTAANTAANAACTNCA ANANCAGTGCATGGTGGTGGAGTAAGGANTCAGAAAGNTANCATGGNAAT CTNCAAGAGTNCGAGCTNCAATCCNGTGAATCTNANTCAANCTTANNGAT NCNGTCCACC WIMP 100 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTHG- 3’and 5' -AAGCTTCACTAGC- 3' >Wimp100 TTTTTTTTTTTGGTGCAGGAGGAGGTAATGCTGGAGCTTTATGTGGAGGA GCTTTTGGTTTTGGTACTCCAGTATGGTGTTGGTGATGGCTGCTGCCATG GCTTTTCCCTTTCTTCTTGGCTAGTGAAGCTTAATCAAGCTTATCGATAC CGTCGACC 246 WIMP 101 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5' -AAGCTn£% 3’ and 5’ -AAGCTTCACTAGC- 3’ >Wimp101 TCGATTAAGCTTCACTAGCCATGATTACCCAATGCCCGATGGACAGTGGA CAAATACATGCATTGATACAGGTAGTGGCACACTACACCATTCTCCGTTT CTTTTCCCAAAAAAAAAAAG WIMP 102 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTnfiF 3’ and 5' -AAGCTTCACTAGC- 3’ >Wimp102 TTCGATTAAGCTTCACTAGCCACTCGAGGAAACAAATCTTCCAAGTTTGC TAAACTAGTCATTCTGGTATATAGCATCGACTTGCTGGGAATATGCATGT TGGGTGCAACTGATTTGATATATTTCAGATAATGATCACGGTTTTCGTTT TGGGAAAAAAAAAAAG WIMP 103 - PUTATIVE IDENTITY - UNKNOWN POOR SEQUENCE WIMP 104 - PUTATIVE IDENTITY - UNKNOWN POOR SEQUENCE 247 WIMP 105 - PUTATIVE IDENTITY - FLAGELLIN PRIMER COMBINATION 5’ -AAGCTUEF 3’ AND 5’ -AAGCTTACGATGC- 3’ >WIMP105 TTCCTATGGCTTTNACAGTAAACACCAACGTAGCATCGTTGAACGTCCAG AAGAACCTGGGTCGNGCCTCCNTNGCTCTTTCGACCTCGATGACTCGTCT GTCCTCCGGTCTGAAAATCAACAGCGCTAAAGACGACGCTGCCGGCCTGC AAATCGCTACCAAGATCACTTNGCAGATCCGTGGCCAGACAATGGCGATC AAAAAC WIMP 106 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTHEF 3' AND 5’ -AAGCTTACGATGC- 3’ >WIMP106 TTTTTTTTTTTGATGAATTTCAATGTATATACATGTCAAGTGAACTGAAG AACCTGGAAAGTCTTATCGAGCCTCAGGCAGGCTAATTGTCAATTCGTTG TTAGAATATATAGTTGATGTGTTGTGAAAGGCAACAAAATTGCTAGATCT CCGACGACTAAGACTAGAGAATGCAGGTTCTTTTGGAGAAGGAAGTGATG CATTGTCATTGCTCAACATCTGAACCACTAATGGCATCGTAAGCTTAATC AAGCTT WIMP 107 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTUEF 3’ AND 5’ -AAGCTTACGATGC- 3' >WIMP107 TGCAGGAATTCGATTAAGCTTACGATGCCATTTTAATCGGCAAATCTCTG CCATATGTTGTTATCTGAGAGGAATTTAAGCATGGTTGCTCGACGTTGTT TATGTTCACAGTTTGATATTATACCAATCAAATTACAATAGGATTGTACT TCCCAAAAAAAAAAA 248 WIMP 108 - PUTATIVE IDENTITY - METAL-BINDING PROTEIN PRIMER COMBINATION 5’ -AAGCTuG- 3’ AND 5’ -AAGCTTAGCAGCA- 3’ >WIMP108 AAGCTTAGCAGCAGCAGCACCAGCACCAGCTGAGGAAACTACAGAAGAAA CCACGGTGGTAGAGCTCAGGAAAATGGATTTTTATAATTATTATTCACCA