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Discovery and Characterization of a Novel Microtubule Associated Protein Involved in Cellulose Biosynthesis Alex Selvanayagam, Rajangam Doctoral Thesis in Biotechnology Stockholm 2008 Sweden Discovery and Characterization of a Novel Microtubule Associated Protein Involved in Cellulose Biosynthesis Alex Selvanayagam, Rajangam KTH – Royal Institute of Technology Swedish Center of Biomimetic Fiber Engineering (Biomime™) School of Biotechnology AlbaNova University Center Stockholm Sweden © Alex S. Rajangam, 2008 ISBN 978-91-7178-980-8 KTH – Royal Institute of Technology Swedish Center of Biomimetic Fiber Engineering (Biomime™) School of Biotechnology AlbaNova University Center Stockholm Sweden Printed at Universitetsservice US-AB Box 700 14 100 44 Stockholm Sweden Dedicated to my dear mom, Mrs. M. Aboorvaseeli Kagoo, living in me ±ýÉ¢ø Å¡Øõ À¡ºÁ¡É «õÁ¡ ¾¢ÕÁ¾¢. Ó. «â÷Åº£Ä¢ ¸¡Ì×ìÌ ºÁ÷ôÀÉõ கற்க கசடறக் கற்பைவ கற்றபின் நிற்க அதற்குத் தக - தி க்குறள் 391 So learn that you may full and faultless learning gain, Then in obedience meet to lessons learnt remain. - Thirukural 391 (Translation by Dr. Rev. G. U. Pope) Alex S. Rajangam (2008), Discovery and Characterization of a Novel Microtubule Associated Protein involved in Cellulose Biosynthesis, School of Biotechnology, KTH – Royal Institute of Technology, Stockholm, Sweden. ISBN 978-91-7178-980-8 Abstract Cell walls are a distinct feature of plants and their chemical constituents, cellulose, hemicelluloses and lignin, are economically valuable. Plant fibres rich in cellulose, which mainly resides in their cell wall, are traditionally used in making paper and textiles. The changing global economic situation and environmental concerns have imparted necessity for renewable, but at the same time value added cellulosic materials. The Department of Wood Biotechnology, KTH together with its collaborators, have established EST libraries and performed transcript profiling during wood development in poplar, a tree considered as a model for wood development. The majority of the genes upregulated during cellulose biosynthesis encode proteins with known or predictable functions, such as carbohydrate active enzymes (CAzymes). However, some of them encode proteins with unknown functions. Characterization of these genes will potentially give additional opportunities to modify fibre properties. This thesis describes the discovery and characterization of a highly upregulated gene with a previously unknown function in poplar xylem, here denoted PttMAP20. Following its early discovery by mRNA profiling, the characterization was initiated with a thorough bioinformatic analysis, and the knowledge obtained was used to devise techniques for further functional analysis. Specific antibodies were raised, affinity purified and characterized. The antibodies were used as a tool for screening recombinant expression in E. coli and for the cellular localization of the protein in plant tissues, visualized with confocal and transmission electron microscopy. A purification protocol was developed for the expressed protein, followed by biochemical characterization. Appropriate model systems were used in both in vivo and in vitro studies. Fluorescently labelled protein transiently expressed in tobacco leaves was used for localization studies and the same system was used to characterize the molecular properties of the protein. Phenotypes arising from overexpressing the PttMAP20 gene were traced in the model plant Arabidopsis. All the results obtained so far indicate that PttMAP20 is a novel microtubule associated protein that binds to a cellulose biosynthesis inhibitor, DCB (2,6-dichlorobenzonitrile) and is required during cellulose biosynthesis in secondary cell walls. Keywords: MAP, unknown gene, cellulose biosynthesis, Populus, microtubule, DCB Copyright © Alex S. Rajangam, 2008, ISBN 978-91-7178-980-8 Abstract in Swedish Alex S. Rajangam (2008), Upptäckt och karaktärisering av ett nytt mikrotubuliassocierat protein som ingår i biosyntesen av cellulosa, Bioteknologi, Kungliga Tekniska Högskolan, Stockholm, Sverige. ISBN 978-91-7178-980-8 Sammanfattning Cellväggen är ett utmärkande drag hos växter, och dess beståndsdelar, cellulosa, hemicellulosa och lignin, är ekonomiskt värdefulla. Växtfibrer består till stor del av cellulosa, som huvudsakligen återfinns i fibrernas cellväggar. Dessa används traditionellt inom pappers- och textilindustrin. Den föränderliga globala ekonomin tillsammans med en växande miljömedvetenhet har tydliggjort behovet av förnyelsebara cellulosabaserade material, som samtidigt medför högre mervärde. Avdelningen för träbioteknik, KTH, har tillsammans med samarbetsparter skapat ett EST-bibliotek och genomfört en transkriptanalys av vedbildningen hos asp, ett trädslag som används som modell för vedbildning. Majoriteten av de gener som är uppreglerade under cellulosabiosyntesen kodar för proteiner med kända eller förutsägbara funktioner, som t.ex. kolhydrataktiva enzymer (CAzymes). Vissa gener kodar dock för proteiner med okänd funktion. Karaktäriseringen av dessa enzymer kan ge nya möjligheter att modifiera en fibers egenskaper. I denna avhandling beskrivs upptäckten och karakteriseringen av en kraftigt uppreglerad gen med tidigare okänd funktion från xylemet hos asp, här kallad PttMAP20. Efter den ursprungliga upptäckten med hjälp av mRNA profilering påbörjades karaktärisering med bioinformatisk analys, och med hjälp av de vunna kunskaperna utformades metoder för vidare funktionell analys. Specifika antikroppar producerades, affinitetsrenades och karakteriserades. Antikropparna användes som screeningverktyg i E. coli och för cellulär lokalisering i växtvävnad, visualiserat med konfokal- och transmissions-elektronmikroskopi. Ett reningsprotokoll utvecklades för det heterologt uttryckta proteinet, följt av biokemisk karakterisering. Lämpliga modellsystem användes för både in vivo och in vitro studier. Fluorescerande protein, kortvarigt uttryckt i tobaksblad, användes för lokaliseringsstudier och samma system utnyttjades för att bestämma de molekylära egenskaperna hos proteinet. Fenotyper från överuttryck av PttMAP20-genen analyserades i modellorganismen Arabidopsis (backtrav). Alla resultat hitintills indikerar att PttMAP20 är ett nytt mikrotubuliassocierat protein som binder till en inhibitor till cellulosabiosyntesen, DCB (2,6-diklorobenzonitril), och som krävs för biosyntesen av cellulosa i sekundära cellväggar. Copyright © Alex S. Rajangam, 2008, ISBN 978-91-7178-980-8 Abstract in Tamil «¦ÄìŠ ¦º. Ã¡ƒ¡í¸õ (2008), ¦ºÂø¸½¢ì¸À¼¡¾ ¦ºøÖ§Ä¡Š ¯üÀò¾¢ìÌ ¬Ã¡öóÐ «¾ý ¦ºÂøÀ¡ðÊ¨É ¸ñÎôÀ¢ÊòÐ §¾¨ÅôÀÎõ ƒ£¨É ¯Ú¾¢ÀÎòÐ¾ø, ŠÜø «·ô À§Â¡¦¼ìÉ¡Äƒ¢, ÌíÄ¢¸¡ ¦¼ìÉ¢Š¸¡ ŠÜÄ¡ý (KTH), Š¼¡ì§¸¡õ, ŠÅ£¼ý, ISBN 978-91-7178-980-8 ÍÕì¸×¨Ã ¦ºøÄ¢¨É ¸Å÷óÐûÇ ¦ºø ÍÅ÷ ¾¡ÅÃí¸Ç¢ý ¾É¢ò¾ý¨Á, «¾ý §Å¾¢Â¢Âø ¯ð¦À¡ÕðÇ¡É ¦ºøÖ§Ä¡Š, ¦†Á¢¦ºøÖ§Ä¡Š ÁüÚõ Ä¢ìÉ¢ý ÀÄÅ¢¾í¸Ç¢ø ÁÉ¢¾É¡ø ¯À§Â¡¸ôÀÎò¾ÀÎ¸¢ÈÐ. ¦ºøÖ§Ä¡Š ¿¢¨Èó¾ ¾¡ÅÃí¸Ç¢ý ¿¡÷¸û ¸¡¸¢¾õ ¾Â¡Ã¢ò¾ø, Ð½¢¸û ¯üÀò¾¢, ÁÃì¸ð¨¼ ¿¢¨Ä¸û ¬¸¢Â ÀÂý¸¨Ç §À¡ýÚ ÀÄ ÀÂý¸¨Ç ¦¸¡ñÎûÇÐ. ŠÅ£¼ý, À¢ýÄ¡óÐ, ¸É¼¡ §À¡ýÈ ¿¡Î¸û «¾ý À¡Ð¸¡ì¸ôÀÎõ ¸¡Î¸Ç¢ø ¯ûÇ ÁÃí¸Ç¢¨É ¦ºøÖ§Ä¡…¢ý ãÄô¦À¡ÕÇ¡¸ ÀÂýÀÎò¾¢ Á¢Ì¾¢¨Â ²üÚÁ¾¢ ¦ºöÐ ¦ºøÅõ ®ðÎ¸¢ÈÐ. ±¾¢÷ÅÕõ ¸¡Äí¸Ç¢ø ¦ºøÖ§Ä¡…¢ø Ò¾¢Â ¾ý¨Á¸¨Ç ÒÌòÐÅ¾ý ãÄ§Á, ¦ºøÖ§Ä¡¨… ±üÚÁ¾¢ ¦ºöÔõ ¿¡Î¸û ¾í¸û ¦À¡ÕÇ¡¾¡Ã ¿¢¨Ä¸¨Ç ¾ì¸¨ÅòÐ¦¸¡ûÇ ÓÊÔõ. §Å¸Á¡¸ Á¡È¢ÅÕõ ¯Ä¸ô¦À¡ÕÇ¡¾¡Ãõ, Áì¸û ¦¾¡¨¸, ¾ðÀ¦ÅôÀ Ýú¿¢¨Ä §À¡ýÈ ÀÄ¾ÃôÀð¼ §¾¨Å¸Ç¢ý ¸¡Ã½Á¡¸×õ ÒÐÅ¢¾Á¡É ÀÂýÀ¡Î¸¨Ç ¦ºøÖ§Ä¡…¢ø ±üÀÎò¾§ÅñÊÔûÇÐ. ŠÅ¢¼É¢ÖûÇ 'KTHÂ¢ý ×ð À§Â¡¦¼ìÉ¡Äƒ¢ Ð¨È', ¾í¸û ÜðÎ ¿¢ÚÅÉí¸Ù¼ý §º÷óÐ À¡ÒÄ÷ ±ýÛõ ÁÃò¾¢ø ¦ºøÖ§Ä¡Š ¯üÀò¾¢Â¡Ìõ §À¡Ð §¾¨ÅôÀÎõ «¨ÉòÐ ƒ£ý¸¨ÇÔõ (ÁÃÀÏ) «¾ý ¯üÀò¾¢ ¾¢Èý («Ç×) ¦¸¡ñÎ ÅÃ¢¨ºôÀÎò¾¢ÔûÇÐ. «ó¾ ƒ£ý¸Ç¢ø ¦ÀÕõÀ¡Ä¡É¨Å ¦ºøÖ§Ä¡Š ÁüÚõ ¦ºø ÍÃ¢ø ¯ûÇ ÁüÈ Á¡× ¾ý¨Á¨ÂÔõ «¨¾ º¡÷óÐ §Å¨Ä ¦ºöÔõ ±ý¨…õ (¦¿¡¾¢) ÒÃ¾í¸¨ÇÔõ ¯üÀò¾¢ ¦ºö¸¢ÈÐ (CAzymes). «ó¾ ÀðÊÂÄ¢ø ÓüÈ¢Öõ ¦ºÂø ¸½¢ì¸À¼¡¾ ƒ£ý¸Ùõ «¼í¸¢ÔûÇÐ. þó¾ Ó¨ÉÅ÷ ¬Ã¡öîº¢Â¢ø Á¢Ì¾¢Â¡É «ÇÅ¢ø ¯üÀò¾¢Â¡Ìõ ÓüÈ¢Öõ ¦ºÂø ¸½¢ì¸ôÀ¼¡¾ ´Õ ƒ£¨É (¾üºÁÂõ PttMAP20 ±ÉôÀÎõ ƒ£ý) ¬Ã¡öóÐ «¾ý ÒÃ¾õ ±ýÉ §Å¨Ä¨Â ¦ºö¸¢È¦¾ýÚ ¸ñÎÀ¢Êì¸ ÓÂüîº¢òÐûÇÐ. PttMAP20 ƒ£ý ¯üÀò¾¢ ¦ºöÔõ mRNAÅ¢ý «ÇÅ¢¨É ¸ñÎ ¦¸¡ûÙõ ¦¾¡Æ¢øÑðôÀò¾¢ý ÓÄõ «Ð ¦ºøÖ§Ä¡¨… ¯üÀò¾¢ ¦ºöÔõ ±ý¨…õ¸¨Ç ´òÐ §Å¨Ä ¦ºöÅÐ ¸ñÎôÀ¢Êì¸ôÀðÎûÇÐ. §ÁÖõ ¾¸Åø ¦¾¡Æ¢ÑðôÀò¾¢ý ÅÊÅ¡É À§Â¡þý·À÷§ÁðÊìŠ…¢ý ¯¾Å¢Ô¼ý PttMAP20 ƒ£É¢ý ¦ºÂø¸û ¸½¢ì¸ÀðÎûÇÐ. PttMAP20 ƒ£ýÛì¸¡É «ñðÊÀ¡Ê (±¾¢÷ôÒºì¾¢ ÒÃ¾õ) ¯üÀò¾¢ ¦ºöÐ PttMAP20 ƒ£É¢ý ÒÃ¾ò¾¢¨É ¸ñÎÀ¢ÊìÌõ Àø§ÅÚ ¦¾¡Æ¢øÑðôÀ §Å¨ÄÀ¡Î¸ÙìÌ ¯À§Â¡¸ôÀÎò¾ôÀðÎûÇÐ. §ÁÖõ PttMAP20 ¦ºøÖìÌû ±í§¸ þÕì¸¢ÈÐ ±ýÚ ÀÃ¢§º¡¾¢ì¸ §¾¨ÅÂ¡É ¯Â¢÷ôÀÊÅí¸û ÀÂýÀÎò¾ôÀðÎûÇÐ. «¾¢¸ ºì¾¢Å¡öó¾ ¨Áì§Ã¡Š§¸¡ôÀ¢ý ãÄõ «¾ý ¦ºÂø¸û ¸ñÎÀ¢Êì¸ôÀðÎûÇÐ. PttMAP20 ƒ£¨É À¡ìÊÃ¢Â¡Å¢ø ì§Ä¡ý (ÁÃÀÏ Á¡üÈõ) ¦ºöÐ «¾ý ÒÃ¾ò¨¾ ¯üÀò¾¢ ¦ºöÐ «¾¢¿Å£É Ó¨ÈÂ¢ø PttMAP20 ÒÃ¾ò¨¾ ÁðÎõ Íò¾¢¸Ã¢ì¸ôÀðÎûÇÐ. Íò¾Á¡É PttMAP20Â¢ý §Å¾¢Â¢Âø ¾ý¨Á¨ÂÔõ ÌÈ¢ôÀ¡¸ ¨Áì§Ã¡ðÎÒÔÇ¢ø ´ðÎõ ¾ý¨Á ÀÃ¢§º¡¾¢ì¸ôÀðÎûÇÐ. §ÁÖõ Á¡¾¢Ã¢ ¯Â¢÷ôÀÊÅí¸Ç¢ø PttMAP20 ÒÃ¾ò¾¢¨É «¾¢¸ «ÇÅ¢ø ¯üÀò¾¢ ¦ºöÐ «¾É¡ø ±üÀÎõ Á¡üÈí¸û ¸ñ¼È¢ÂôÀðÎûÇÐ. PttMAP20 ÒÃ¾õ Êº¢À¢ (DCB, ¸¨Çì¦¸¡øÄ¢) ±ÉôÀÎõ §Å¾¢Â¢Âø ¦À¡Õ§Ç¡Î À¢¨ÉÔõ ¾ý¨Á ¦¸¡ñ¼¾¡¸×õ ¸ñ¼È¢ÂôÀðÎûÇÐ. Êº¢À¢ ¦ºøÖ§Ä¡Š ¯üÀò¾¢¨Â ¾ÎìÌõ ±ýÀÐ ±ü¸É§Å ¸ñÎÀ¢Êì¸ÀðÎûÇÐ. §Áü¸ñ¼ ¬Ã¡öîº¢Â¢ý ãÄõ PttMAP20-¦ÂýÛõ ƒ£ý ¦ºøÖ§Ä¡Š ¯üÀò¾¢Â¢ý §À¡Ð §¾¨ÅôÀÎõ ´Õ ƒ£ý ±ýÀÐõ, ¨Áì§Ã¡ðÎÒÔÇ¢ø ´ðÊ §Å¨Ä ¦ºöÔõ ±ýÀÐõ, ¦ºøÖ§Ä¡Š ¯üÀò¾¢¨Â ¾¡ìÌõ Êº¢À¢¨Â ´ðÎõ ÒÃ¾õ ±ýÀÐõ ¸ñÎÀ¢Êì¸ÀðÎûÇÐ. ±¾¢÷ÅÕõ ¸¡Äò¾¢ø PttMAP20 ƒ£¨É §¾¨Åì§¸üÀ Á¡üÈ¢ «¾ý ¦ºøÖ§Ä¡Š ¯üÀò¾¢ ¾¢Èý ÁüÚõ §Å¾¢Â¢Âø ¾ý¨Á¨Â Á¡üÈ¢Ôõ, Êº¢À¢-Â¢É¡ø ¯ÕÅ¡Ìõ ÍüÚôÒÈ Í¸¡¾¡Ã §¸Î¸¨Ç Á¡üÈ ÅÆ¢Å¨¸ìÌõ. Copyright © Alex S. Rajangam, 2008, ISBN 978-91-7178-980-8 Table of Content PROLOGUE ......................................................................................................................... 1 1. INTRODUCTION........................................................................................................ 3 1.1. CELLULOSE ....................................................................................................... 3 1.2. ECONOMIC IMPORTANCE OF CELLULOSE ............................................ 3 1.2.1. Textiles .......................................................................................................................... 4 1.2.2. Paper and pulp ............................................................................................................. 4 1.2.3. Biofuel ........................................................................................................................... 6 1.2.4. Other uses ..................................................................................................................... 6 1.3. SCIENCE OF CELLULOSE .............................................................................. 7 1.3.1. Chemistry of cellulose................................................................................................... 7 1.3.1.1. Polymerisation .................................................................................................... 8 1.3.1.2. Crystallinity ........................................................................................................ 9 1.3.1.3. Polymorphy......................................................................................................... 9 1.3.2. Biology of cellulose biosynthesis ................................................................................ 10 1.3.2.1. Cellular location ............................................................................................... 10 1.3.2.2. Molecular biology ............................................................................................ 11 1.3.2.3. Enzymology....................................................................................................... 12 1.3.2.4. Allied components of cellulose biosynthesis ..................................................... 13 Sucrose synthase - Sourcing glucan ............................................................................. 13 Cytoskeleton- a key element of cellulose synthesis .....................................................14 Membrane bound cellulase - KORRIGAN .................................................................. 19 GPI- anchored protein- COBRA .................................................................................. 19 Membrane bound protein of unknown function - KOBITO ........................................ 20 1.3.2.5. Inhibitors of cellulose biosynthesis................................................................... 20 1.3.2.6. Model of the biosynthetic machinery ................................................................ 21 1.3.3. Physics of cellulose biosynthesis ................................................................................ 23 1.3.3.1. Polymerization of biopolymer .......................................................................... 23 1.3.3.2. Modelling the movement of CS Complex .......................................................... 24 1.4. CELL WALL – THE DOMAIN FOR CELLULOSE BIOSYNTHESIS ..... 26 1.4.1. Primary and secondary cell wall ................................................................................ 26 1.4.2. Cellulose biosynthesis in secondary cell wall............................................................. 27 1.4.3. Co-expressing unknown genes in cellulose biosynthesis ............................................ 27 2. OBJECTIVES OF THE PRESENT INVESTIGATION ....................................... 29 3. MATERIALS AND METHODS .............................................................................. 31 3.1. TRANSCRIPT PROFILING ............................................................................ 31 3.1.1. Poplar expression profiling on microarrays .............................................................. 31 3.1.2. Quantitative PCR ........................................................................................................ 31 3.2. BIOINFORMATICS ......................................................................................... 31 3.2.1. Sequence retrieval ...................................................................................................... 31 3.2.2. Phylogenetic analysis ................................................................................................. 32 3.2.3. Gene mapping ............................................................................................................. 33 3.2.4. Expression profiling of TPX2 genes in Arabidopsis ................................................... 33 3.3. PROTEIN BIOCHEMISTRY .......................................................................... 33 3.3.1. PttMAP20 antibody production .................................................................................. 33 3.3.2. Protein extraction from wood tissues ......................................................................... 33 3.3.3. Immunoblotting ........................................................................................................... 34 3.3.4. Recombinant PttMAP20 in E. coli .............................................................................. 34 3.3.5. PttMAP20 orthologs gene synthesis, expression and protein purification ................. 35 3.3.6. Binding of PttMAP20 to MT ....................................................................................... 35 3.3.7. Co-precipitation assay of binding of PttMAP20 to poplar MT .................................. 36 3.3.8. Assembly of BMT in the presence of DCB .................................................................. 36 3.3.9. Labeling of PttMAP20 by [3H]DCPA ........................................................................ 36 3.4. IMMUNOLOCALIZATION EXPERIMENTS.............................................. 37 3.4.1. Confocal microscopy, poplar tissues .......................................................................... 37 3.4.2. Confocal microscopy, tobacco.................................................................................... 37 3.4.2.1. Deletion series of PttMAP20, cloning .................................................................... 38 3.4.3. Transmission electron microscopy (TEM), poplar tissues ......................................... 39 3.5. PHENOTYPIC CHARACTERIZATION ....................................................... 39 3.5.1.1. Overexpression of PttMAP20 in Arabidopsis ............................................................. 39 4. RESULTS ................................................................................................................... 41 4.1. DISCOVERY AND EXPRESSION ANALYSIS OF PttMAP20 ................... 41 4.2. BIOINFORMATIC CHARACTERIZATION ............................................... 43 4.2.1. Identification and sequence analysis of PttMAP20 orthologs .................................... 43 4.2.2. Conservation of the extended TPX2 domain of M20L ................................................ 45 4.2.3. Identification of proteins containing a TPX2 domain ................................................ 46 4.2.4. Phylogenetic analysis of TPX2 domain ...................................................................... 47 4.2.5. Genomic organization of TPX2 genes ........................................................................ 49 4.2.6. Gene structure determination and comparison of M20L genes.................................. 51 4.3. BIOCHEMICAL CHARACTERIZATION.................................................... 54 4.3.1. Antibody development and recombinant expression in E. coli ................................... 54 4.3.2. PttMAP20-MT binding studies ................................................................................... 54 4.3.3. DCB Binding Studies .................................................................................................. 56 4.3.3.1. DCB binding of PttMAP20 and its orthologs ................................................... 57 4.3.4. In situ and in vivo localization .......................................................................... 59 4.3.4.1. Poplar immunolocalization, in situ............................................................................. 59 4.3.4.2. Transient expression in tobacco, in vivo .................................................................... 61 4.4. PHENOTYPIC CHARACTERIZATION ....................................................... 63 4.4.1. Overexpression in Arabidopsis .................................................................................. 63 5. DISCUSSION ............................................................................................................. 