ACAAGATATGAGCACTATTCACCACCTCCTCAGATCTTCAGTGACGAGAA CCCCAATGCATGTTCTGTTATGTAAGACAGTTTTGAAAATGAAGGGCAAG ATGGGATGGGAAAAAATAATTACGGGCGATAAATTTGCTGCTTATAAATA ATATATATATATATATATCCTGCTTCCATGTGCAAAAAAAAAAAG BLASTX homology >gi|6469127|emb|CAB61745.1| (AJ275311) farnesylated protein [Cicer arietinum] Score = 52.3 bits (123), Expect = 2e-06 Query: > 44 EETTVVELRKMDFYNYYSPTRYEHYSP ------- PPQIFSDENPNACSVM 172 EET WE++K ++Y Y + + P PPQIFSDENPNACSVM Sbj ct : 5 2 EETKWEMKKNEYYYKYGTEVFAYPDPAYPLQAYPPQIFSDENPNACSVM 1 O 1 NORTHERN HYBRIDIZATION (32 4 812 h Band 108 249 WIMP 109 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCT11G- 3’ AND 5’ -AAGCTTAGCAGCA- 3’ >WIMP109 CTTTTTTTTTTTGGCAAAAATACCGT CGATAATAAAGTAGTAAATCACCTTAGAAAACATCCTTAAGCATCCAGAT ACCATAAAATTAGACAAAAACATAAAATGAGATCTTTCGGAAACTTTAAA AAAACCCAAAAGAGCAACAATTTCTCAATCATTCAGAAATCTCAACATCA CCATCAGTAATCTCCTGCTGCTAAGCTTAATCAAGCTT WIMP 110 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTHEF 3’ AND 5’ -AAGCTTAGCAGCA— 3’ >WIMP110 CTTTTTTTTTTTGGGCTGAAATGGCAGGAATTGCAATCAAATGTTGCTCA CAGATCCCCCACTTAATTATATATTGTGTTGTAGAGGAGAAAAAATAAAA ACACAAATGGCAGCATGACATGCACAAAGAGGGTTTTGCTGTTTTTGTGC GTCATTCTGGCTGCTGCTGCTGCTGCTAAGCTTATCAAGCTT 250 WIMP 111 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTuG- 3' AND 5’ -AAGCTTAGCAGCA- 3’ >Wimp111 TTTTTTTTTTTGGAAAGAGACTAAAACTTTGATTCAAACATTTGGGACAA AATTTAGACTAGAATATGATGATAAATACAACTAAGATTCCTAAAATTAC ACTGATTAATTAATATAGCAAAAACAAAACTCACACCCCTAATTATACAC CAATTCTGCTGCTAAGCTTAATCAAGCTT WIMP 112 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5' -AAGGTuG- 3’ AND 5’ -AAGCTTAGCAGCA- 3’ >Wimp112 TCGATTAAGCTTAGCAGCAGATATATGTACAGTATTTTTGTTTCCAAATG GGGATTGGATGGTAGGTCGTGCTATGGCTTTACTGTTTGTTTAGTTGTCA TTGTTACCCTAATTGTTTATTATTCAAGGTCTACTGTTTCTTCCAAAAAA AAAAA WIMP 113 - PUTATIVE IDENTITY - HYPOTHETICAL PROTEIN PRIMER COMBINATION 5’ -AAGCTnG~ 3’ and 5’ -AAGCTTCGTACGT- 3’ >Wimp113 .AAGCTTCGTACGTTCAGAGGAAGATGATATGGATACATCCTAGGAGCAAA (3GCAAGTGAAGCGTTTTTATGATAAGCTTTTAAAGAGCATCCTTTGTGAA 'TGGATGTTCTTCAATGGTCTGAACAGTATTGTTACTCCTACATCTGAAAT TPCACTTCAATATACCGTGAACTTAAACTTTTTGAAGACAATGCATAGCTG IXCATGTCACTCTACAACATTGGCAAGGTGTGTGGTTTCCCATTGCATAAA (3CTGTTTAATCTCAGCTGTAATGACTTGAAATGGTCACTCTGATGATCTT .AAGAGCGTATTTCTTTGTTTCCCCAAAAAAAAAAA 251 WIMP 114 - PUTATIVE IDENTITY — RIBOSOMAL PROTEIN 4OS PRIMER COMBINATION 5’ -AAGCTnG- 3' and 5’ -AAGCTTCGTACGT- 3’ >Wimp114 TTTTTTTTTTTGGGGATTCTAAAACTTCAAAACTTGAATGGAAAATTAT TAAAATCTTGGACATAACGAAATTTCAAAATATAATAGCTAAGTAATAGA TTCTTGTTCGGGCAGAGAGCGAAAAGAACATGGTTCACGAAAATGAGAGC AGTTGGCCACTTTCTTGCACTAACAATGTTTAAAGGACAAAAACAAAACT CTAATGCAGTTACTGCTGGCGGAGTTCAGTCTTCTTCTTTTGCCAAAGCC TGTCCACGGCACTGTCAGCATCACCCTGGGCACGTACGAAGCTTAATCAA GCTT WIMP 115 - PUTATIVE IDENTITY UNKNOWN PRIMER COMBINATION 5’ -AAGCTNE% 3’ and 5' -AAGCTTCGTACGT- 3’ >Wimp115 TTTTTTTTTTTGAACATACACAAAGAATCCTATTCTTGATCTGAATCTCG AGTTCTTGGATTTTAATCTTATTGAACAAATTAATCAAAGCTTGATCAGC TTCCTTTTAACACCTCCAAACGTTGAAGAATCGCAAATAACCAAGTAGTA GTCTTTTACTTGTGATCATGATGAGCTTGATTTGATGACAGTACGTACGA AGCTTAATCAAGCTT 252 WIMP 116 — PUTATIVE IDENTITY — RIBOSOMAL PROTEIN 4OS PRIMER COMBINATION 5’ -AAGCTn£# 3’ AND 5’ -AAGCTTAGCAGCA- 3’ >Wimp116 TGAAAATGAGAGNAATTGGTCANTTTCTTGCACTAACAATGTTTATAGGA CANAAACAAAACTCTAATGCANTTACTGCTGGCGGAGTNNATTCTTCTTC TTTCGCCANAGCCTGTCCANNGCACNGTCAGCATCACCCTGNGCACGTAC GAAGCNTAATCAANCTTATCGATACNG WIMP 117 — PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTnG- 3’ AND 5’ -AAGCTTAGCAGCA- 3’ >Wimp117 TTTTTTTTTTTGGAATAAAACACTCAACAGCTATATATGACAGCCAATT AAGACAACAACTAACAATAGATAGGTGATTATTACACAAGCCAGCCAATC GGGGGTTAAGATTTTTGCTTCTTGTCCATTACAAATGTCTAGATCTTTGA TTTCTATATACGTACGAAGCTTAATCAAGCTTCNAAAAGAGGAAAANCAA GNACGGCCNGGCNGCTTTTCNTCCCCCTTTCNCCCCCCCCNTNATCCCCT AGGNCTNTNTGTTTNCGGAATTCGAACCCCTTTTTTNCNNTCCATTTNCC CC WIMP 118 PRIMER COMBINATION 5’ -AAGCTnG- 3’ AND 5’ -AAGCTTAGCAGCA- 3' >Wimp118 TGCAGGAATTCGATTAAGCTTCGTACGTGCTAAATTCTAGCTGATCAACT TGTGCTGTATTAAAGACTCGGATGATATCCTGAATCCATCAAGGCCTGTT ATATTACATACGTTTTTTCCAAAAAAAAAAA 253 WIMP 119 PRIMER COMBINATION 5’ -AAGCTn£F 3’ and 5’ -AAGCTTACGATGC- 3’ >Wimp119 TTTTTTTTTTTCAACCATGATTATACAAAGACGATGTCTGACCAATACAG TATGCAACATCATTCAGCTTAAGAGCTAAACAGATCTTCATTTCCTATAG CTCACTACTTGATATACTGAGAAAGCCACCTAAAACCCTCTCCATAGCCC ATTTTGCGGACAATGCTACACATGAATACCTCAAGGGGACGGACATTTGA GTCGACCAAGTTCACCTTACCCTTGCCAGTGGTGAAGTTGGTGAGACCCA GGTTGTAACGCAACTCATCTTCCGAGGCAGCATATGGGATATCAATCTTG TTGCCCAAAACAAGAAA BLASTX homology >gi|3334323Isp|004834|SARA_ARATH GTP-BINDING PROTEIN SARlA Score = 139 bits (347), Expect = 9e-33 Query: < 317 FLVLGNKIDIPYAASEDELRYNLGLTNFTTGKGKVNLVDSNVRPLEVFMCSIVRKMGYGE 138 FL+LGNKIDIPYAASEDELRY+LGL+NFTTGKGKVNLVDSNVRPLEVFMCSIVRKMGYGE FLILGNKIDIPYAASEDELRYHLGLSNFTTGKGKVNLTDSNVRPLEVFMCSIVRKMGYGE 183 Sbjct: 124 Query: < 137 GFRWLSQYIK 108 GF+W+SQYIK Sbjct: 184 GFKWVSQYIK 193 Band 119 254 WIMP 120 PUTATIVE IDENTITY — UNKNOWN PRIMER COMBINATION 5’ -AAGCTn£F 3’ and 5’ -AAGCTTACGATGC- 3' >Wimp120 TTCGATTAAGCTTCACTAGCCACTCGAGGAAACAAATCTTCCAAGTTTGCTAAACTAGT CATTCTGGTATATAGCATCGACTTGCTGGGAATATGCATGTTGGGTGCAACTGATTTGA TATATTTCAGATAATGATCACGGTTTTCGTTTTGGGAAAAAAAAAAAG WIMP 121 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTULF 3’ and 5' -AAGCTTACGATGC- 3’ >Wimp121 TTTNCTCTTTCGACCTCGATGACTCGTCTGTCCTCCGGTCTGAAAATCAA CAGCGCTAAAGACGACGCTGCCGGCCTGCAAATCGCTACCAAGATCACTT NGCAGATCCGTGGCCAGACAATGGCGATCAAAAACGCCAACGACGGTATG TCCCTGGCGCAAACCGCTGAAGGCGCACTGCAAGAGTCGACCAACATTNT GNAGCTGTATGCGTGAACTGGCTGNCCAGTCGCGAAACGACAGCNACAGT GCCACCGACCGTGAAGCGCTGAACAAAGAATTNATCATGGCTTTAACAGT AAACACNAACGTAGCATNNATG WIMP 122 PRIMER COMBINATION 5’ -AAGCTMCF 3’ and 5’ -AAGCTTACGATGC- 3’ >Wimp122 TAGCAGCAGCAGCATAACTGGCATTGACACCCATCCAATGTCCAATGATA ACAATATTGCAACCGGTACACTTAGCAGCAAAGGCATGATTGTCAATGGC TGCAATGAGGTTGTCAAGCAAGAAGTGCTGAAGAACACTTGATGCATTTC TAGAGCACGGAAAGTAAAGATGTAATTTCATTATTTTAGATGGGGTTTCA TTTTTCAAGCTGAGGCAGATAA 255 WIMP 123 PRIMER COMBINATION 5’ —AAGCTMCF 3’ and 5' -AAGCTTACGATGC- 3’ >Wimp123 AAGCTTAGCAGCAGAAGCTTTGGAAGACATCACTGCTCTCTTCTATGATG AAGAGCGCAATGAGATCTATACAGGCAATAGGCTTGGTCTAGTTCATGTG TGGTCTAACTGATTTTTTGACAAATCCTTGTTTGCTTAAGGTTGTTAATG TTTAATCAGATGGTTGAATGAGCATGCTGCCTGGATGTGAGAGTCCCCTG A WIMP 124 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTUCF 3’ and 5’ -AAGCTTACGATGC- 3’ >Wimp124 TTTNCTCTTTCGACCTCGATGACTCGTCTGTCCTCCGGTCTGAAAATCAA CAGCGCTAAAGACGACGCTGCCGGCCTGCAAATCGCTACCAAGATCACTT NGCAGATCCGTGGCCAGACAATGGCGATCAAAAACGCCAACGACGGTATG TCCCTGGCGCAAACCGCTGAAGGCGCACTGCAAGAGTCGACCAACATTNT GNAGCTGTATGCGTGAACTGGCTGNCCAGTCGCGAAACGACAGCNACAGT GCCACCGACCGTGAAGCGCTGAACAAAGAATTNATCATGGCTTTAACAGT AAACACNAACGTAGCATNNATGAACGTTCAGAAGAACCTGGGNCGCGCCT CCGACGCTTNTTTCGACCTCNATGACTCGCTGTTCTCCGGTCTGAAAATC