65 6. CONCLUSION .......................................................................................................... 69 7. LIST OF ABBREVIATIONS ................................................................................... 71 8. ACKNOWLEDGEMENTS ...................................................................................... 73 9. REFERENCES........................................................................................................... 77 APPENDICES .................................................................................................................... 95 PROLOGUE Cellulose, we can’t imagine many things in life directly or indirectly that does not involve it. We eat it, we wear it, we live in it, we sit on it, we write on it, we burn it… but when it comes to science, wondering how it is produced, then we have to say: an untold story. Scientists have been working to bring light in this area with little success. The challenge lies in the complexity of cellulose and its biosynthesis, which have not yet been possible to solve with the available technology. This was the situation when I started this thesis work and – surprisingly – has not changed much during all these years. However, we have now entered the post-genomic era that will allow faster progress than before as it has become a matter of retrieving the pieces of the puzzle by various, often high throughput analyses and putting these pieces together. I started this work when the first plant genomes from Arabidopsis, rice and Populus were progressing beyond the first drafts. At the same time, data were also emerging from the transcript profiling in the same organisms. Populus is now considered a model for wood formation and wood is the richest source for cellulose. The information on the mRNA transcript abundance during wood development in poplar, which was obtained by a Swedish collaboration between KTH, Stockholm and UPSC, Umeå and their research networks, is the base stone of this thesis. These data identified many unknown genes that are highly expressed during wood formation. Particularly interesting were such unknown genes, which were co-regulated with genes known to be involved in cellulose biosynthesis. This thesis focuses on one of the most upregulated unknown genes identified by expression profiling in hybrid aspen and I have structured this thesis in the same fashion as the function of the unknown gene was slowly revealed using various biotechnical tools. 1 2 1. INTRODUCTION 1.1. CELLULOSE Cellulose is a linear organic polymer made up of glucose units. It is the main constituent of the cell walls (or the extracellular matrix) of all land plants, higher algae, oomycetes and some bacteria. Cellulose is the most abundant organic material in nature and represents a renewable, biodegradable, biocompatible and derivatisable natural raw material for various industrial uses. Wood, biologically defined as the prevalent secondary xylem tissue of trees, is the most abundant source of cellulose. Cellulosic materials have contributed to the development of human life in many ways. For example right from the times of cave men it has been used as firewood and later in making potters wheels that directly helped the transition of human life into a developed civilization. There is a wide variety of cellulose-based materials which serve many basic needs in the present day societies (Figure 1). Figure 1: Examples of some sources and uses of cellulose: Pictures showing representative species acting as a source of cellulose; plant secondary xylem cells (hybrid aspen), Oomycete sporangium (Saprolegnia sps.), Cyanobacteria body filaments (Nostoc sps.) and bacterial spores with biofilms (Staphylococcus aureus). 1.2. ECONOMIC IMPORTANCE OF CELLULOSE Cellulose and its derivatives are used in various applications and have become inevitable for human kind. The following section gives a brief summary of some of the economically important cellulose-based products. The discussion highlights the increasing global demand and depleting natural resources that provide cellulosic raw material. The global trends in cellulose production economy are inducing a paradigm shift towards ‘value added’ cellulosic products in industrialized countries. In addition 3 to economic driving forces, this shift also reflects social and the environmental aspects of the future. 1.2.1. Textiles Textiles comprise clothing and household materials made with any kind of fibre or yarn, including plant fibres. Cellulose is the main constituent of plant fibres. The plant yarns used for clothing are mainly derived from cotton, jute, flax and hemp while coconut coir, grasses, straw, bamboo fibres etc are used for making mats, rugs, mattresses and similar products for household use. To understand the magnitude of the fibre market, the world’s leading economy, USA, can be used as an example. The US market alone had a turnover of US$ 1000 billion in 2007 comprising of Industrial, Man-Made Fibres, Greige Goods, Finished Fabrics, Apparel, Home Furnishing and Carpets & Rugs. It had a deficit of more than US$ 80 billion in the textiles market (US Department of Commerce) (Figure 2). Figure 2: Economy of US textile industry (a) US textile and apparel price in 2007 (Bureau of Labour Statistics (BLS), US) and (b) increasing imports of textile and apparel from 1992 to 2007 (US Department of Commerce). 1.2.2. Paper and pulp Paper is still the main material used globally to disseminate information and knowledge and to provide raw material for packaging and household uses. Historically linens, rags and other fibre-rich plant materials were used for paper making while presently the tree boles are the predominant raw material. The long fibres from softwood (conifers) and short fibres from hardwood (deciduous) trees determine the strength, opacity and surface smoothness of paper (Williams, 1994). Softwood trees such as spruce (Klinga et al., 2007), pine (Todorow, 1984), fir (Comeau et al., 2003), larch (Valade, 1998) and hemlock (King et al., 1998) and hardwood trees such as eucalyptus (Dias et al., 2002), aspen (Zarges et al., 1980) and birch (Suryawanshi, 2004) are extensively used to extract pulp. There are also non-woody annuals and agricultural fibres such as e.g. cereal straws (Schott et al., 2001), bagasse (Gavelin, 1979; Granick, 1979), bamboo (Sood et al., 2005), flax (Sain and Fortier, 2002), hemp (Kovacs et al., 1992), jute (Bhushan et al., 1998) and many others that are used by the forest deficient regions (Gutierrez et al., 2004). By 2004, the world’s production of paper and paperboard was as high as 359,602 million tons contributing several thousands of billions of dollars to the world economy (Pulp & Paper International). 4 Figure 3: Wood and Paper Exports and Imports: Ballooned and inflated territory size shows the proportion of worldwide wood and paper net exports (a) and imports (c) and the graphs shows the region wise contribution to the net wood and paper exports (b) and imports (d). Source: www.worldmapper.org, SASI Group (University of Sheffield) and Mark Newman (University of Michigan). Finland is the biggest exporter of wood and paper in the world, followed by Sweden and Canada while developed countries with larger populations such as Japan, United States and Great Britain are the top three importers (Pulp & Paper International). Interestingly, while the US is the biggest producer, it is also the biggest consumer which creates a need for import as well (Figure 4). The increasing trend of per capita consumption in developing nations, for example China and Korea shows a growing demand for paper and paper products in a global perspective. Figure 4: Worlds Paper Production and Per Capita Consumption in 2004. Source: Pulp & Paper International 5 1.2.3. Biofuel Cellulose, in the form of wood, is the oldest source of energy for the mankind. The global environmental concerns and the depleting resources of fossil fuels have created prospects for the production of biofuels, including cellulosic ethanol (Lynd et al., 1991). Cellulosic ethanol is generally produced from non food crops (dedicated crops) and inedible waste products (residues). Corn stocks, switchgrass and soybean stocks are some dedicated crops and residues such as sawdust and bagasse are some of the commonly used biomass for biofuel production. The biochemical conversion of biomass to ethanol currently involves three basic steps, thermo chemical treatments of raw lignocellulosic biomass, enzymatic treatment and fermentation mediated by bacteria or yeast (Service, 2007). Starch was used as the biomass in the past but cellulose is slowly replacing it. It is expected that cellulosic ethanol will reduce the burden of the increasing price of the gasoline. This is illustrated by Table 1 summarizing the goals and impacts of cellulosic ethanol set by US Department of Energy. Cellulosic Ethanol Goals and Impacts (Adapted from Smith et al., 2004) Factors Today - (Starch) Interim - Term (Starch and Cellulose) Long - Term (Cellulose ) Billion gallons Fossil fuel displaced (Greene, 2004) CO2 reduced 4 2% 20 10% 30 to 200 15 to 100% 1.8% 9% 14 to 90% Feedstock Starch (14% energy yield) Waste cellulose Cellulosic energy crops (>37% energy yield) Process Starch fermentation Acid decrystallisation: Transition to enzymes Cellulases Single-sugar metabolism Multiple microbes Some energy crops Enzyme decrystallisation and depolymerisation Large, central processing Distributed or centralized, efficient processing plants Little cellulose processing Deployment Large, central processing Cellulase and other glycosyl hydrolases Sugar transporters High-temperature functioning Multisugar metabolism Integrated processing Designer cellulosic energy crops Carbon sequestration through plant partitioning Table 1: Cellulosic Ethanol Goals and Impacts. Source: Data reproduced from the U.S. Department of Energy Office of Science report (http://genomicsgtl.energy.gov/). 1.2.4. Other uses Raw material: Cellulose is used as the raw material to make synthetic materials such as rayon, cellophane, cellulose acetate, cellulose nitrate, plastics, varnishes, lacquers, inks, adhesives, photographic films, magnetic tapes, artificial sponges, explosives and many other products. All these synthetic products have a very big market in the materials, information technology and allied fields. 6 New applications: Many cellulose based materials are used in medical technology. Due to its biocompatibility, cellulose is very well suited as bandages, wound dressings and sutures. More recently the discovery of artificial circulation tubing and valves with cellulose based materials has brought a new dimension to this field (Klemm et al., 2001; Klemm et al., 2004). Enzyme technology, fibre modification and biomimetic studies are extensively carried out with the cellulose based materials. For example, the XET technology, recently developed at KTH, relies on the principle of natural binding property of cellulose and xyloglucan and allows specific surface modification of cellulose without compromising the mechanical performance of the material (Teeri et al., 2007; Zhou et al., 2007). This technology is presently being commercialized by KTH and Sweden-based companies. 1.3. SCIENCE OF CELLULOSE As early as 1838, Payen coined the name cellulose (Payen, 1838). In the following section, cellulose science is discussed from three different viewpoints, chemistry, biology and physics of cellulose and its synthesis. The aim is to highlight aspects that are already understood and those that remain in the dark and the overview might envision the discovery of new undefined processes. 1.3.1. Chemistry of cellulose Cellulose is an unbranched polymer of glucopyranose units joined by β-(1,4) linkages. Glucose is a six carbon sugar and as one of the carbons has an aldehyde group it is called as aldohexose. Glucose is produced by autotrophic organisms by the process of photosynthesis. The photosynthetic reaction leading to the aldohexose can be briefly outlined with the following reaction; CO2 and H2O forms C6H12O6 and O2. Figure 5: D-Glucose and its derived polymer. D-Glucose shown with Fisher projection, chemical structure of it’s α and β configurations are shown in chair model and their respective polymers; starch and cellulose. Glucose has both D and L stereoisomeric forms and the D-glucose is present in the biological systems. When glucose moieties polymerize they are linked with either α- or β-glucosidic bonds. The anomeric configuration is determined by the position of the hydroxyl (OH) group at the anomeric carbon (marked as 1 in Dglucose and α & β D-glucopyranose in Figure 5), an OH group in the cis position, below the plane of the sugar ring (when drawn in a Haworth projection) leads to the α 7 configuration while an OH group in the transposition, above the plane of the sugar ring leads to β. The position of OH group in the anomeric carbon of the glucose unit determines the fate of the glucose unit. The chemistry and structure of starch and cellulose determine their physical properties such as crystallinity which in turn reflects their biological roles. Starch and glycogen, storage polymers in plants and animals (respectively), are generally loosely packed for rapid solubilisation when energy is needed. In contrast, cellulose is packed as ordered sheets of crystalline material which is responsible for the physical strength of plant cell walls (Figure 5). 1.3.1.1. Polymerisation In a cellulose chain, every second glucose unit is rotated to 180° and this makes two anhydrous glucose units, a cellobiose molecule, rather than glucose the smallest repeating unit of cellulose. The β-configuration of the bonds joining the Dglucose units allows cellulose to form very long and straight polymers unlike starch which has α-configuration leading to a curved structure. Starch also contains branches (Figure 5). The degree of polymerization (DP) of cellulose can be as high as 25000, as in an algae Valonia ventricosa (Mcdonough, 1983) but it is significantly different between different organism and cell types. Figure 6: Schematic illustration of a cellulose molecule forming molecular fibrils: Chemical structure of cellulose molecule showing the functionally (β 1-4 glucose) and structurally (cellobiose) fundamental units integrating into a microfibril. The structure of the cellulose chain is stabilized by intramolecular hydrogen bonds between the glucose units of the same chain. Every cellulose molecule has a reducing and a non-reducing end. This is since the terminal glucose in one end of the chain is free to convert from the cyclic hemiacetal form into the open chain aldehyde form, which can reduce Cu2+. This end is hence called the reducing end. The linearity of the cellulose enables them to align and stack together to make microfibrils (Figure 6). The cellulose chains in a microfibril are connected by intermolecular hydrogen bonds between the adjacent cellulose chains. The resulting sheets of microfibrils are also stabilized by van der Waals bonds and hydrophobic interactions. The 8 polymerisation of the cellulose is postulated to happen at the same time when the polymers stack into well ordered crystals. This has been studied with Calcofluor white staining that specifically blocks the inter-polymer hydrogen bonding (Haigler et al., 1980). 1.3.1.2. Crystallinity The supermolecular structure of cellulose can be characterized by the degree of crystallinity, crystallographic parameters, crystalline dimensions, structural indices of amorphous domains, dimensions of fibrillar formation and other factors (O'Sullivan, 1997). X-ray diffraction pattern of cellulose suggest that a cellulose microfibril consist of 70% highly crystalline material and the rest is composed of para-crystalline regions that are sometimes described as amorphous regions (Fink et al., 1987). The crystalline regions consist of well ordered layers of cellulose chains while the cellulose chains in amorphous regions are more disordered. Naturally occurring cellulose in the cell walls of plant fibres has lower density as compared to pure cellulose implying that it contains a larger fraction of less ordered, amorphous cellulose. For example the density of crystalline cellulose in cotton fibre is 1.55g/cm3 while single cellulose crystal is 1.59 g/cm3 (O'Sullivan, 1997). 1.3.1.3. Polymorphy When cellulose has been studied with various modern techniques it has been shown to exist in 7 different polymorphic forms: Cellulose I (Iα and Iβ), Cellulose II, Cellulose IIII, Cellulose IIIII, Cellulose IVI and Cellulose IVII. However, only three of these forms, Cellulose I (Iα and Iβ) and Cellulose II, exist in nature (Figure 7). These polymorphic forms are interconvertible. Cellulose I is the native form of cellulose in most organisms while a few organisms, such as Acetobacter xylinum, have cellulose II naturally (Kuga et al., 1993). The Iα and Iβ allomorphs are found in different ratios in different cellulosic materials and differ with respect to physical properties brought by their crystal packing, molecular conformation and hydrogen bonding (Nishiyama et al., 2003). In cellulose I the glucan chains occur in sheets of cellulose chains that are organized parallel to each other, their reducing end pointing to the same direction (Stipanovic and Sarko, 1978) and the individual chains are antiparallel relative to each other in cellulose II (Kolpak and Blackwell, 1975). Cellulose II is the most extensively studied cellulose allomorph since it’s the most thermodynamically stable form due to an extra hydrogen bond per glucose residue compared to Cellulose I. It is formed from Cellulose I by regeneration, solubilising in a solvent followed by reprecipitation by dilution in water, or by mercerization which is done by swelling the native fibres with sodium hydroxide treatment (Atalla et al., 1976). Other crystalline forms of cellulose can be obtained by physical and chemical treatments and often they have properties that are not found in native cellulose. Cellulose IIII and Cellulose IIIII are formed by treatment of cellulose I and cellulose II, respectively, with liquid ammonia or some amines and subsequent removal of ammonia. The polymorphs IVI and IVII are prepared by heating celluloses IIII and IIIII respectively to 260 ºC, in glycerol (O'Sullivan, 1997 and references therein). Recently a non-crystalline form of cellulose called as nematic ordered cellulose (NOC) has been obtained and it is used to visualize the single glucan chains with high-resolution transmission electron microscopy (HRTEM); the strand is visible because of the surrounded uranyl acetate negative stain (Kondo, 2001). 