AACNAGCCCTTCAAGACNACGCNTNTCGGTCTGCAANTTGCTACCCAAGA CACTTTGCAGATNCGTGGCCAGACAAATGGCGANTCAAAAA 256 WIMP 125 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5' -AAGCTn£F 3’ and 5’ -AAGCTTAGCAGCA- 3’ >Wimp125 TAGCAGCAGCAGCATAACTGGCATTGACACCCATCCAATGTCCAATGATA ACAATATTGCAACCGGTACACTTAGCAGCAAAGGCATGATTGTCAATGGC TGCAATGAGGTTGTCAAGCAAGAAGTGCTGAAGAACACTTGATGCATTTC TAGAGCACGGAAAGTAAAGATGTAATTTCATTATTTTAGATGGGGTTTCA TTTTTCAAGCTGAGGCAGATAA WIMP 126 - PUTATIVE IDENTITY - HYPOTHETICAL PROTEIN PRIMER COMBINATION 5’ -AAGCTu£F 3’ and 5’ -AAGCTTAGCAGCA- 3’ >Wimp126 AAGCTTAGCAGCAGAAGCTTTGGAAGACATCACTGCTCTCTTCTATGATG AAGAGCGCAATGAGATCTATACAGGCAATAGGCTTGGTCTAGTTCATGTG TGGTCTAACTGATTTTTTGACAAATCCTTGTTTGCTTAAGGTTGTTAATG TTTAATCAGATGGTTGAATGAGCATGCTGCCTGGATGTGAGAGTCCCCTG AAACTGTAACATTTACCATACTTGTACCATGTCATATTTCTTTTTGGTTT TGGAAATTGATTTCCTTGTGTCTGTTTCCCTCGAAAAAAAAAAA WIMP 127 — PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTMCF 3’ and 5’ -AAGCTTAGCAGCA- 3' >Wimp127 AAGCTTAGCAGCAGCAACTTCAACAGCAGCAGGAGCAACAGCAGCAGCAA CAAGGTGGGGATTTGAAAATGAGAGGATCATTGACTTCTTCAAGCCAGAA AGATAATGGATCTGAGGCTAATTCCTCTTCATCAAAGGATTGAAAATGAG AAGGCAGCTACTTAATGTTCTCTACTGTTGTCTACATCCTAGTTGCAATC TGTTGTCACATAGGATATTTTCCCTTCCTTTTATCTCCAGTCCTTCTAAT ATACATTACCGAACATTTCTACCGAAAAAAAAAAAGC 257 WIMP 128 PRIMER COMBINATION 5’ -AAGCT1flZ- 3’ and 5' -AAGCTTAGCAGCA- 3’ > Wimp128 TTTTTTTTTTTCGATACACAGCAAAACTAACGAAGATATTAAATTATAAA TAAGAGGACAAAAGGAAGAAAAAGAAAGGTCCATCATAATCCGATCTCTC TTACACACAAACAAACAAATAGAGACAGGGGGGGTGGATAAAGACAAGCT CTTTTCATTTAATTTGTGGGGTGGCTACGGAAAGATCCCAAAGCCTTAAT GGCTCCAGCTCTCAGAATGTACTGGTGGTATGCAGCTGCTGCTAAGCTTA ATCAAGCTT BLASTX homology >gi|8071628|gb|AAF71820.1lAFl41900_1 (AF141900) putative aquaporin Score = 55.0 bits (130), Expect = 2e-07 Query: < 248 SLAAAAYHQYILRAGAIKALGSFRSHPTN 162 +LAAAAYHQYI LRA AI KALGS FRS+PTN Sbjct: 251 ALAAAAYHQYILRAAAIKALGSFRSNPTN 279 258 WIMP 129 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTnfiF 3’ and 5’ -AAGCTTAGCAGCA- 3' >Wimp129 TTTTTTTTTTTCAATGAAACGCAGGTGGTAATGGAAAGCTATAAGATAAT AATATATTTGGAAATTACAATAAAGAAAAGAAAAGAGTGTGATATCGTCT TTCTGCTGCTAAGCTTAATCAAGCTT WIMP 130 - PUTATIVE IDENTITY — RUBISCO ACTIVASE PRIMER COMBINATION 5’ -AAGCTNLF 3’ and 5’ -AAGCTTCGTACGT- 3’ >Wimpl30 TTTTTTTTTTTCAAAAAATACCAATAGAAGAGATTATTCAATAATATACT CAATTGACTATAAAAAAGAAGTCAGCTGTTATATATACCTTGATATTAGG CAGGCTCATGAAGTTCTTGGAGATGTGAACAACAAGCTTGTCCATGAAAG CAGGAGCAATGTAGAAACCATCCATGTTGTTGTCCAAGTTGTACCTAAAG CACAGTCACCAAGTCATGTTCATAATCAAACTTGTCCACAACTTCAGACG AAATGGAAATCATAATTCATAAGTATTAGCTAAACAGCGAGAATAATCTA CAATAATTCCCATATATAAAAGTATGGTACTTACGTACGAAGCTATCAAG CTT WIMP 131 PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTnC- 3’ and 5’ -AAGCTTCGTACGT- 3’ >Wimpl31 TTTTTTTTTTTCAAGTCAATTTGCTGTCACGCACACATGCAATATAGATT ATAATTGCAACGTGACAATGGTGTGTAACATCAAGCCAATTCAACAAAAC TTGTTCTATTTTAATTCGGCTCAACTATACAACGATCATCTATTTGGCCT TTACAGAGTATTTTGCTCGCAAAGCAAGACCTAATCCAATACGTACGAAG CTTAATCAAGCTT 259 WIMP 132 - PUTATIVE IDENTITY — UNKNOWN PRIMER COMBINATION 5’ -AAGCTnAr 3'and 5’ -AAGCTTACGATGC- 3’ >Wimp132 AAGACCAACTTTGTCTTTGAATTCTCATAGATCAGTCATATTAAATATGA AAAGTTAAGCACAGCTTAGACATGATAAAAACAAGGCACAGTTAGAACAA GACAGCCTTCTACATTGCAATAGCACCCGTCACTTTCTGGTGACAAGGGT TAGCCTGCAACTATTTAAGCCTACATGTAGATATCTACAGTATATGTGTC TGAGTACTTGTCAACCTGTATCTGCATCGTAAGCTTAATCAAGCTTATCG ATACCGTCGACC WIMP 133 SEQUENCED TWICE - POOR SEQUENCE WIMP 134 — PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5' -AAGCTnAr 3’and 5’ -AAGCTTACGATGC- 3' >Wimp134 AAGCTTACGATGCTGTAGATTTTCTGTTCTGAGAATTTGATTGGATTGTA ACAAATTTTCCTTTGAATTATAGAGATCCACCTCTTCGGTTTTCTAAAAA AAAAAG WIMP 135 — PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTHG- 3’and 5’ -AAGCTTGGTGAAC- 3’ >Wimpl35 AAGCTTGGTGAACANAANATATTATTTTGCCAAGTATATTANTTNTAAAA NGTCGTNGTCTCTGGCNTANTATCANTNACTNTACCATCCAANGCNNANT ANNCNAGNTAGCCTCATATGTATTTAATANCANTACTGGGACCATTGTNT ANCTGCGAAATCAGTACCTGATNATGATTTCTAACTTTATTTCAAAAAAA AAAA 260 WIMP 136 PRIMER COMBINATION 5’ ~AAGCTuG- 3’and 5’ -AAGCTTGGTGAAC- 3’ >Wimp136 TTTTTTTTTTTGGGGAAGAATGGTATTTGCGAAGACCGGAACAGAAAAGC TAATGATAATTGAATAGTTGGACCTAATCCTTATACTTCATCTCAGACAT CAGAAAGCCAGGCATCTCAAAGGAGCCTGAGAACTGTTCAACATCAGCCT TGAGGGCCTCGATTTCCTTATTGTTCACCAAGCTTAATCAAGCTT BLASTX homology >gi|7433539|pir|lB71400 glycine hydroxymethyltransferase (EC 2.1.2.1) - Arabidopsis thaliana Score = 63.2 bits (151), Expect = 4e—10 Query: < 181 MVNNKEIEALKADVEQFSGSFEMPGFLMSEMKYKD 77 +VNNK+++ LKADVE+FS S+EMPGFLMSEMKYKD Sbjct: 437 