9 Figure 7: Transformation of cellulose into its various polymorphs 1.3.2. Biology of cellulose biosynthesis Although cellulose is one of the simplest polysaccharides known, nonenzymatic, chemical synthesis has not been very successful. Cellulose-producing organisms have cellulose only in their cell walls or as an extracellular matrix. The important common feature among them is that cellulose is always located outside the cell membrane. This feature leaves only two options for the location of the cellulose producing protein machinery; either the cellulose synthesizing enzyme can be inside the cell and export the product outside the membrane via vesicular transport or the enzyme/s could be present on the membrane and produce the product directly into the exterior of the cell. The later option is more likely considering the high amount of cellulose produced, the velocity of the synthesis and the crystallinity of cellulose in various organisms. Figure 8: Structure of cellulose synthase rosette. Six ‘lobed’ rosette from (A) an Algae, Micrasterias denticulata (Picture from the Giddings et al., 1980) and (B) a Plant, Vigna angular (Picture from the Kimura et al., 1999). A: The P-face of a complex consisting of hexagonal array of rosettes and best preserved rosette are composed of six particles seen in cropped and enlarged (x10) image (scale bar 0.1μm x 200,000) and B: Immunolocalization of cellulose synthase rosette with antibody conjugated with gold label (scale 0.1 μm; bar in inset = 30 nm). 1.3.2.1. Cellular location The present evidence suggests that cellulose is synthesized by particulate complexes called the cellulose synthases (CS) which are found in the cell membrane. 10 The components in the CS complex are still not evident except for the protein referred to as the catalytic subunit of cellulose synthase (CesA). Cellular location of the CS has been studied using various microscopic techniques. The first speculations that CS might be located in cell membrane were presented as early as 1958 (by Roelofsen). The first proof was the visualization of an organised macromolecular structure in the plasma membrane in Oocystis apiculata, an alga. As this structure was attached to the growing end of the cellulose chain, it was called the Terminal Complex (TC) (Brown and Montezinos, 1976). CS have since also been visualized in other cellulose producing organisms such as bacteria (Zaar, 1979), mosses and ferns (Rudolph and Schnepf, 1988), algae (Mizuta and Brown, 1992b) and many vascular plants (Emons, 1994). The morphology of the CS in the membrane studied in plants suggests that it holds a rosette shape. The rosettes are believed to consist of a number of catalytic subunits (CesA) and the number is predicted with the size of the rosette (with microscopy) and the size of cellulose microfibril. Microscopic images of the CS rosettes labelled with antibodies in Vigna angular suggest that each rosette complex has six lobes (Kimura et al., 1999). Such six lobed rosette complex has been reported in many other plant species (Figure 8). 1.3.2.2. Molecular biology The first cellulose synthase gene was identified in Acetobacter xylinum (Saxena et al., 1990; Wong et al., 1990) and later in a cotton fibre cDNA library (Pear et al., 1996). Sequence comparisons revealed that these genes encode glycosyl transferases that belong to the Cazy family GT2 (www.cazy.org/fam/GT2.html). The first structure of a GT family 2 was gotten from the x-ray crystal diffraction of a bacterial extracellular SpsA protein from Bacillus subtilis. This SpsA protein is not a cellulose synthases but it belongs to the same family as the CesA proteins and their catalytic mechanism and catalytic residues are thus expected to be similar. Figure 10: General membrane topology of cellulase synthase protein. The transmembrane domains are shown with dark grey vertical bars in the plasma membrane (2+6); Zn, zinc-binding region; VR1, Variable region 1; CSR, class-specific region or hyper variable region. Approximate positions of acceptor and donor binding residues D and QxxRW motif are shown. The topology of the CesA protein Topology models for CesA protein have been postulated based on the available protein sequence alignments. Conserved regions are annotated with putative functions, mostly relying on the available enzyme characteristics of SpsA protein of the GT family 2. The latest version of the predicted topology model for CesA protein sequence in general is illustrated in Figure 10 (Taylor, 2008). The catalytic site of the CesA protein is predicted to face the cytoplasmic side of the plasma membrane and is 11 flanked by two putative transmembrane regions (containing 2 or 6 transmembrane helices). The central cytoplasmic region is highly conserved in CesA protein except in the hyper variable regions (HVR) (Vergara and Carpita, 2001; Taylor, 2008). Three well conserved aspartic acid residues (D) are found, which probably are the putative substrate and the acceptor binding sites (Nagahashi et al., 1995). The amino terminus holds a RING-type zinc finger, a cysteine-rich domain containing four CxxC motifs, which is generally known to mediate protein-protein interactions (Saurin et al., 1996). In cotton, a mutation in the zinc domain resulted in the destabilization of the rosette, which suggests that the domain might be necessary for the rosette stability (Kurek et al., 2002). Cellulose synthase super family Genomic sequence data, especially of the cellulose producing organisms, have been used to identify all the CesA genes present in an organism. Many genes that are similar to CesA genes were found and named as CS-like genes (Csl). Csl families have the catalytic motifs D, D, D, QXXRW like CesAs and other GTs, but do not have the N-terminus zinc domain (Richmond and Somerville, 2000, 2001). It is hypothesized that Csl might be involved in synthesis of non-cellulosic polysaccharides. 1.3.2.3. Enzymology The synthesis of the individual glucan chains of cellulose is catalyzed by a glucosyltransferase (EC 2.4.1.12), also referred to as UDP-glucose-β-glucan glucosyltransferase, UDP-glucose-β-D-glucan glucosyltransferases and UDP-glucosecellulose glucosyltransferase. Glucosyltransferases catalyse the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. In cellulose synthesis, UDP glucose is the sugar moiety that is transferred to the growing cellulose polymer which acts as the acceptor molecule (Glaser, 1958; Lin et al., 1990). Figure 9: General mechanism of the inverting glycosyltransferases (Picture adapted from Charnock et al., 2001). Glucosyltransferases catalyse glucan synthesis by two main mechanisms with respect to the configuration of the reactant and the product. Retaining 12 glucosyltransferases retain the configuration of the anomeric carbon of the sugar after the reaction while the inverting mechanism does not. Family 2 glycosyl transferases are inverting enzymes. The inverting mechanism is a single displacement reaction which starts with a nucleophilic attack by the acceptor molecule at the anomeric carbon of the donor. The nucleotide diphospho sugar is the donor in the case of cellulose synthesis (Figure 9). Sugar hydroxyl acceptors are required to be activated by deprotonation by the catalytic carboxylate base. Another catalytic carboxylate of a member of family GT2 simultaneously coordinates a divalent metal ion on the sugar donor (Charnock et al., 2001). In order to explain how the glucan units in the growing chain can be rotated relative to one another, it has been hypothesized that the glucosyltransferase might have two active sites (Albersheim et al., 1997). However the model suggesting the presence of two active sites is not evident at least seen from the accumulating data of the protein sequences of CesAs from different organisms (Coutinho et al., 2003). 1.3.2.4. Allied components of cellulose biosynthesis Cellulose biosynthetic machinery is probably composed of several components besides the CesA proteins. Those components might be directly or indirectly involved in the structural, functional, biochemical and in other ways responsible for the functioning of the cellulose biosynthetic machinery. Sucrose synthase - Sourcing glucan The cellulose biosynthesis requires a constant supply of UDP-glucose which can be formed by two different metabolic pathways; 1. a reaction catalyzed by UDPGlucose pyrophosphorylase (Kleczkowski et al., 2004) and 2. a reaction catalysed by sucrose synthase (SuSy, EC 2.4.1.13) forming sucrose and NDP-glucose from NDPglucose and NDP-fructose (Haigler et al., 2001). Expression profiling studies in poplar have suggested that the sucrose synthase mediated reaction might dominate during wood formation (Hertzberg et al., 2001). There are soluble SuSys; membrane bound SuSys (Delmer et al., 1995; Guerin and Carbonero, 1997; Etxeberria and Gonzalez, 2003; Anguenot et al., 2006) and SuSys associated with cytoskeleton (Winter et al., 1997; Azama et al., 2003). The membrane or cytoskeleton bound SuSys might be involved in cellulose biosynthesis (Amor et al., 1995). Both the soluble and the membrane bound SuSys activity are shown to be controlled by calcium dependent phosphorylation (Huber et al., 1996). Various isoforms of SuSy have been found; Arabidopsis has 6 isoforms and T-DNA insertion mutation studies have shown that all of them have different expression patterns and cellular location. More interestingly, none of these mutants, even the double mutants resulted in a change in phenotype (Bieniawska et al., 2007). A SuSy has been localized in the secondary cell walls of cotton and Zinnia (Salnikov et al., 2001b; Salnikov et al., 2003) and decreased cellulose was reported in antisense SuSy construct in carrot (Tang and Sturm, 1999). The suppression of SuSy in cotton repressed the cotton fibre cell initiation and development (Ruan et al., 2003) while overexpression of SuSy in Arabidopsis has resulted in altered plant growth and fibre development and noticeable decrease in cellulose content (Park et al., 2004). Transgenic cotton with over expressed SuSy exhibit increased cellulose deposition in the secondary cell walls which results in high quality bigger fibres, shown in Figure 11 (Haigler et al., 2007). In maize, SuSy is shown to oligomerise and bind to F-actin that is dependent on the hyper sucrose concentration and hyper phosphorylated state while the opposite is shown for SuSy’s association with plasma membrane (Duncan 13 and Huber, 2007). Though these cases might suggest the connection of sucrose synthase with the cellulose biosynthesis, a direct connection is still awaiting experimental demonstration. Figure 11: Over expressed sucrose synthase in cotton. Light microscopy pictures showing the cross section of cotton fibres of transgenic cotton with increased sucrose synthase (SuSy). The over expressing SuSy cotton lines shows increased secondary cell wall and crushed lumen (Pictures adapted from Haigler et al., 2007). Cytoskeleton- a key element of cellulose synthesis Actin, tubulin and intermediate filaments are the three types of structures found in the cytoskeleton. Cytoskeleton helps cells to maintain their shape, size, movement and intracellular transport. However the morphology of a plant cell is determined by its cell wall where cellulose is the major constituent. It has been known for a long time that there is a connection between cellulose synthesis and the cytoskeleton (Paredez et al., 2006). Since the cytoskeleton is evidently connected to cellulose biosynthesis so would the factors associated with the cytoskeleton, at least indirectly. Both MF and MT networks are extremely dynamic and the associated factors are involved with the maintenance of these networks. The versatility of the two networks, especially during cell differentiation, is imposed by its associated factors. These associated factors might act as an interphase between the network and its function. The following section summarizes the reported connection of the cytoskeletal components such as actin and tubulin with cellulose biosynthesis. Cortical actin microfilaments. Actin microfilaments (MF), which are made up of two strands of actin homopolymers, are a part of the cytoskeleton. Actin is involved in cellular organelle movements and cell morphogenesis (Hussey et al., 2006; Higaki et al., 2007; van der Honing et al., 2007). Interestingly, the MF network seems to be interdependent with another cytoskeletal network, the Microtubuli (MT) (see below). In particular, there are reports that suggest that actin filaments orient the organization of MT arrays (Cyr and Palevitz, 1995). In Hydrocharis root hair cells it has been shown that the MT and MF networks were co-localized and MTs were closer to plasma membrane than MF (Tominaga et al., 1997). Earlier MF network has not drawn much attention with reference to the regulation of cellulose biosynthesis. However, increasing evidence of MT in the regulation of cellulose biosynthesis has imparted attention to MF as well. Microscopic examination has shown that the MF network rearranges itself during the differentiation of the tracheary elements in Zinnia mesophyll cells (Kobayashi et al., 1987). Similar changes were also noticed in the differentiating tobacco protoplast 14 cells (Hasezawa et al., 1989). Cotton fibres and Zinnia tracheary elements formed disorganized cellulose microfibrils when treated with cytochalasin that specifically disrupts actin microfilaments (Seagull, 1990). Mutual alignment of the actin microfilament and the cellulose microfibril in the apical cells of Sphacelaria rigidula that lacks cortical MTs would in fact suggest that the actin might be involved in the oriented deposition of cellulose microfibril (Karyophyllis et al., 2000). The cell wall strength of fucoid algae, Pelvetia compressa is found to be dependent on F-actin (Bisgrove and Kropf, 2001). In another electron microscopic study, four different species of algae, the differentiating cells of Sphacelaria rigidula, the apical cells of Dictyota dichotoma, the subapical cells of Choristocarpus tenellus and the meristematic epidermal cells of D. dichotoma, the innermost layer of the cortical actin microfilaments were found to be in the same orientation as the newly formed cellulose microfibril (Katsaros et al., 2002). The cell wall-plasma membranecytoskeleton (WMC) structural continuum has been studied with protoplast preparation methodology and the actin network undergoes reorganization when this continuum is disturbed. This suggests that the F-actin network has a major role in this continuum (Wojtaszek et al., 2007). These isolated findings in fact show the connection between the orientation of the cellulose microfibrils and the actin filament but details of this connection are still not established. Since the actin networks are also involved in organelle trafficking, the cellulose-actin connection could be indirect. On the other hand, the actin network is also involved with the cytoplasmic streaming, a constant motion of cellular components, which might be involved in handling the force required, or formed, in the movement of the cellulose synthesizing machinery. Figure 12: Actin network. Cartoon showing the actin microfilament network interconnected with the αactin and the actin polymer showing the Filamentous actin (F-actin) subunits of Globular actin (G-actin). The microfilaments constantly polymerize and depolymerise even during active cell division and cytoplasmic streaming. The proteins associated to MF might be involved with actin stability, cellular signalling involving calcium, phosphoinositides, pH or reversible phosphorylation and other functions (Ayscough, 1998). Several actin associated proteins have been reported in plants such as annexin, auxilin, CAP protein, myosin, profilin, plastin and spectrin. Genes homologous to these have been found to have high level of expression during the secondary cell wall formation in Populus (Hertzberg et al., 2001). The possible interesting proteins with reference to cellulose-MT association are MT-actin cross-linking protein (F22D22.1), membrane binding integrin (K16L.22.7) and a microtubule associated protein (MAP), MAP2 that binds both MT and MF (Hussey et al., 2002a). In a separate study integrin has been shown to bind to an ultrathin film of cellulose (Goennenwein et al., 2003). In cotton a kinesin, GhKCBP, is found to be colocalized both with MTs and MFs (Preuss et al., 2003). MAP190, from Tobacco BY-2 Cells, was found to bind both to MT and MF in vitro (Igarashi et al., 2000). The potential roles of actin associated factors have not been explored with respect to cellulose biosynthesis. 15 Cortical microtubules Microtubuli (MT) represent the major cytoskeletal network composed of polymerized units of heterodimers of α and β tubulin. The cytoskeletal MT is considered to be important for the cell development and its maintenance (Lloyd and Chan, 2004). MT is a dynamic network that has constantly growing and disassembling termini (for more details about the MT, see Figure 13). MT has a major role in cell division, especially the regulation of the proper movement and the division of chromosomes between the sister cells. The network is stationary in animal cells where the non growing end is anchored at the organizing centres such as centriole, which is absent in plant cells (Hussey et al., 2002b). In plant cortical MTs, the MT organizing centers (MTOC) are dispersed throughout the cortex and since the nucleation and orientation is not coupled the orientation is autonomous (Cyr and Palevitz, 1995). The cortical MT network has changing patterns that coincide with changing cellular morphology. For instance, in the fibre cells the cortical MTs tend to spiral by growing transversely through the length of the cell, a feature noticed in the cellulose deposition as well (Baskin, 2001). The alignment hypothesis proposing that the orientation of the cellulose microfibrils depends on the MT orientation was first put forward by Green (1962). He found that cellulose microfibrils in the algae, Nitella axillaris aligned with other long polymers, which were sensitive to colchicine (Green, 1962). Interestingly the discovery of the MT happened later (Ledbetter and Porter, 1963) and much later the cellulose synthesizing machinery, referred as rosette, was discovered at the plant cell plasma membrane (Mueller and Brown, 1980). In 1974, Brent Heath suggested that the cellulose orientation-MT connection could also be indirect implying that the MT consistently determined the cellulose deposition (Heath, 1974). The alignment hypothesis is still being questioned with various findings. Glaucocystis spp. that has cisternae between the MTs and cell membrane has an innermost cellulose layer that is not parallel with the underlying MT (Robinson, 1977). It has also been reported that the cortical MT in the root hairs of Equisetum hyemale have an axial alignment whereas the synthesized cellulose microfibrils have changing orientations in the different layers (Emons, 1982). Figure 13: Model showing the tubulin dimer formation and its polymerisation. α -/β tubulin dimerization mediated by prefoldin and cytosolic chaperonin; A and B, sequester of α -/β tubulin respectively, C-E, tubulin folding cofactor (TFC) and Arl2 mediates the dimer formation. The polymerizing MT (MT) has growing positive (+) and negative (-) ends created by tetrameric complex made of two γ-tubulin (γTuSc) units with Spc97 and Spc98. Katanin is thought to be associated with the negative end of the MT (Picture adapted from Mayer and Jurgens, 2002). 16 Although the alignment hypothesis was not proven until recently there were several reports that favoured it. In Oocystis solitaria, the inhibition of the MT resulted in the change of the cellulose orientation and when the inhibition was reversed the orientation was reversed as well (Quader and Robinson, 1979). In Closterium acerosm it was shown that the microfibrils had a disorganized orientation in the primary cell wall and with a patched alignment in the secondary cell wall, both could be related with its MT network (Hogetsu, 1983). Visualizing the fluorescently labelled cellulose synthase complex in Arabidopsis hypocotyl cells, Paradez et al. (2006) made a breakthrough discovery (Figure 14), the CS machineries moved in the same track as the underlying MT! Never before had the movement of CS machinery been demonstrated and in fact until then it only existed as a hypothesis based on the stationary cellulose microfibril. In the same experiment, it was also noted that the CS complex could retain its linear track, at least temporarily, even when MT is destabilized during the synthesis which could be due to the orientation of the already orderly placed product, the cellulose microfibril (Paredez et al., 2006). In Eucalyptus grandis, the overexpression of β-tubulin gene have directly influenced the cellulose microfibril angles in the secondary cell walls (Spokevicius et al., 2007). In Arabidopsis, Morlin, 7-ethoxy-4-methyl chromen-2-one, has been shown to cause swollen root phenotype and it is inferred that Morlin inhibits MT dynamics and CS complex movement (DeBolt et al., 2007b). Figure 14: Colocalization of cellulose synthase complex and MT in Arabidopsis etiolated hypocotyl cells : Stacked live time phase images of (A) CS6, representing the complex, with yellow fluorescent tag (YFP), (B) MT with CFP labelled Tua (α-tubulin marker) and (C) the overlay of CS (green) and MT (red) labelling. The white arrows point to the CS and MT motion seen in the video images found in the publication, available online (Picture adapted from Paredez et al., 2006). Microtubule associated proteins (MAP) are working as an ally of MT for various functions such as the stability of MT, cellular transport, cytoskeletal roles and several other functions (Hamada, 2007). MAPs have attracted more attention since the MT-cellulose alignment hypothesis became evident. One subgroup of mictotubule associated proteins contains filamentous/cross linking MAPs. MAP65, a 65kDa family protein, was first found in tobacco and has been shown to bundle MT during cell elongation (Jiang and Sonobe, 1993). MAP65 forms fine filaments that bridge the cortical MT and determine the stability of MT network. Several MAP65 and MAP65 like proteins are found in Arabidopsis (Hussey et al., 2002b). Another filamentous MAP of 200kDa, known as MOR1 is also a bundling protein first found in tobacco suspension cultures. The mutation of MOR1 17 conferred a temperature-dependent shortened phenotype with highly disorganised cortical MT (Whittington et al., 2001). Another group of microtubule associated proteins comprises membrane binding MAPs. The connection of MT and the cell membrane might be important with reference to membrane bound CS complex and its possible connection with the MT network. A 99kDa MAP in tobacco plant is found to connect the MT with the plasma membrane radially and this MAP was later found to be a phospholipase D (PLD) (Gardiner et al., 2001). Using a specific inhibitor of PLD, it was found that the elongation of the root was highly reduced, which could be due to the effect of MT disruption. This inhibition could be reversed by n-butanol, effective activator of PLD (Gardiner et al., 2003a). In Arabidopsis, AtEP1 has been shown to bind both to the plus end of the MT and to the motile, pleiomorphic tubulovesicular membrane networks that surrounds organelles and merge with endoplasmic reticulum (Mathur et al., 2003). It is possible that cellulose synthase complex is indeed assembled in Golgi and transported to the plasma membrane and MAPs like AtEP1 might have an intermediate role in the transport. MT is formed by tubulin heterodimers (α and β tubulin), but with a birefringence analysis it is understood that tubulin can also exist as long and cyclic oligomers. Such oligomer formation is dependent on the MT associated proteins such as Tau, MAP2 and other MAPs (Mithieux et al., 1985). Finally, some MAPs have been identified that confer phenotypes with altered cell structure. Reverse genetics is an important tool to identify the function of a gene by mutating it by gene specific deletions or insertions. Here we discuss the mutants with visible phenotypes that concern MAPs and with altered cell structure which would imply that the cellulose content in the cell wall is altered. Fragile mutants called fra1 and fra2 were found to contain mutation in the gene coding for kinesinlike protein and katanin-like proteins respectively (Burk and Ye, 2002; Zhong et al., 2002). In fact the fra1 mutation had occurred due the mutation in N-terminal microtubule-binding domain. WVD2 and WDL1 phenotypes that resulted in a helical phenotype and impaired anisotropic cell expansion in Arabidopsis indeed affect MAPs (Yuen et al., 2003; Perrin et al., 2007). Another helical phenotype was reported in the Arabidopsis mutant tortifolia1 (TOR1) or spiral2 (spr2). TOR1 protein is a 94kDa MAP that contains both coiled-coil region and HEAT repeat domain which are the characteristics of possible protein-protein interaction (Buschmann et al., 2004). The sku6-1 mutant of Arabidopsis resulted in altered leaf petioles with right handed axial twisting and skewed roots. The SPR1 gene codes for 12kDa protein and with fluorescent labelled studies in transgenic seedlings SPR1 is found to be localized to MT. Further studies have shown that it might be involved in MT polymerization dynamics that influence cell expansion and axial twisting (Sedbrook et al., 2004). AtMAP65-3 is another MAP that results in defective root and cytokinetic phenotype that resulted from defects in phragmoplast. AtMAP65-3 is found to bind only to mitotic MT arrays (Muller et al., 2004). The following plant MAPs conferred phenotypes that resulted from their overexpression and RNAi downregulation. AtMAP18 specifically binds to the nongrowing end of the MT and is believed to control the destabilization of the MT. The overexpressed and the RNAi lines of AtMAP18 had resulted in destructive root phenotype. In both cases, the cortical MT array is highly altered resulting in a reduction of cell length and size of the hypocotyl and cotyledon. The cellular localization of AtMAP18 is found to be patched throughout the cortical MT, the aspect reflecting its location only in the non-growing end of the MT (Wang et al., 2007). Plant specific AtMAP70-5 increases the length distribution profile of MT and 18 when over expressed it perturbs the polarity by inducing extra poles for growth. Both over expressing and RNAi lines have resulted in dwarfing of the inflorescence stems and of the right handed helical growth (Korolev et al., 2007). In general there are more than 70 MAPs reported in Pfam by February, 2008 (http://www.sanger.ac.uk/Software/Pfam/) and interestingly some of them are found to have unknown functions (Appendix Table 3). In general the MAPs found so far that influence the cell structure have not been studied for cellulose characteristics. The altered metabolic events in the mutants need to be deciphered in order to get a clear picture of the MAPs molecular function with reference to cellulose biosynthesis. Membrane bound cellulase - KORRIGAN KORRIGAN is an endo-1,4-β-glucanase (cellulase). It was first found in a cellulose deficient mutant called kor which lacked cell elongation in the absence of light (Nicol et al., 1998). Another allele of kor had defected cell plate formation and reduced cellulose content in the primary cell wall (Zuo et al., 2000; Lane et al., 2001; Sato et al., 2001). Recombinant kor orthologs from pea and poplar were shown to exhibit 1,4-β-D-glucanase activity on both carboxyl methyl cellulose (CMC) and amorphous cellulose (Molhoj et al., 2002; Master et al., 2004). Since, in Agrobacterium tumefaciens and Acetobacter xylinum, 1,4-β-glucanase has been shown to be essential for the normal cellulose biosynthesis (Standal et al., 1994; Matthysse et al., 1995), the role of KORRIGAN in cellulose biosynthesis is possibly significant. In Populus tremula x tremuloides, PttCel9A, a KOR1 ortholog, is found to be highly upregulated during the secondary cell wall formation (Hertzberg et al., 2001). In Populus tremuloides, PtrKOR was found to have high expression in tension wood, low expression in the reaction wood and co-expression with CesA genes (Bhandari et al., 2006). The cellular localization of the KOR is not yet clear; Cterminal GFP fusion studies in tobacco BY-2 cells shows the KOR in intracellular organelles in interphase cells and in the phragmoplast (Zuo et al., 2000). N-terminusKOR fusion protein was found to be localized in the endosomes and Golgi membranes and also in some motile compartments near plasma membrane (Robert et al., 2005). Sitosterol cellodextrin has been identified as the possible primer for the cellulose synthesis and with connection to this, KORRIGAN was proposed to cleave away the glucan units from the primer after the crystallization (Peng et al., 2002). Fackel, hydra1 and sterolmethyltransferase 1/cephalopod are the sterol mutants in Arabidopsis and it has been shown that they have abnormal cellulose content (Schrick, 2004). Though these data might suggest the possibility of the KORRIGANs role in the cellulose biosynthesis, clear evidence of its cellular function with reference to cellulose biosynthesis is not evident. GPI- anchored protein- COBRA COBRA is a glycosyl-phosphatidylinositol (GPI)-anchored protein. It is found in plasma membrane and longitudinal cell wall of elongating roots through some MT dependent mechanism (Schindelman et al., 2001; Roudier et al., 2002; Roudier et al., 2005). The mutants of COBRA have reduced cellulose content and also had swelling resulted from disorganised cellulose deposition (Roudier et al., 2005). Interestingly there is only one COBRA gene, COB, but there are 11 COB-like genes; defect in COB gene affect only the second phase of the anisotropic cell expansion during cell elongation but COB-like members affect other cell walls (Li et al., 2003; Jones et al., 2006). 19 Membrane bound protein of unknown function - KOBITO KOBITO gene mutation results in cellulose deficient and dwarfed mutants that exhibited random cellulose microfibril orientation of the new layer of the cell wall (Pagant et al., 2002). Two other alleles of KOBITO mutant were found; one conferred cellulose deficiency referred to as elongation-deficient mutants (eld) (Lertpiriyapong and Sung, 2003) and another one was found in a screen for abscissic acid-insensitive mutants (abi8) (Brocard-Gifford et al., 2004). KOBITO is a protein with unknown function and predicted to be a type II membrane protein with the N-terminus exposed to the cytosol. KOBITO is found in the membrane of the cells in the root elongation zone. It has punctuated pattern in the intracellular distribution in cell division zone, root elongation zone and cell walls (Pagant et al., 2002; Brocard-Gifford et al., 2004). The possible role of KOBITO in the cellulose biosynthesis is still unclear. 1.3.2.5. Inhibitors of cellulose biosynthesis Many compounds have been found to inhibit cellulose biosynthesis and can thus be used as a tool to study the cellulose biosynthesis. Cellulose biosynthesis inhibitors (CBI) are chemically unrelated compounds but affect cellulose biosynthesis directly or indirectly (Sabba and Vaughn, 1999). There are compounds like colchicine that disrupts MT and that has been shown to alter cellulose microfibril (Emons et al., 1990). However, colchicine is considered as MT disrupting compound and used for MT-CS interaction studies. Dichlobenil, isoxaben, phthoxazolin, triazenofenamide are some more specific CBIs that are available and here only two of the important and widely used CBIs are discussed. Dichlobenil or DCB The most extensively studied CBI is dichlobenil, 2,6-Dichlorobenzonitrile (DCB). The cellular process such as DNA synthesis, transcription, protein synthesis and cytoplasmic streaming are unaffected by DCB treatment (Meyer and Herth, 1978; Galbraith and Shields, 1982). Cellulose is deposited in the cell plates in the later stage while callose is the main constituent that is needed in early stage of the cell plate formation (Samuels et al., 1995). DCB has been shown to prevent the completion of cell plates in Allium cepa root cells (Buron and Garciaherdugo, 1983; Gonzalezreyes et al., 1986; Vaughn et al., 1996), Nicotiana tobaccum mesophyll protoplasts (Meyer and Herth, 1978), Nautilocalyx epidermal cells (Venverloo et al., 1984) and Tradescantia stamen hair cells (Mineyuki and Gunning, 1990; Sabba and Vaughn, 1999). In Gossypium hirsutum DCB treatment results in stunted fibers rich in juxtaposed electron-translucent and -transparent areas that can be labeled with callose antibodies (Vaughn and Turley, 2001). In onion root tip cells DCB treatment increases the amount of callose (Vaughn et al., 1996) while DCB treatment of petunia and lily pollen tubes affects cellulose synthesis and not callose, the main constituent of the pollen tube (Anderson et al., 2002). From these studies it is evident that the effect of DCB promotes or affects callose, β-1,3 glucan, when it inhibits cellulose synthesis. DCB treatment has been found to reduce the number of TCs in protonemata of Funaria (Rudolph et al., 1989). In wheat, the Rosette-TC structure is not altered but TCs accumulate by DCB treatment (Herth, 1987). With mutant studies it has been shown that cells of red algal members Rhodella and Porphyridium, normally lacking cellulose in their cell wall, can adapt to DCB by altering their cell wall chemistry (Arad et al., 1993; Arad et al., 1994). In Vigna radiata, the DCB treatment changes 20 the cell wall morphology (Satiatjeunemaitre, 1987; Vaughn and Turley, 2001). In Vaucheria, an algae, the DCB treatment removes the small filaments found between presumptive cellulose-synthesizing terminal complexes (TCs) and disarrange the TC rows (Mizuta and Brown, 1992a). It has been shown, in an agronomic study, that every other crop has different tolerance to DCB. The increasing order of magnitude of resistance to DCB is noted in Corn>Sorghum>Cotton>Turnip (Hardcastle and Wilkinson, 1971). The inhibitory concentration of DCB has been reported to be several fold higher in two algal species, Porphyridium and Rhodella, compared to other vascular plants in general (Arad et al., 1994). Photoaffinity analog of DCPA, [3H]DCPA, a structural and functional analog of DCB(Cooper et al., 1987), had specifically labeled a ∞18kDa protein in Cotton and ∞12kDa proteins in Chara and tomato (Delmer et al., 1987). In Acetobacter xylinum, DCB induces the formation cellulose II with normally formed cellulose I (Yu and Atalla, 1996). Isoxaben N-[3-(1-ethyl-1-methylpropyl)-1,2-oxazol-5-yl]-2,6-dimethoxybenzamide is commonly referred to as AE F150944 or isoxaben and has been experimentally proven as an effective CBI (Vaughn and Turley, 2001). Isoxaben is a pre-emergence herbicide, used before crop development and has been shown to selectively inhibit the synthesis of acid-insoluble cell wall polysaccharides (Heim et al., 1990; Scheible et al., 2001). It has been shown that the Arabidopsis Ixr1 mutants (conferring AtCesA3) are resistant to isoxaben (Scheible et al., 2001). Later it was found that ixr2-1 carries a mutation in an amino acid close to the C-terminus of CesA6, which could be the target of the isoxaben (Desprez et al., 2002). In Zinnia elegans it has been shown that crystalline cellulose synthesis is specifically inhibited by isoxaben and suggests that the CS rosette is disturbed by isoxaben application (Kiedaisch et al., 2003). In isoxaben habituated cells of Phaseolus vulgaris, high activity of xyloglucan endotransglucosylase (XET) activity is reported which might be due to the decreased cellulose content (Alonso-Simon et al., 2007). 