LVNNKDLDQLKADVEKFSASYEMPGFLMSEMKYKD 471 NORTHERN HYBRIDIZATION . "1 ”Band 136 261 WIMP 137 - PUTATIVE IDENTITY — UNKNOWN PRIMER COMBINATION 5’ -AAGCTuG- 3’ and 5’ —AAGCTTCCTGCAA- 3’ >Wimpl37 NTNNATTCNANTAANCTTCCTGCAAACATTCTAGANGATGCTGACTGCGC GGATNTCGATCTCCTGAAACCCACCCATTGAATGGTTGAAATATCTTGGA ANTCGCCCACGTTACNNGCGTGGGTNNTTCAANTNNCTAGGAGCGANATN NGGNGGATGNTCANTATNGTTTTTGCTATNCNGNANCNNGCCTTNCTGGG CACCTGTCTNATGTCATCGTGACCGTCCGGTTCAGGGCNGGNAGNGTTNC CCCNACCANTTCCNGNGANCATCAGNTGANTNTCCC WIMP 138 — PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ —AAGCTnG~ 3’ and 5’ -AAGCTTCCTGCAA- 3’ >Wimp138 AAGCTTCCTGCAAGGAAAAGAGTATGTGCAAGCTTTTGTTAGCAAGGACG ACAGTGCTCGTGCTGTTGATGCTGCTGCATGAAATCTGCTTTCGCTGGTG TAAATCAAACTGAAATATTAGCATGATCATATTTTCAAAATAAAATACCC TGTCTTGGAAAACAGAATTTAAAACCTTCCTAACTGTGATAACTATATCT ATAACCTATATGGCTGCTTGTGCTTGTGCCAAAAAAAAAAA WIMP 139 PRIMER COMBINATION 5’ -AAGCTHG~ 3’ and 5’ -AAGCTTCCTGCAA- 3’ >Wimpl39 GGTAATCCAGGTTCAGGTGTGCTCACTAATCTACTCTTAAGCATAACAAT AGAATATGATAACTTAGGTGTAAACCTTAGCAAGTGGTGTATCTTCTATT CTTGGTTTCATTCCAAAAAAAAAAA 262 WIMP 140 — PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ -AAGCTuG- 3’ and 5’ -AAGCTTCCTGCAA- 3’ >Wimpl40 AAGCTTCCTGCAAGTTGGTGGGTGATTGCCCGTTGTAATAATGTATGGGG TCTTAGAATTGGAGTGAGGTTGTAATCATTTCATAAGAAAACATTTTGTA AAGAGTGTTTCTGCCTTCTCTGCCTCCAAAAAAAAAAA WIMP 141 - PUTATIVE IDENTITY - UNKNOWN PRIMER COMBINATION 5’ —AAGCTUCF 3’ and 5' -AAGCTTGGTGAAC- 3’ >Wimpl4l CTTTTTTTTTTTCGACAGCTAAAAACCAGTAACAAATCAATAACCAAGC TCCCAACTTTCCTGAGCTTCAGAATCAGCTTTAGCTTATCCTGGCAAAGA TTAAAAAAAGAAACTACAACAAATATGACATCTCCTACACGGCAGTTGTC AGTATTCAGTACCCATACACTACTCAATTTAGTTCACCAAGCTTAATCAA GCTTAAAACTTGNACCCCCTCAAGGGGAAAACCCTTTTNGGGNAAAGCCC CTNNTAAANCCCCCACCNTTTTTGGGGGAGGNGCNNACCCNTTCGANCCG GGGCCCCTTATTNTGGGACGGNNTNCAAAGGGAAAAAANGGGCGGGCCGN TTTTNTCCCCTTCNCCCCCCTANACCCCCGGCNTGGNNTTCGGAATTNNA ACCCTTTTTTTAAACCNTTTNCCCNAAAACCCNANNCCNTTNAAGGANAA TTNATCCCNCCNCCCTGGGGNTCTTTTNCCANCC 263 APPENDIX B RESULTS OF DATABASE SEARCHES 264 BE 02 .w.z 89:2 6:3 SAN .w.z or 3 BE 02 .w.z .w.z BE oz 2 3 mg: 02 E8536 mcoz E8556 0:02 .m.z E. 3 3E 02 28553 0:02 65 .m.z 9. 9 c3053 Eacmtom méw com coon NV Amuse 5 BE 02 Eonchm 0:02 use. 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