1.3.2.6. Model of the biosynthetic machinery The cellulose biosynthetic machinery is not yet deciphered completely. Earlier the available information of cellulose biosynthesis from cytological, molecular biological and enzymological studies has been used to build models. The cartoon of a cellulose synthase complex and its allied components in Figures 15 and 16 have been made based on the following knowledge. The only components of the cellulose biosynthetic machinery that have been experimentally verified so far are the CesA proteins. The cellular function and protein sequence information of CesAs suggest that they have both transmembrane, extracellular regions and cytosolic regions. In many hypothetical models, the location of the catalytic site is suggested to be cytosolic; meaning that the polymerisation will take place in the cytosol and the crystallization will involve an intrusion process through the plasma membrane (Brown and Saxena, 2000). Many isoforms of CesA are reported, different in different species and with immunolocalization labelling with CesA isoform specific antibodies it has been demonstrated that three distinct isoforms are essential for proper assembly and function of the complex (Gardiner et al., 2003b; Taylor et al., 2003; Taylor et al., 2004; Desprez et al., 2007; Persson et al., 2007). It is suggested that six ‘lobes’ of a rosette may synthesize six cellulose chains which then co-crystallize to form an elementary microfibril with 6 x 6 chains and that six such elementary microfibrils form a cellulose microfibril shown in Figure 15 (Herth, 1983). This hypothesis is now 21 widely accepted and also agrees with the dimensions of the cellulose crystals in higher plants. The N-terminus zinc domain of the CS proteins are involved in the stabilization of the complex by dimerizing the CesA isoforms which when disturbed affects the stability and function of the rosette (Kurek et al., 2002). Figure 15: Model suggesting the stoichiometry of CS complex (Doblin et al., 2002) and CS isomers pattern in the rosette (Taylor, 2008). As mentioned before the CS complex motion follows the underlying MT track (Eckardt, 2003; Paredez et al., 2006). Though the connection between the CS complex and the MT is not yet established, the classical rail-train model could be revisited with a careful caution that the connections are not established and could be indirect. The requirement of sucrose synthase for cellulose synthesis and its cellular localization studies in fact suggest that it is present in the cell membrane. The co-localization of CesA and SuSy is reported and also its co-expression pattern with CesAs suggests that it is in the membrane and accessible to the cellulose synthesizing machinery (Salnikov et al., 2001a; Albrecht and Mustroph, 2003). KORRIGAN is expected to be present in the cell wall face of the membrane, though its role in the cellulose deposition is not yet clear. The actin microfilaments have not yet been considered to have an important role in cellulose biosynthesis but together with their associated protein and factors, the connection to MT, its cellular function in cytoplasmic streaming and trans-membrane signal transduction; actin might have indirect roles. The following section about physics of the cellulose synthesis considers the whole machinery as one single motion factor. Figure 16: Model suggesting the organization of the possible participants of the cellulose biosynthesis. 22 1.3.3. Physics of cellulose biosynthesis This section gives a summary of biophysical studies done to understand the CS complex motion and its cytoskeleton composed of protein polymers. Mechanical work in the cells is done by specialized motor proteins that operate in continuous mechanochemical cycle. For example, the pulling and pushing of MT and MF could derive energy from the constant polymerisation and depolymerization events (Mogilner and Oster, 2003). In case of the CS complexes, their motion is not yet understood properly. Earlier it was believed that processive molecular motors such as kinesin, that transport vesicles and organelles using MT, were acting as the driver of CS complex motion (Heath, 1974). Afterwards it was believed that MT themselves created a shear force that drove the complex in the fluid mosaic membrane (Hepler and Palevitz, 1974). Later it was understood that the energy created from the polymerization itself could be enough to drive the complex (Herth, 1980). The membrane flow hypothesis was postulated suggesting that CS complex is free to move in the plane of the plasma membrane and the polymerization could drive the CS complex (Mueller and Brown, 1982). This hypothesis could be justified with the results of the experiments of Calcofluor treatment where the cellulose polymerization could be effectively blocked with Calcofluor treatment (Haigler et al., 1980). 1.3.3.1. Polymerization of biopolymer Increasing evidence of the connections of the MT and CS complexes can be taken into account to describe the CS complex movement with MT stability. The stability of the MT network would directly influence the CS machinery and so could the MF network since it is in turn expected to influence the MT network. The stability of the MT and MF could be understood based on their polymerization and depolymerization events that are dependent on a number of other proteins and biochemical factors. The polymerization of organic polymers such as cellulose, MT and MF could be explained with brownian polymerization ratchet (BPR) that is used to explain cellular motions. Brownian movement is a random movement of a particle in a liquid or gas and this could be used to explain the cellular particle movement in the cellular fluid (Peskin et al., 1993). It is believed that thermal collision constantly batter cellular components and that cell have naturally adapted to harness this random motion into directed biological processes. The chemical energy that is converted into the mechanical pushing force by filament polymerization is stored in the difference in chemical potential, ∆G (Gibbs free energy), between the free monomer (tubulin, glucan) and the polymer (tubulin dimer or oligomer, actin polymer and glucan chain). If the free energy of the bound and unbound state of monomer is at equilibrium then ∆G = 0 and kon = koff. Therefore the polymerization is spontaneous when the free energy of monomers in solution is greater than the monomers integrated into the polymer (Mogilner and Oster, 1996, 2003). The cellulose synthesis could be explained with two different models of BPR; 1. BPR for rigid filaments (Peskin et al., 1993) and 2, BPR for semi-flexible filaments (Mogilner and Oster, 1996). In Peskins model, the rigid filaments is fixed at one end and there is diffusive force (F) that acts from the opposite side of the fixed end and the force is resisted by the filament with a backward motion and this excursion distance accommodate a new monomer. For every addition the required force must be exerted by a distance of δ (equal to the size of the monomer, here it’s a glucan size) and thus the work done (W) = F δ. The Mognilner and Oster model for semi-flexible filament, accounts the biological property of the fixed load and the filament. Here the 23 model take into account the stiffness of filament and does not depend on the diffusion coefficient as in the Peskin model. Figure 17: The energies of unbound and bound state in the polymerization ratchet. E- Energy, ∆G – change in Gibbs free energy, ∆G* - energy barrier between the transition of free to bound state and kon and koff – rate constant of the unbound and bound state (Mogilner and Oster, 2003). 1.3.3.2. Modelling the movement of CS Complex A model suggesting the CS complex locomotion was recently proposed by Diotallevi and Mulder, 2007. The model considers the relevant physical components to obtain a heuristic explanation for the propulsion process which had been used to reproduce the experimentally calculated CS complex speed and also has implemented a stochastic stimulation to explain its motion. The model considers the CS complex as a planar membrane bound object facing towards the cell wall. The cellulose polymer is modeled as inextensible semiflexible chains of beads. The stochastic simulation of the motion of six cellulose filament have been made and interestingly this simulation have shown that the CS complex move away from the direction of the synthesis and also, that the complex rotate in its place in order to release the twist made by the cellulose chain (see Figure 18 below). The model suggests the cellulose polymerization and the crystallization as the combined driving forces. This model also takes into account the polymer flexibility and the membrane elasticity as force transducers. The interesting finding of this model is that it was possible to calculate the speed of cellulose synthesis to be in the range of Vp ≈ 10-9 – 10-8 m/s which is consistent with the experimental value of 5.8 x 10-9 m/s (Hirai et al., 1998; Diotallevi and Mulder, 2007). There are other mathematical and geometrical models suggesting the orientation of cellulose deposition. A geometrical model by Emons et al. (1994) quantitatively relates the deposition of cellulose microfibrils to the density of the CS complex in the plasma membrane, the distance between the microfibrils and the geometry of the cell (Emons, 1994; Emons and Kieft, 1994; Emons and Mulder, 1998). The cellulose microfibril deposition is believed to follow the notion of winding tapes densely around a cylinder. The helicoidal cell wall of the microfibril orientation changes by a constant angle from one lamella to another (Emons and Mulder, 1998). 24 Figure 18: Stochastic simulation (four snap shots A, B, C and D) showing the motion of six filament of CS complex. Movement of the CS complex shown in terms of distance from position of snap shot A to snap shot D considering the membrane to be static. The rotation of the complex is shown with the displacement of a glucan chain (Glucan chain A in picture), with its connection point to the terminal complex (Picture adapted from Diotallevi and Mulder, 2007). 25 1.4. CELL WALL – THE DOMAIN FOR CELLULOSE BIOSYNTHESIS Cellulose producing organisms mainly deposit cellulose in their cell walls and large cell walled organisms such as tree boles are used as the source of cellulose. Therefore the plant cell wall is an ideal domain to study various aspects of cellulose biosynthesis. The availability of the genomic data for the model plants Arabidopsis, rice and poplar is a valuable resource for the cell wall studies. Poplar is widely used as a model for wood formation in trees. Wood formation involves complex biological processes such as cell division, cell expansion, secondary cell wall deposition, lignification and programmed cell death (Mellerowicz et al., 2001). In the following section, cellulose biosynthesis in primary and secondary cell wall is discussed. Studies of the non-cellulosic content of the plant cell wall such as hemicelluloses and lignin are not described, considering the scope of the thesis. 1.4.1. Primary and secondary cell wall First formed cell wall in a plant cell is called the primary cell wall. When the cell undergoes specialization many new layers of walls are deposited called secondary cell wall. It is noted that the primary cell wall is less organised compared to the secondary cell walls which have defined orientation at various stages of their development. Secondary cell wall biosynthesis is a complex process that involves coordinated regulation of several diverse metabolic pathways. It has three layers, S1, S2 and S3, which are different from each other with respect to the cellulose microfibril orientation (Figure 19). The S2 layer has an acute angle (less than 90˚) relative to the long axis of the cell and makes the bulk of the secondary cell wall (Mellerowicz et al., 2001). The bulk of economically important biomass is produced in the secondary cell wall. Figure 19: Cartoon showing the types of cell wall in a xylem cell. 26 1.4.2. Cellulose biosynthesis in secondary cell wall The most significant event during secondary cell wall formation is the increased cellulose biosynthesis and lignification. Secondary cell wall biosynthesis can be induced in Arabidopsis by decapitating its inflorescence (Lev-Yadun, 1994). The plant, when grown in short day conditions can produce secondary cell walls in the hypocotyl and its anatomy is similar to the angiosperms wood (Chaffey et al., 2002). Therefore, Arabidopsis can also be used to study some aspects of wood formation (Nieminen et al., 2004). A set of three isoforms of CesAs can be grouped as primary cell wall specific and as secondary cell wall specific. For example in Arabidopsis CesA1, 3 and A6-like are primary cell wall specific while CesA4, 7 and 8 are secondary cell wall specific (Taylor, 2008) and the secondary cell wall specific CesAs are CESA4, 7 and 8 (Oh et al., 2003; Brown et al., 2005a; Persson et al., 2005). PttCesA1, A3-1 and A3-2 are suggested to be specific for secondary cell walls (Djerbi et al., 2005). Both α-tubulin and β-tubulin, that forms MT, are found to be upregulated in the xylem compared to bark tissue (Oh et al., 2003). Expression analysis has been performed to identify the genes that were co-expressing with irx1 and irx3 and those that were identified are a member of COBRA family, COBL2 (At5g15630), several other genes of glucosyltransferases and hydrolases and many unknown genes (Brown et al., 2005b). A sucrose synthase is expressed in synchrony with the secondary cell wall specific CesA’s (Hertzberg et al., 2001). KOR and the UTP-glucose-1-phosphate uridylytransferase, another pathway to provide UDPglucose, were found to be upregulated during the early secondary cell wall formation (Hertzberg et al., 2001). 1.4.3. Co-expressing unknown genes in cellulose biosynthesis With reference to cellulose biosynthesis, almost all the expression profiling experiments performed had not only identified the expression pattern of cellulose synthesis and related genes, but also a huge number of genes with unknown functions. Recent annotation of the Arabidopsis genome suggests that of the 27,139 identified functional genes, 7592 (28%), are predicted as proteins of either hypothetical or unknown function (http://arabidopsis.org/). In an EST classification of 102,019 ESTs in poplar 22% of genes were reported to be unclassified and another 23% of the genes were having the lowest blast score (Sterky et al., 2004). In Eucalyptus, a transcript analysis done with xylem to leaf subtractive library reveals that more than 17% of the genes expressed preferentially in xylem have an unknown function and 44% of the genes give no hits in other available gene databases. In a cDNA-amplified fragment length polymorphism (AFLP)-based transcriptome analysis in hybrid aspen, apart from cellulose synthesis related genes such as sucrose synthase genes, CS genes, other GTs and GHs, kinases at least 20 genes were found to have either hypothetical or unknown function (Prassinos et al., 2005). The significance of the unknown genes in the cellulose biosynthesis is inevitable and by exploring its function the dark areas of cellulose synthesis can be solved. 27 28 2. OBJECTIVES OF THE PRESENT INVESTIGATION The general objective of the present investigation was to characterize a gene of unknown function that was found to be highly expressed during the secondary cell wall formation in Populus tremula × Populus tremuloides (hybrid aspen). The specific aims were: 1. To analyse the sequence of the unknown gene with appropriate bioinformatic tools. 2. To determine its expression pattern at transcript level and identify the cellular localization of the protein in vivo. 3. To establish recombinant expression of the protein and perform biochemical characterization assays in vitro. 4. To investigate phenotypes of plants overexpressing the gene. 29 30 3. MATERIALS AND METHODS 3.1. TRANSCRIPT PROFILING 3.1.1. Poplar expression profiling on microarrays Expression data from microarray experiments were obtained from UPSCBASE (www.upscbase.db.umu.se/), experiment number 30 (courtesy of Göran Sandberg) using the following PU numbers PttCS1: PU07560; PttCS3: PUPU07525, PU07543, PU30161 (mean values); PttCS9: PU06755; PttMAP20: PU00458, PU02741. The data was normalized against a sample containing equal amount of RNA from all tissues and organs analyzed and average level of expression was calculated in cases when a gene was represented by several spots on the microarray. 3.1.2. Quantitative PCR Stem samples from P. tremula were collected from a natural population of ca 5 m high trees growing outside Umeå, immediately frozen in liquid nitrogen and stored at -70°C. Tangential sections (30 µm) was collected across the wood forming tissues using a cryomicrotome (Uggla et al., 2001) and pooled into 8 samples. Total RNA extraction, cDNA synthesis and SYBR green qPCR were performed using BioRad Aurum Plant Mini Kit, Bio-Rad iScript cDNA synthesis kit and Bio-Rad SYBR Green qPCR mix respectively following manufacturer’s instructions. All qPCR data was expressed as –ΔCt values (where ΔCt is CtCONTROL GENE - CtTEST GENE). The data were normalized by using two housekeeping genes, ubiquitin (Pt794626, fgenesh4_pg.C_scaffold_13026000001) and elongation factor 1β (Pt815174, estExt_fgenesh4_pg.C_LG_I1178). Mean Ct values for these two genes were used when calculating ΔCt values. Each sample was analyzed in 3 technical replicates. Primers used for gene expression analysis were designed from P. trichocarpa gene model sequences (see Appendix Table 1 for a list of all primers). The PCR program included initial denaturation at 95°C for 3 min; 40 cycles of 10 s at 95°C, 30 s at 55°C, 30 s at 72°C and a final melt curve analysis (incremental heating from 54°C to 95°C). 3.2. BIOINFORMATICS 3.2.1. Sequence retrieval Genome sequences and their allied gene predictions were retrieved for Arabidopsis (v 5, January 2004, tigr.org), rice (v 4, January 12, 2006, rice.tigr.org) and Populus trichocarpa (v 1.1, 2006, jgi.org, DoE Joint Genome Institute and Poplar Genome Consortium). A pre-release of Medicago truncatula genome (December 14, 2006), including gene predictions, was downloaded from http://www.medicago.org/. Zea maize contigs were analyzed and downloaded from www.plantgdb.org in April 2007. Animal TPX2 proteins (Q2U500, A2APB8, Q6P9S6, Q6DDV8, Q643R0, Q805A9, Q6NUF4, Q5ZIC6, TPX2, Q5RAF2, Q96RR5 and Q8BTJ3) were downloaded from the Pfam website and redundant sequences were ignored. The homologs of PttMAP20 and its orthologs in Arabidopsis, Cotton and Rice were fished out from the web resources from http://www.ncbi.nlm.nih.gov/. 31 Similarity search and bioinformatics Sequence similarity searches for mRNA and EST support for genes were primarily conducted using NCBI's online Blast using full-length protein sequences. Similarity searches were performed using the blast http://www.ncbi.nlm.nih.gov/blast/) and TBlastn (http://www.incogen.com/public documents/vibe/details/NcbiTblastn.html) (Altschul et al., 1990; Altschul et al., 1997) and the EST sequences were retrieved from the NCBI database dbEST (http://www.ncbi.nlm.nih.gov/dbEST/). The HMMER package (Eddy, 1998) was used for the alignment and identification of TPX2 domains, using the Pfam model PF06886.1. The Pfam web site, http://pfam.sanger.ac.uk/, was used for general domain architectural analysis. The assembly of ESTs to putative transcripts was done with CAP3 (Huang and Madan, 1999). Multialignments were computed using Kalign (Lassmann and Sonnhammer, 2005), Muscle (Edgar, 2004) and MAFFT (Katoh et al., 2002; Katoh et al., 2005). Visualization of the alignments was done using TeXShade (Beitz, 2000). The quality of conservation of a sequence alignment of plant TPX2 domains was plotted using the EMBOSS plotcon software, http://bioweb.pasteur.fr/seqanal/interfaces/plotcon.html, with a standard window size of 4 for both DNA and protein sequences. The full-length protein coding sequence of PttMAP20 was obtained by sequencing the cDNA clones A068p39u and A089p68u on both strands using a primer walking with unlabelled universal and gene-specific primers. The GenBank accession number of the PttMAP20 sequence is DQ661742. The PttMAP20 sequence was also used as the query to search the PDB for structural homologues by using the software HHpred (Soding, 2005; Soding et al., 2005) (http://protevo.eb.tuebingen.mpg.de/hhpred). Phosphorylation sites were predicted by NetPhos 2.0 (Blom et al., 1999) available at http://www.cbs.dtu.dk/services/NetPhos/ and MotifScan of SwissProt available at http://myhits.isb-sib.ch/cgi-bin/motif_scan. Gene models for Oryza sativa were downloaded from TIGR release 4 (http://rice.tigr.org/tdb/e2k1/osa1/) and Arabidopsis thaliana models came from TAIR (genome release 6, www.arabidopsis.org). Poplar sequences included ESTs and contigs from PopulusDB (Sterky et al., 2004) and P. trichocarpa gene models from JGI (http://genome.jgi-psf.org/Poptr1/ Poptr1.home.html). TPX2-containing proteins from Xenopus, Gallus, mouse and human where retrieved from UniProt (Wu et al., 2006). Proteins containing the TPX2 domain were identified using HMMSEARCH in the HMMER package (http://hmmer.wustl.edu) and the TPX2 model from Pfam (Finn et al., 2006). Protein hits with an E-value of less than 10^-9 were considered significant. The hit on At5g28646 had an E-value of 0.00036 and was studied more closely, which revealed that this was an incorrect prediction of Q84ZT9_WAVEDAMPENED2. All Populus hits had significant hits to transcripts from PopulusDB. Alternative transcripts were removed, three in Oryza and four in Arabidopsis, in favor of longer gene models. The multiple alignment of PttMAP20 and the Arabidopsis and Medicago truncatula homologs was performed by Kalign (Lassmann and Sonnhammer, 2005), available at (http://msa.cgb.ki.se/cgi-bin/msa.cgi). FGENESH (Salamov and Solovyev, 2000) was used to predict genes and gene products in the region 14899001-14909001 on the Arabidopsis chromosome 5. 3.2.2. Phylogenetic analysis Phylogeny studies were done using MrBayes (Ronquist and Huelsenbeck, 2003). Each analysis had four MCMC chains running for 4x106 iterations, default thinning and the first 10% iterations removed as burnin. The mixed amino acid model 32 with gamma-distributed rate-change over sites was chosen. Analysis of possible adaptive evolution was performed with codeml in the PAML package (Yang, 1997). 3.2.3. Gene mapping Mapping of the transcripts or proteins to genome sequences and the determination of exon/intron-structure was done using Exonerate (Slater and Birney, 2005). Promoter analyses were conducted using TSSP (Solovyev and Shahmuradov, 2003). Phylogenetic footprinting for motif finding was done with MEME (Bailey and Elkan, 1994; Bailey and Gribskov, 1998) with settings chosen to look for up to five motifs, present in some but not necessarily all sequences. Local genomic alignments were computed using DBA (Jareborg et al., 1999). 3.2.4. Expression profiling of TPX2 genes in Arabidopsis The expression profiling data for the Arabidopsis TPX2 genes were extracted from the Gene Atlas performed with ATH1 (22K full genome Arabidopsis Affymetrix GeneChip) available online (http://www.arabidopsis.org/). 3.3. PROTEIN BIOCHEMISTRY 3.3.1. PttMAP20 antibody production Antibodies were raised against the full length PttMAP20 protein expressed in E. coli using the expression vector pAFF8c-3c encoding N-terminal polyHis6 and albumin binding domain (ABP) tags in frame with the inducible T7 promoter (Larsson et al., 2000; Graslund et al., 2002). The full-length cDNA sequence was cloned using the following primers: forward 5’-GCAGATACAGCGGCCGCAATGGGAAAGCACACAAAAAT CTG-3’ and reverse 5’-GGCGGCGCGCCTTAATTAACAGCTTCCAGGCATGATC-3’, followed by insertion into the poly-cloning site of pAff8c-3c and transformation and expression in E. coli BL 21-CodonPlus (DE3)-RIL (Stratagene). Protein expression was carried out as described by (Larsson et al., 2000), followed by purification of the recombinant protein under denaturing conditions using the polyHis6 tag using TALON spin™ (BD Bioscience, US) columns following the manufacturer’s recommendations. The purified protein fraction was buffer exchanged from the elution buffer containing 6 M Urea to PBS [0.02 M sodium phosphate and 0.5 M sodium chloride (pH 7.0)]. Rabbit IgG antibodies were raised by Agrisera AB (Vännäs, Sweden). The antiserum was affinity purified using the recombinant protein cross-linked to a HiTrap™ NHSactivated HP column (Amersham Biosciences, Sweden). 3.3.2. Protein extraction from wood tissues Poplar trees (P. tremula X P. tremuloides, clone T89) were grown under natural light in the green house to a height of 5 m (Uggla et al., 2001). For xylem protein extracts, stem pieces of aspen were frozen in liquid nitrogen immediately after harvest and stored at –80°C. The frozen stem pieces were thawed to room temperature to peal off the bark. The xylem tissue was scraped from the stem surface and rapidly dropped into liquid nitrogen. Subsequently, the scraped xylem tissue was ground separately to a thin powder under liquid nitrogen and mixed in 1:1 ratio (w/v) with the extraction buffer [100 mM of MOPS, 2 mM of EDTA and 2 mM EGTA (pH 7.5)]. Xylem crude extract was homogenized at 120 bar for 15 minutes in a cell disruption bomb (Parr Instrument Co., Moline, IL, USA). The homogenized slurry was centrifuged at 4000g for 20 min at 4°C and the resulting supernatant was used as the 33 crude extract of essentially soluble proteins (referred to as the supernatant). The protein concentration in the supernatant was measured using the BioRad Protein assay (BioRad Laboratories, CA, USA) with BSA as standard. The extraction represents the crude extract of soluble proteins. 3.3.3. Immunoblotting The protein samples, after lysing in SDS-PAGE buffer at 95°C for 5 minutes, were electrophoretically separated on 10% SDS-PAGE gel (NuPAGE, Invitrogen) run at 200 V for 1 hour. 20 μg of total protein in the supernatant fraction was applied per lane and 10 µl per lane of the solubilized proteins in the pellet fraction. After the electrophoresis, the gel was equilibrated in a transfer buffer (Tris 3.0 g/l, Glycine 14.4 g/l, Methanol 20%) and the same buffer was used for transferring the proteins onto a PVDF membrane (Immuno-Blot™ PVDF membrane, BIO RAD) at 100 mA and 4°C, overnight, using a semidry blotting apparatus (Transblot® SD Transfer Cell, Biorad) and). The membrane was then blocked in 5% non-fat milk and TBS-Tween [0.02 M Tris-HCl, 50 mM sodium chloride and 0.1% tween (pH 7.4)] for 12 hrs at 4 °C with slow shaking. The blocked membrane was incubated with the primary antibody (0.5 μg/ml) in TBS-Tween overnight with slow shaking at 4°C, followed by washing twice with TBS-Tween for 1 hour. The secondary antibody reaction and the staining were carried out using the ECLTM anti-rabbit IgG-HRP-linked antibodies according to the manufacturer’s recommendations (ECL Western blotting detection reagents and analysis system, Amersham Biosciences). The Western blot image was captured using the luminescent image analyzer (LAS-1000plus, Fuji). 3.3.4. Recombinant PttMAP20 in E. coli Full length PttMAP20 cDNA was amplified using the forward primer (introducing an N-terminal polyHis6 tag) 5’-CCCTCTAGAAAATGCATCATCATCA TCATCACGAGAAAGC-3’ and the reverse primer 5’-ATATAGGTACCCAGTTAATTAA CAGGCTT-3’ and cloned in the vector pET-28a-codon + (Novagen), followed by transformation into E. coli BL 21-CodonPlus (DE3)-RIL cells (Stratagene). An overnight culture was used to inoculate 500 ml of Luria Broth (LB) and grown at 37 °C, 140 rpm in a baffled conical flask. The T7 expression cassette was induced by IPTG (0.1 mM) at A600nm= 0.8. The cells were harvested after 2 hours by centrifugation at 4.000 x g for 20 min at 4°C and resuspended in 20 ml of buffer A [50 mM sodium phosphate and 300 mM sodium chloride (pH 7.0)]. The resuspended cells were freeze-thawed at -80°C - 0°C. Lysozyme (0.01 mg/ml) was added to the sample followed by incubation on ice for 1 hour. The sample was then sonicated twice for 2 min on ice, with a 10 min pause in between, at 30% duty cycle and an output control 7 (Sonifier 250, Branson USA). The sample was centrifuged at 10.000 g for 20 min at 4°C and filtered with 0.45 µm filters (Acrodisc 32 mm syringe, Pall Newquay, UK). The following purification steps were done using Äkta FPLC (Amersham Biosciences, Sweden). The extracted soluble proteins were applied to hand packed columns containing 5 ml Ni Sepharose™ High performance matrix (Amersham Bioscience, Sweden), pre-equilibrated with 5 column volumes of Buffer B [500 mM Imidazole, 50 mM sodium phosphate and 300 mM sodium chloride (pH 7.0)]. Unbound proteins were washed with 2 column volumes of Buffer A and the bound proteins were eluted with a linear gradient of increasing concentration of Imidazole using Buffer B. The purity of the eluted protein was checked on a 10% SDS-PAGE (NuPAGE, Invitrogen) and the selected fractions were pooled and buffer 34 exchanged using a PD 10 column (Amersham Bioscience, Sweden) to 50 mM HEPES containing 300 mM sodium chloride (pH 7.0). The high molecular weight background protein species seen in the elution of the affinity purification were separated using a size exclusion chromatography. HiLoad 16/60 Superdex 75pg (Amersham Bioscience, Sweden) was used to prepare pure fractions of the PttMAP20 protein. 3.3.5. PttMAP20 orthologs gene synthesis, expression and protein purification Arabidopsis (At5g37478), Cotton (DW520361) and Rice (Os09g13650.1) genes were customs synthesized (Sloning Bio Technology GmbH, Germany) and cloned in the vector pET-28a-codon + (Novagen). All the clones were transformed into the E. coli BL 21-CodonPlus (DE3)-RIL (Stratagene). The following expression and purification protocol was used for all the four genes mentioned above. An overnight culture was used to inoculate 500 ml of Luria Broth (LB) and grown at 37 °C, 140 rpm in a baffled conical flask. The T7 expression cassette was induced by IPTG (0.1 mM) at A600nm = 0.8. The cells were harvested after 2 hours by centrifugation at 4000 x g for 20 min at 4°C and resuspended in 20 ml of buffer A [50 mM sodium phosphate and 300 mM sodium chloride (pH 7.0)]. The resuspended cells were freeze-thawed at -80°C - 0°C. Lysozyme (0.01 mg/ml) was added to the sample followed by incubation on ice for 1 hour. The sample was then sonicated twice for 2 min on ice, with a 10 min pause in between, at 30% duty cycle and an output control 7 (Sonifier 250, Branson USA). The sample was centrifuged at 10.000 g for 20 min at 4°C and filtered with 0.45 µm filters (Acrodisc 32 mm syringe, Pall Newquay, UK). The following purification steps were done using Äkta FPLC (Amersham Biosciences, Sweden). The extracted soluble proteins were applied to hand packed columns containing 5 ml Ni Sepharose™ High performance matrix (Amersham Bioscience, Sweden), pre-equilibrated with 5 column volumes of Buffer B [500 mM Imidazole, 50 mM sodium phosphate and 300 mM sodium chloride (pH 7.0)]. Unbound proteins were washed with 2 column volumes of Buffer A and the bound proteins were eluted with a linear gradient of increasing concentration of Imidazole using Buffer B. The purity of the eluted protein was checked on a 10% SDS-PAGE (NuPAGE, Invitrogen) 3.3.6. Binding of PttMAP20 to MT Protein binding to taxol (Paclitaxel) stabilized bovine MTs (BMT) was assayed using the MT Binding Protein Spin Down Assay Kit of Cytoskeleton, USA. 20 µL of the MT suspension was mixed with 1 µg of pure recombinant PttMAP20, followed by centrifugation through a cushion of 60% glycerol at 100,000 x g. Control experiments were performed with BMT alone, BSA instead of PttMAP20 and with PttMAP20 without BMT. The pellet and supernatant fractions were carefully separated after centrifugation and the proteins in both fractions were analyzed by 10% SDS-PAGE (NuPAGE, Invitrogen). The influence of DCB on the binding of PttMAP20 onto BMT was tested by incubating 1 µg of pure recombinant PttMAP20 with 0- 200 µM of DCB at room temperature for 20 min, whereafter the solutions were added to pre-assembled BMT and subjected to the spin down binding assay. 35 3.3.7. Co-precipitation assay of binding of PttMAP20 to poplar MT PMT were obtained by in vitro assembly of tubulin monomers contained in 50µg of xylem protein extract (supernatant fraction, see above) following the instructions of the spin down assay kit BK029 of Cytoskeleton Inc. In brief, to assemble aspen microtubules, 20 µl of the xylem protein extract (supernatant fraction, 9.4 µg / µl) was mixed with 2.5 µl of the Cushion Buffer (BK029, Cytoskeleton) and incubated at 35˚C for 20 min. The synthesized PMT were then stabilized and diluted with 200 µl of the pre-warmed general tubulin buffer (BK029, Cytoskeleton), supplemented with 10 µl of taxol, at 35˚ C for 30 min. All experiments hereafter were performed at room temperature unless otherwise indicated. 50 µg of xylem protein extract (supernatant fraction) was used as a source of native PttMAP20. Alternatively, 0.5 µg of the recombinant PttMAP20 was used. Affinity purified rabbit anti-PttMAP20 antibodies at a dilution of 100 μg/ml were added to the xylem protein extract or the recombinant PttMAP20 and incubated for 5 min, followed by an incubation with anti-rabbit Alexa Fluor®-488-fluoro-nanogold™ Fab fragment (Nanoprobes, NY, USA) at a dilution of 1:1000 (T6199, Sigma) for another 5 min in dark (hereafter referred to as the MAP20 sandwich). In a separate vessel, the in vitro assembled PMT preparations were incubated with mouse anti-αtubulin antibodies (T6199, Sigma) at a dilution of 1:1000 for 5 min, followed by the addition of Rhodamine (TRITC)-conjugated affinity pure rabbit anti-mouse IgG antibodies (Jackson, PAUSA) at a dilution of 1:1000 for another 5 min in dark (PMT sandwich). The PMT sandwich and the MAP20 sandwich were pooled (1:1 v/v) and incubated in dark for 1 hour. The microtubules carrying the antibodies and PttMAP20 were then pelleted using the microtubulin binding protein spin down assay kit (Cytoskeleton Inc, USA). The specificity of the spin down assay was controlled by spinning down a mixture of all of the other components but the PMT. The resultant pellets were washed briefly with 100 µl of TBS (pH 7.0) by pipetting, resuspended in 10 µl of TBS-Tween (pH 7.0) and observed using fluorescence microscopy (Olympus BX-51, Japan). The images were captured using four different filters: a 550 nm excitation – 570 nm emission filter (U-MW1G2, Olympus, Japan) to detect the red fluorescence corresponding to the microtubulin labeling, 495nm excitation - 520nm emission filter (U-MW1BA2, Olympus, Japan) to detect the green fluorescence corresponding to the PttMAP20 labeling and a multiple wavelength filter (DAPI / FITC / TRITC N61000V2, Olympus, Japan) to detect both signals simultaneously. 3.3.8. Assembly of BMT in the presence of DCB 100 µg batches of monomeric α and β tubulins in 20 µL of the General Tubulin Buffer (Cytoskeleton, USA) were mixed with increasing concentrations of DCB (0.75 μM –7500 μM) dissolved in DMSO and incubated on ice for 10 min. A final concentration of 10 mM GTP was then added to initiate polymerization of BMT, followed by analysis of the extent of polymerization using the MT Binding Protein Spin Down Assay Kit (Cytoskeleton, USA). 3.3.9. Labeling of PttMAP20 by [3H]DCPA [3H]DCPA was custom synthesized by PerkinElmer, USA, as previously described (Cooper et al., 1987). The labeling experiments were performed at room temperature in a final volume of 20 µL. Standard reaction mixtures consisted of 50 mM HEPES pH 7.0, 300 mM NaCl, 1 µg of pure PttMAP20 and 0.75 μM [3H]DCPA 36 dissolved in 10% ethanol (final concentrations). In control experiments, [3H]DCPA was replaced by ethanol. Competition experiments were carried out by supplementing the standard reaction with a final concentration of 2 µM, 20 µM, 200 µM, 2 mM or 20 mM DCB (in methanol) or DCPA (in ethanol). Controls were performed by replacing DCPA and DCB by ethanol or methanol, respectively. The mixtures were incubated on ice for 5 minutes, subjected to UV illumination (250 nm) for 10 min and analyzed by SDS-PAGE. After Coomassie Blue staining, the gels were incubated for 30 min in the Amplify™ reagent (Amersham Biosciences, Sweden) and dried. The radioactive PttMAP20 bands corresponding to the different reaction mixtures were detected by autoradiography using sensitive X-Ray films (Hyperfilm, Amersham Biosciences, Sweden), excised from the gels and deposited in bottles containing 4 mL scintillation liquid. The amount of [3H]DCPA bound to PttMAP20 was determined by measuring the corresponding radioactivity using a liquid scintillation counter. 3.4. IMMUNOLOCALIZATION EXPERIMENTS 3.4.1. Confocal microscopy, poplar tissues Poplar trees (P. tremula X P. tremuloides, clone T89) were grown under natural light in the green house to a height of 5 m (Uggla et al., 2001). Free hand transverse and longitudinal radial sections were prepared from aspen stems, between the 10th and 15th nodes from the top. The sections were quickly transferred to the fixative solution (50 mM PIPES, 5 mM MgSO4, 5 mM EGTA and 4% paraformaldehyde; pH 7.0) and fixed for 30 min at room temperature. The fixative was replaced by 6 M urea and the sections were incubated for 30 min at room temperature. All steps thereafter were done on a slow shaking table. The sections were washed three times for 5 min with PBS (pH 7.4) containing 0.1% Tween-20 and blocked with 5% non-fat milk in PBS for 30 min at 25°C in a Petri dish. The sections were transferred to eppendorf tubes containing the PttMAP20 primary antibody (see supplemental methods) (100 μg / ml, the pre-immune serum) or anti-α-tubulin antibodies (T6199, Sigma, 24 μg / ml) in a volume of 30 μl of PBS/ 3 sections and incubated for overnight at 4°C. The samples were then washed three times with PBSTween for 5 min to remove the excess antibodies, followed by blocking with 5% nonfat milk for 10 min at room temperature. All the steps thereafter were done in dim light. The sections were transferred to the secondary antibody solution (Alexa Fluor®-488-fluoro-nanogold™-Fab (Nanoprobes, NY, US), diluted 1:10 (v/v) in TBS (0.02 M Tris-HCl and 50 mM sodium chloride (pH 7.4)) and incubated overnight at 4°C. The sections were then quickly quenched in 0.5% Toluidine blue for 15 sec to block the background signals from lignin, washed 4 times in PBS-T for 10 min and mounted on to the slide with Vecta shield (Vector Laboratories) for confocal microscopy (LSM510, Zeiss). Fluorescence signals were observed using an excitation wavelength of 488 nm and a 505-530 nm emission filter. The image transmitted with the visible light channel was superimposed onto the fluorescence image to reveal anatomical details of the tissues examined. 3.4.2. Confocal microscopy, tobacco Confocal microscopy was performed 3-5 days after agro infiltration on a Zeiss Confocal microscope, LSM 510 META. Sections of leaves were mounted on slides in a water/glycerol solution. Images were acquired with an argon/krypton laser. Images 37 were acquired with the following settings for the microscope: For YFP, excitation of laser line 514 nm, emission filter BP 530-600 nm using a Plan-Apochromat 63x/1.4 oil objective lens. DNA constructs: Constructs for PttMAP20 localization studies in tobacco were generated by PCR amplification of MAP20 cDNA sequence using Phusion DNA polymerase (Finnzyme) with primers MAP20 forward (5’-CACCATGGAGAAAGC ACACACAAAATCTGC-3’), N-MAP20 reverse (5’-TTAATTAACAGGCTTCCAGGCATGA TCATG-3’) for inclusion of the stop codon and MAP20 forward, C-MAP reverse (5’ATTAACAGGCTTCCAGGCATGATCATGGGG-3’) for product lacking the stop codon. PCR products were cloned into Gateway pENTR/D-TOPO vector using the directional TOPO cloning kit (Invitrogen), according to the manufacturer’s instructions. MAP20 ORF was then recombined using the Gateway LR Clonase Enzyme Mix (Invitrogen) into the destination vectors pEarleyGate 101 and 104 to create a C-terminal and N-terminal fusion, respectively. Constructs for MAP20 Nand C-terminus epitope tagging with myc tag were generated using primers N-myc forward (5’-TTATTAGGTACCATGGAACAAAAGTTGATTTCTGAAGAAGATTTGGAGAAA GCACACACA-3’), N-myc reverse (5’-TTATTATCTAGATTAATTAACAGGCTTCCA-3’) and C-myc forward (5’-TTATTAGGTACCATGGAGAAAGCACACACA-3’), C-myc reverse (5’-TTATTATCTAGATTACAAATCTTCTTCAGAAATCAACTTTTGTTCATTAACA GGCTTCCA-3’). The PCR fragments were cut with KpnI/XbaI and cloned directionally into KpnI/XbaI linearized pAI301, a binary vector consisting of pGA581 with (An, 1987) the CaMV 35S promoter, a multiple cloning site and the NOS terminator inserted in the HindIII and EcoRI sites. All constructs were sequenced. The Nicotiana benthamiana plants were grown in environmentally controlled growth cabinets under a 16-h photoperiod-8-h dark regime at 25°C. Agroinfiltration was carried out in planta on 2 weeks-old plants by injecting agrobacteria in the apoplastic space of the leaves (as described by Voinnet et al., 2003). Briefly, Agrobacterium tumefaciens strain C58C1 was grown at 28°C in LB medium supplemented with 50 µg/ml kanamycin and 5 µg/ml tetracycline to stationary phase. Bacteria were sedimented by centrifugation at 5000 x g for 15 min at room temperature and resuspended in infiltration solution (10 mM MES and 10 mM MgCl2) supplemented with 150 µg/ml acetosyringone. Cells were left in this medium for 2-3 h and then infiltrated into the subaerial part of fully expanded N. benthamiana leaves, using a 2 ml needleless syringe. After agroinfiltration, tobacco plants were put back in the growth chambers. All the Agrobacterium strains harboured the pCH32 helper plasmid (Hamilton and Baulcombe, 1999). 3.4.2.1. Deletion series of PttMAP20, cloning Deletion constructs were generated by PCR amplification of MAP20 cDNA sequence using Phusion DNA polymerase (Finnzyme) with primers N-NO-TPX2 forward (5’-CACCATGGAGAAAGCACACACA-3’), N-NO-TPX2 reverse (5’-TTTGAAT TCCTGGGGCTTAGTG-3’) for the deletion construct with the N-terminus alone; TPX2 forward (5’-CACCATGAAACTCCACACTGGAC-3’) and TPX2 reverse (5’-TCTGTCA AAGTAAGGCATCAATTGAG-3’) for the deletion with the TPX2 domain alone; C-NOTPX2 forward (5’-CACCATGCCTTACTTTGACAGACC-3’) and C-NO-TPX2 reverse (5’ATTAACAGGCTTCCAGGCATGA-3’) for the construct with the C-terminus alone; NNO-TPX2 forward and TPX2 reverse for the construct with N-terminus and TPX2 domain; TPX2 forward and C-NO-TPX2 reverse for the deletion with the C-terminus and TPX2 domain. The PCR products were cloned into Gateway pENTR/D-TOPO vector using the directional TOPO cloning kit (Invitrogen), according to the 38 manufacturer’s instructions. The entry vector was then recombined using the Gateway LR Clonase Enzyme Mix (Invitrogen) into the destination vectors pEarleyGate 101 to create a C-terminal fusion to YFP. All the constructs were checked by sequence analysis. 3.4.3. Transmission electron microscopy (TEM), poplar tissues Developing wood tissue segments of Poplar trees (P. tremula X P. tremuloides clone T89) cultivated under natural light in the green house to a height of 5 m were dissected from the stems (Uggla et al., 2001). The tissue samples were pre-treated with 6 M urea for 60 min (Shi et al., 1994), washed 3 times for 10 min with 25 mM potassium phosphate buffer (pH 7.2), fixed overnight at 4°C in 4% (v/v) paraformaldehyde, 0.05% (v/v) glutaraldehyde in 25 mM phosphate buffer and washed again 3 times for 10 min with the buffer. The samples were then dehydrated through a graded ethanol series (50%, 70%, 80%, 90%, 95% and 99.5%) 3 times in each, for 10 min. LRW resin (TAAB Laboratories, Reading, UK) was then added dropwise to absolute ethanol to reach a final concentration of 10% and the infiltration of resin was performed overnight. This was followed by one 50% and two 100% LRW changes and overnight infiltration step for each change. The material was transferred to the gelatin capsules and polymerized for 8-24 hrs at 60° C. Ultrathin sections (80 to 100 nm thick) were cut with an ultramicrotome (MT-7000; RMC, Tucson, AZ) and transferred to 200-mesh nickel grids (TAAB) coated with Formvar. The sections were blocked with a blocking solution (1 % BSA in PBS) for 15 min at room temperature and incubated for 2 hrs at room temperature in either the antiMAP20 antibody solution (0.1 mg/ml in BSA/PBS) or the pre-serum. The samples were then washed with PBS two times for 10 min and incubated 1 hr at room temperature with 10-nm colloidal gold–conjugated anti-rabbit antibody (BB International Ltd), diluted 20 times in BSA/PBS. Finally, the samples were washed with PBS two times for 10 min and with distilled water for 5 min, contrasted by adding 5% Uranium Acetate and observed using a Jeol 1230 transmission electron microscope. 3.5. PHENOTYPIC CHARACTERIZATION 3.5.1.1. Overexpression of PttMAP20 in Arabidopsis In order to generate transgenic Arabidopsis plants overexpressing PttMAP20 fused to the myc epitope at the C-terminus with the 35S promoter, wild type ecotypes (Col-0) were transformed using the floral dip method (Clough and Bent, 1998) and selected on kanamycin-containing media. Transformation was verified by phenotype and PCR analysis using primers specific for PttMAP20 (C-myc forward and C-myc reverse described above). The expression was verified by RT-PCR carried out as follows. Total RNA was isolated from poplar leaf, xylem and phloem tissues using RNeasy plant minikit from Qiagen and 0.2 micrograms was used for RT-PCR using SuperScript III from Invitrogen. The following primers were used for PttMAP20 detection: N-NO-TPX2 forward (5’-CACCATGGAGAAAGCACACACA-3’), N-NOTPX2 reverse (5’-TTTGAATTCCTGGGGCTTAGTG-3’). The housekeeping ribosomal RNA gene (23S-40S) was used as a reference: 23S-40S forward (5’-CTGGTC GCAAGCTCAAGTCCCAC-3’) and 23S-40S reverse (5’-GACCTTGGCTTCTCCTTCTTCTC TTTG-3’). 39 40 4. RESULTS 4.1. DISCOVERY AND EXPRESSION ANALYSIS OF PttMAP20 Data from microarray analysis of different poplar tissues and organs show that a small cytosolic protein is preferentially expressed in developing secondary xylem tissues (Figure 20A). This gene was denoted Unk1 but was later renamed PttMAP20 (see below). The new name will be used throughout the thesis. The expression pattern of PttMAP20 is remarkably similar to three previously identified xylem associated PttCESA genes (Hertzberg et al., 2001; Djerbi et al., 2004). To confirm this pattern of expression, high-resolution quantitative PCR expression analysis (qPCR) of PtMAP20 and PtCESA1, PtCESA3-2 and PtCESA9-2 was conducted across the wood forming tissues. Tissue samples were dissected into 8 fractions from functional phloem towards maturing wood cells using tangential cryosectioning (Uggla et al., 2001) giving a spatial resolution of gene expression of about 90µm in the radial direction (Figure 20B, side panel). Indeed, PtMAP20 expression was very low in functional phloem tissues and primary walled cambial cells while it increased in expanding cells and most dramatically at the onset of secondary wall biosynthesis (Figure 20B). The peak values of PtMAP20 expression in fractions 4 - 6 corresponded to the peak rate of fiber wall thickening as measured from SEM images of the corresponding tissue. Moreover, the qPCR analysis confirmed a very tight co-expression between PtMAP20 and the secondary wall specific CESA genes. The only deviation from this pattern was observed during late maturation of wood fibers where the PtMAP20 expression dropped to a lower level somewhat earlier that of the CESA genes (Figure 20B). Figure 20A: Co-expression of poplar secondary cell wall associated CESA genes and MAP20. Relative mRNA abundance of PttCESA1, PttCESA3, PttCESA9 and PttMAP20 in different tissues and organs as analyzed on cDNA microarrays. 41 Figure 20B: The relative mRNA abundance of PtCESA1, PtCESA 3-2, PtCESA 9-2 and PtMAP20 as quantified by qPCR in eight samples across developing xylem tissues, from functional phloem towards maturing wood cells. The approximate sampling positions are indicated on an anatomical section as follows: functional phloem (1), cambial meristem (2), expanding wood cells (3), secondary cell wall forming and maturing wood cells (4-8). Scale bar =50µm. Closer analysis of gene expression across the meristematic tissue undergoing cell division and primary cell wall synthesis shows generally low relative expression of PttMAP20 throughout the cambium. Higher level of expression is detected in only one zone which probably corresponds to early differentiating vessel elements as suggested by the concomitant induction of PttCESA1 (Figure 20C) (data from Schrader et al., 2004a; Schrader et al., 2004b). The overwhelming majority of the PttMAP20 ESTs originate from wood related cDNA libraries (Appendix table 4 and 5) and the relative level of PttMAP20 transcripts is clearly the highest in xylem relative to other poplar tissues (Figure 20D). The qPCR analysis and the abundance of PttMAP20 transcripts in EST libraries (www.populus.se) all indicate a high level of expression of PttMAP20 in woody tissues in poplar. Thus, it can be concluded that MAP20 is a highly expressed gene specifically associated to secondary wall formation and tightly co-regulated with secondary wall CESA genes. C D Figure 20C: Relative level of expression of PttMAP20 across the cambial meristem, numbered from the phloem mother cells (1) to the oldest xylem mother cells (12) on the X-axis (Schrader et al., 2004a; Schrader et al., 2004b), 20D: Relative level of expression of PttMAP20 in different poplar tissues. Data courtesy of Göran Sandberg, Umeå Plant Science Center, Sweden. 42 4.2. BIOINFORMATIC CHARACTERIZATION 4.2.1. Identification and sequence analysis of PttMAP20 orthologs The cDNA clone, used in the microarrays experiments, of PttMAP20 encoded the full length gene. At the initial phase of the discovery, the PttMAP20 sequence blast analysis didn’t yield any hit. The protein sequence was predicted to be a soluble coiled coil. The TPX2 domain in the PttMAP20 protein sequence was revealed in the blast searches only later. Due to the possible involvement of PttMAP20 in cellulose biosynthesis, putative orthologs (as defined by Fitch, 1970) were searched for corresponding genes in all published plant genomes and in the NCBI dbEST (Table 2). The current annotations of the gene models Os09g13650.1 in rice and At5g37478 in A. thaliana had to be adjusted since sequences homologous to the other TPX2 proteins were detected beyond the current gene models (see Materials and Methods). Reannotation of the rice gene was facilitated by an EST sequence, NM_001069361.1 and in both cases the new gene models were more similar to PttMAP20 than the current gene models. Appendix Table 2 summarizes the gene models identified in different plant species. These gene models are hereafter designated as ‘Map20-Like’ (M20L). None of the animal TPX2 proteins were found to show any significant similarity with PttMAP20 beyond the TPX2 domain (data not shown). Putative PttMAP20 orthologs were easy to detect among monocots and dicots by simple analysis of sequence similarity. In gymnosperms, however, thorough phylogenetic analysis was required in order to find putative orthologs of PttMAP20. In this way, 11 sequences in Pinus and 10 sequences in Picea (with ESTs from P. engelmanii, P. glauca and P. sitchensis) were found to encode a TPX2 domain. Multiple alignments of the MAP20-like proteins were computed using MUSCLE, MAFFT and KALIGN, but no reliable full-length alignment could be found. In particular, there was little consensus at the N- and C-terminal ends of the proteins (see Figure 21 and Appendix Figure 1 for MAFFT and MUSCLE alignments, respectively). However, the programs did agree on a strongly conserved 81-residue region containing the TPX2 domain, here called the extended TPX2 domain. It was verified that no other known domain structures are located in the regions flanking the TPX2 domain of the M20L proteins. Table 2. A list of MAP20-Like gene models (M20L) in different plant species Species Origin Protein acc Gene prediction PtMAP20 P. tremula x P. tremuloides Accession eugene3.00440209 (592874) (denoted estExt_fgenesh4_pg.C_440200 in the Populus genome release 1.1) POPLAR.3073.C1 PopulusDB Arabidopsis thaliana At5g37478 Gene prediction Oyza sativa Os09g13650.1 Medicago truncatula AC147472 Brassica napus Gossypium hirsutum Glycine max Hordeum vulgare CN734834 DW520361 BQ612497 CX630106 Gene prediction Genome scaffold dbEST dbEST dbEST dbEST PttMAP20 AtM20L, b At5g37478_alt b OsM20L Malus domestica CV083029 dbEST MdM20L Triticum aestivum Zea maise BJ280729 AY110515 dbEST dbEST TaM20L d ZmM20L Populus trichocarpa a a MtM20L BnM20L GhM20L GmM20L HvM20L c Identical to PtMAP20; bNew gene model; cSingle EST support, may be short on 5’ side; dAmbiguous codons were resolved using the shorter AI795385. 43 Figure 21: Sequence alignment of M20L proteins made with the MAFFT alignment tool. The TPX2 domain is marked with a black bar and the extended TPX2 domain by a dotted line under the sequences. Notes above the TPX2 domain indicate sites where residues are lost relative to the Pfam domain model of TPX2. The identity or similarity to a consensus sequence is indicated in blue and red (respectively), the last codon of an exon in orange and the exon/intron boundary in frame 2 in yellow. 44 4.2.2. Conservation of the extended TPX2 domain of M20L To understand the conservation of the TPX2 domain in plants, a multiple alignment was made with DNA sequences of the extended TPX2 domain within the M20L sequences. The similarity plots were made with both DNA and protein sequences of the extended M20L TPX2 domain to check the quality of conservation. The similarity score was low for the DNA sequences compared to protein sequences (Figure 22A), mainly due to silent mutations in the 2nd and 3rd nucleotide of the triplet codon (Figure 22B). Figure 22A: A multiple alignment made with DNA sequences of the extended TPX2 domains of the M20L protein sequences. Differences with respect to A. thaliana are noted and the conserved bases are indicated by dots. The third codon position is printed in grey. 45 Figure 22B: Similarity plot graph made with the extended TPX2 domain with reference to relative residue position (both DNA and Protein sequences) using EMBOSS plotcon software using a standard window size of 4. 4.2.3. Identification of proteins containing a TPX2 domain Proteins containing a TPX2 domain were identified in the completely sequenced genomes of poplar (Populus trichocarpa) (Tuskan et al., 2006), Arabidopsis thaliana (Arabidopsis_Genome_Initiative, 2000) and rice (Oryza sativa) (Goff et al., 2002; Goff, 2005), using the Pfam model of TPX2. The fully sequenced animal genomes only contain one gene encoding a single TPX2 domain. In contrast, plant genomes were found to encode rich repertoires of different proteins containing a TPX2 domain, 12 in Arabidopsis, 10 in rice and 19 in Populus. Here we have compared the TPX2 domains in all fully sequenced plant and animal genomes as well as other known TPX2 containing proteins from the Pfam database (Figure 23). Figure 23: Alignment of the TPX2 domains from all available, non-redundant full-length TPX2-related proteins in the fully sequenced genomes of Oryza, Arabidopsis and Populus and one representative TPX2 protein from Xenopus, Gallus, mouse and human. The intensity of the shading is proportional to the percent identity of the aligned sequence. 46 4.2.4. Phylogenetic analysis of TPX2 domain Molecular evolution of the TPX2 domain was studied with phylogenetic analysis of all available TPX2 domain sequences (Figure 23). The subtrees in the phylogeny mostly follow the established species history (Figure 24A), but there are peculiarities to note. First, the M20L orthologs deviate from the established species phylogeny (see e.g. the apple sequence) and the branching is not fully resolved. The lack of phylogenetic resolution is seen also in other parts of the tree, including several branches with low statistical support. However, these problems are to be expected as the phylogeny is estimated from the short TPX2 domain alone, with only 58 informative positions in the alignment. The M20L clade was revisited by reconstructing a phylogeny based on all Angiosperm extended TPX2 domains of M20L whereby the branching was significantly improved (Figure 24B). The phylogenetic tree has two distinct branches, one for animals and one for plants. The plant branch bifurcates into two types of TPX2, one that has a so called KLEEK motif previously identified in the Arabidopsis WVD2 protein (Yuen et al., 2003) and another which does not. Proteins containing a KLEEK motif form a monophyletic clade and have several branches each with its own monocot and dicot sub-branches, as exemplified by WDL5/WDL6 and WDL4 in Figure 24A. Based on our current analysis the M20L genes constitute a clade in the non-KLEEK branch. Notice that gymnosperm sequences are present in both major branches and that there are two likely orthologs to PttMAP20 in Picea. Secondly, Tetrahymena thermophila is found in the plant clade, adjacent to the KLEEK-containing proteins (Figure 5A), which is not reasonable given the species tree. Again this could be due to lack of data, but there is also another interpretation: If T. thermophila is considered an outgroup of the present phylogeny, i.e. we re-root the tree at the base of the thermophila branch, then the first duplication occurred before plants and animals diverged. This implies that animals lost at least one TPX2 gene, while plants took advantage of the redundancy by further evolutionary diversification. This old duplication could reflect different functions in the two main branches of the TPX2 phylogeny. Regardless of where the root of the phylogeny is correctly placed, the expansion of proteins containing a TPX2 domain among plants is in striking contrast to animal TPX2. 47 Figure 24A: Phylogenetic tree made with all the available and newly found eukaryotic proteins containing a TPX2 domain. The Bayesian posterior probability is indicated for weak branches. Probabilities exceeding 0.8 were removed for clarity. The clades with M20L proteins and KLEEK motif are marked. Mutations in genes with reported phenotypes in Arabidopsis are marked in red. 48 Figure 24B: Phylogenetic tree made with extended TPX2 domain. 4.2.5. Genomic organization of TPX2 genes The organization of TPX2 genes was mapped in all the three fully sequenced model plants: the universal model Arabidopsis, the wood model Populus and the grass model Oryza. We could not find a general pattern for the TPX2 gene locations in the three genomes (Figure 25A, B and C). Synteny comparisons suggested that many recent paralogs, e.g. WDL1/WVD2 and WDL6/WDL5 in our phylogeny, could be due to large scale duplications (Table 3). The linkage groups previously found in the three species were used to overlay the location of these gene pairs (Arabidopsis Genome Initiative, 2000; Goff et al., 2002; Goff, 2005; Tuskan et al., 2006). The many recent paralogs, especially in Poplar, could make the gene localization ambiguous, but the highest scoring alignment stood out in cases. When aligning a protein sequence to the genome, the second-best match had incomplete sequence coverage, several or many mutations and significantly lower score compared to the highest scoring match. The TPX2 paralogs in the poplar genome that arose due to the genome duplication are noted in the gene models and their respective linkage groups (Table 3 and Figure 25A). All genes in the TPX2 gene family in P. trichocarpa, except MAP20, seem to come in pairs and they are all more recent than the last species split in this phylogeny. This is consistent with the likely whole-genome duplication event (Tuskan et al., 2006). While there are plenty of duplications in the TPX2 gene family in general, none of the species included in the present study has more than a single copy of M20L genes. Such a singleton representation suggest that M20L genes have characteristics that render them duplication resistant (Paterson et al., 2006) during events of polyploidy. A search for TPX2-containing pseudogenes was conducted in the P. trichocarpa genome using translating Blast and the known TPX2 domains. Even at a 49 relatively low threshold (E=0.001), no hits were found outside the known TPX2 genes. Unless some predicted genes are false positives, the P. trichocarpa does not have any TPX2 containing pseudogenes. Figure 25A: Genomic organization (to scale) of TPX2 genes in the genome of Populus trichocarpa Figure 25B: Genomic organization (to scale) of TPX2 genes in the genome of Oryza sativa 50 Figure 25C: Genomic organization (to scale) of TPX2 genes in the genome of Arabidopsis thaliana. Microarray experiments in Arabidopsis indicate that WDL1 (At3g04630) and WVD2 (At1g3780) have similar expression patterns (Appendix figure 2) with increased expression in inflorescence tissue, consistent with a duplicated regulatory module. Even WDL5 (At4g32330) exhibits somewhat increased expression in inflorescence tissue. Table 3: Gene pairs and their respective linkage groups present in Populus trichocarpa, Oryza sativa and Arabidopsis thaliana Species Duplicated gene pairs Linkage group Populus trichocarpa Pt668651: Pt195236 Pt415809: Pt578210 Pt658783: Pt564607 Pt667581: Pt653406 Pt658207:Pt658207 IV: XVII VI: XVIII VIII: X VI: XVI VIII:X Oryza sativa Os02g10690.1: Os06g40450.1 Os03g58480.1: Os12g38790.1 II: VI III: XII Arabidopsis thaliana AT2g25480.1: At4g32330.1 Q84ZT9: At3g04630.1 II: V III:V 4.2.6. Gene structure determination and comparison of M20L genes A comparison of the exon/intron structures of known M20L genes in A. thaliana, P. trichocarpa, O. sativa, M. truncatula and Z. mays revealed variation in both the exon numbers and the intron lengths (Figure 26A). In order to identify potential transcription factor binding sites (TFBS), 3000 bp of the genome sequence was extracted upstream from the M20L start-of-translation point in O. sativa, A. thaliana, M. truncatula and P. trichocarpa. In M. truncatula the M20L gene was found to be located in a contig (CT963077, from chromosome 3) including 3000 bp upstream of the translation start of the gene. The Zea mays gene had a match against two contigs (ZmGSStuc11-12-04.70298.1 and ZmGSStuc11-1204.9118.1(Spokevicius et al., 2007). As they were seemingly overlapping, they were reassembled (using CAP3) into one contig from which 1975 bp upstream of start-oftranslation was extracted. Putative TATA-boxes upstream of M20L were predicted and the same regions were also used to search dbEST in order to identify likely UTRs. In particular, monocot members showed longer introns. Interestingly, the TPX2 domain intersects three exons in all studied species and the domain covers 48 bp in the first and 63 bp in the last of the three exons. Consequently, the mid exon 51 is 60 bp in all cases. This is another indication of strong selective pressure on the TPX2 domain. In an alignment of the five genomic regions, the pairwise identity was lower than 44% in all cases, indicating considerable divergence on regions without selection. However, two motifs for potential TFBSs were revealed using phylogenetic footprinting (Figures 26B and 26C). Motif 1 (39 bp) was significant in all five species (E=1.7x10-14). In dicots, a slightly wider motif of 43 bp was found (Figure 26C). Motif 2 consisting of 28 bp was also found in all five species (E=2.8x10-5) but this width was reduced by two bases on the 5' flanking region if applying MEME to dicots only. No further significant motifs could be found in all five species. These motifs were, with small differences, corroborated by pairwise genome alignments using DBA. It was furthermore verified that the motifs are noncoding and not covered by any ESTs and are hence not likely to be part of the translated regions of these genes. These motifs are larger than what is common for a TFBS, suggesting that they may represent two regulatory modules. It is interesting to note that although dicots and monocots share little similarity in TPX2 gene structure and protein sequence, their TFBS motifs are well conserved. In Poplar there are 4 putative TATA boxes. Considering EST hits at this region, the TATA box at the position -100 might be important (Figure 26B). A similar arrangement is seen in M. truncatula although with two TATA boxes downstream of the TFBS motifs. In this case, ESTs suggest a promoter, which is close to the conserved TFBS motifs similar to Populus, but this would render both predicted TATA boxes false. The locations of the motifs in Arabidopsis are quite different in that they are distant from each other. Short EST hits are reported for the downstream region of the first motif, but offer no real support for any of the predicted TATA boxes. Looking at the monocots, O. sativa has the motifs in the same order as the dicots and EST hits are found in the downstream region of motif 2, clearly an extension of the first exon. It is reasonable to assume these ESTs represent an UTR and a predicted TATA box is consistent with this hypothesis. The results are similar for Z. mays, with exon-extending and UTR-indicating EST hits, except that the order of the motifs is switched. Figure 26A: Upstream regions with predicted TATA boxes are indicated by a green ‘T’, motif 1 by a red box, motif 2 by a blue box and EST hit regions by dark pink lines. The lines are scaled to actual sequence length. 52 Figure 26B: The positions (in base pairs from the translation start) of exons are marked, to scale, with dark boxes while the lines correspond to introns. The TPX2 regions are marked with dark red lines. Figure 26C: Sequence alignments for the motifs 1 and 2. Blue color indicates identity to the consensus sequence. The left flank of motif 1 shows the extension of this motif in dicots. 53 4.3. BIOCHEMICAL CHARACTERIZATION 4.3.1. Antibody development and recombinant expression in E. coli Polyclonal antibodies were raised in rabbits using recombinant PttMAP20 fused to an albumin-binding protein. In Western blot analysis the resulting antibodies were found to recognize specifically the recombinant PttMAP20 while a single protein band of slightly higher molecular mass (33kD) was detected in crude protein extracts from developing xylem (Supplemental Figure 27A). Since similar discrepancies between the predicted molecular weight and protein mobility in SDSPAGE have also been observed for other MAPs (Noble et al., 1989; Wang et al., 2007), since no other cross reacting bands were recognized in the plant protein extract and since the antibody clearly recognized the recombinant PttMAP20, the antibody was considered specific for PttMAP20. A B 1 kDa 188 98 2 1 2 3 4 5 6 7 8 kDa 191 97 62 49 64 38 51 28 17 39 14 28 6 19 Figure 27: A PttMAP20 in poplar tissue extracts as detected using PttMAP20 specific antibodies in a Western blot. Left lane, SeeBlue® Plus2 prestained protein standard (LC5925, Invitrogen); lane 1 xylem extract; lane 2- recombinant PttMAP20 protein. B. SDS-PAGE analysis of recombinant PttMAP20 expressed in E. coli during different stages of purification. MWL - SeeBlue® Plus2 pre-stained protein molecular weight standard (Invitrogen); lane 1, total soluble protein extract of E. coli cells expressing PttMAP20; 2, PttMAP20 protein eluted from the Ni Sepharose™ High performance matrix; 3, the same sample as in lane 2 after concentration and buffer exchange; lanes 4 – 8, fractions eluted from HiLoad 16/60 Superdex 75pg size exclusion matrix. Lanes 4 – 7, 20µL of protein sample; lane 8, 1µg of representative fraction of purified PttMAP20. 4.3.2. PttMAP20-MT binding studies The presence of a TPX2 domain suggested that PttMAP20 might be a microtubule associated protein. To test this hypothesis, PttMAP20 was expressed in E. coli, purified (Figure 27B) and used for binding studies with in vitro polymerized bovine microtubules (BMT). After incubation with BMT and centrifugation, PttMAP20 was recovered in the pellet fraction together with BMT (Figure 28A lane 4P), while it remained in the supernatant fraction in the absence of BMT (Figure 28A, lane 5S). BMT in the absence of other proteins were found in the pellet (Figure 28A, lane 3P) while BSA (used as a non-binding control protein) remained in the supernatant, whether incubated with or without BMT (Figure 28A, lanes 1S and 2S, respectively). These data indicate that PttMAP20 indeed binds to microtubuli. 54 Figure 28A: Coomassie blue stained SDS-PAGE (color inverted) demonstrating the binding of PttMAP20 to bovine microtubules (BMT) in vitro. 20 µL of supernatant (S) or 5 µL of resuspended pellet (P) (see text) containing the different proteins were loaded in the gel as follows: 1. BSA and BMT; 2. BSA; 3. BMT; 4. BMT and PttMAP20; 5. PttMAP20. Figure 28B: Fluorescent micrographs showing the binding of PttMAP20 onto poplar and bovine MT. Images are shown of in vitro assembled PMT and BMT incubated with no PttMAP20 (A3, D3), with xylem extract containing PttMAP20 (B3, E3) and with recombinant PttMAP20 (C3 and F3). Columns A1C1 and D1-F1 detection of the assembled PMT and BMT respectively by using visible light; columns A2F2 detection of the mouse anti-α-tubulin antibodies labelled with Rhodamine (TRITC)-conjugated affinity-pure rabbit anti-mouse IgG antibodies; columns A3-F3 detection of anti-PttMAP20 antibodies labeled with anti-rabbit Alexa Fluor®-488-fluoro-nanogold™ Fab fragment; columns A4 – G4, an overlay of the images in columns 2 and 3; Control lane G experiment performed without including MT but rest of other components as in lane C and F. 55 Recombinant PttMAP20 was also found to bind to MT assembled in vitro using poplar protein extracts as a tubulin source. Finally, a protein in the xylem extract that reacted with PttMAP20 specific antibodies was found to bind to in vitro assembled poplar MT (Figure 28B). 4.3.3. DCB Binding Studies Since the cellulose synthesis inhibitor, 2,6-diclorobenzonitrile (DCB) has been shown to bind to other plant proteins similar in size to PttMAP20 (Delmer et al., 1987), we investigated whether recombinant PttMAP20 could be labeled with a photoreactive and radioactive DCB analog, 2,6-dichlorophenylazide ([3H]DCPA) (Delmer et al., 1987). Incubation of increasing concentrations of PttMAP20 with 0.75 µM [3H]DCPA led to a gradually increasing signal when the mixtures were irradiated by UV light (Figure 29A, lanes 1-5). Control experiments in which either [3H]DCPA, UV irradiation or both were omitted gave no signal (Figure 29A, lanes 6-8). A Figure 29: Labelling of recombinant PttMAP20 by [3H]DCPA. (A) lanes 1-5: 1 ng, 10 ng, 100 ng, 1 µg and 10 µg (respectively) of purified PttMAP20 were labelled in the presence of 0.75 µM [3H]DCPA as described in Materials and Methods; 6-8, control experiments performed using 1 µg of PttMAP20 but omitting the UV irradiation (lane 6), [3H]DCPA (lane7), or both (lane 8). (B) Inhibition of the binding of [3H]DCPA to PttMAP20 by competition with DCPA. The experiments were performed using 0.75 µM 3 [ H]DCPA, 1 µg of PttMAP20 and increasing concentrations of DCPA as indicated in the plot. The amount of bound [3H]DCPA was determined by measuring the radioactivity in the PttMAP20 bands excised from SDS-PAGE gels (liquid scintillation; see Materials and Methods for experimental details). (C) as in B, but using DCB as a competing molecule. A maximum of 5% variation was obtained for each point in B and C after replicate experiments. The specificity of the labeling was confirmed by competition experiments showing that the labeling of PttMAP20 by [3H]DCPA decreased with increasing concentrations of non-radioactive DCPA (Figure 29B) or DCB (Figure 29C). The amount of [3H]DCPA bound to PttMAP20 dropped by nearly 50% when 2 μM competing DCPA was added in the photoaffinity labeling reaction mixture together with 0.75 µM [3H]DCPA (Figure 29B). A slightly lower competing effect was obtained with DCB in the same conditions (~25% decrease of bound [3H]DCPA in the presence of 2 μM DCB) (Figure 29C), probably since DCPA, but not DCB, is structurally identical to [3H]DCPA. The amount of bound [3H]DCPA dropped by up to 90% in the presence of a 25-fold excess of either of the competing reagents (Figure 56 29B and C), thereby demonstrating the specificity of the labeling. Interestingly, the presence of DCB up to a final concentration of 200 µM did not influence the binding of PttMAP20 onto microtubules (Figure 30A). Furthermore, no effect on MT polymerization was detected in the presence of up to 7500 µM DCB (Figure 30B). A B Figure 30: Effect of DCB on the binding of PttMAP20 onto BMT and on the polymerization of BMT. (A) Increasing concentrations of DCB were incubated in the presence of 1 g recombinant PttMAP20 for 20 min. The mixture were subsequently added to pre-assembled BMT and subjected to the spin-down binding assay (see Materials and Methods). The presence of tubulin and PttMAP20 in the pellet and supernatant was assessed by SDS-PAGE analysis. 1: no DCB; 2-4: 2 µM, 20 µM and 200 µM of DCB, respectively. (B) Increasing concentrations of DCB were mixed with monomeric α and β tubulins. After 10 min incubation on ice, polymerization of BMT was initiated using the spin-down assay kit and the extent of polymerization was determined by SDS-PAGE analysis of the supernatant and pellet. 3-7: presence of 0.75 µM, 7.5 µM, 75 µM, 750 µM and 7500 µM of DCB (respectively); 1: DCB was replaced by buffer; 2: DCB was replaced by methanol. 4.3.3.1. DCB binding of PttMAP20 and its orthologs Since different plants are known to exhibit different sensitivity to the herbicide, some orthologs of PttMAP20, from rice, Arabidopsis and cotton, were recombinantly expressed in E. coli (Figure 31A and 31B). Expression screening were performed with western blotting using the Anti-PttMAP20 antibodies (Figure 31C) and the recombinant proteins were affinity purified with the N-terminus HIS tag (see Materials and Methods; 3.3.5). 31A Figure 31 (A): Sequence alignment of AtM20L, GhM20L, OsM20L and PttMAP20 proteins and the extended TPX2 domains is marked with thick black line 57 31B 31C pET 28a(+) AtM20L GhM20L OsM20L pET 28a(+) AtM20L GhM20L OsM20L Figure 31 (B) Expression screening of pET28 a(+) and AtM20L, GhM20L and OsM20L, all cloned in pET28a(+) and expressed in E. coli BL21 (DE3 Codon+). Cell pellets resulted from same cell density were used to extract the soluble protein and 10µL of samples were loaded on a 12% SDS PAGE: Lane1. 0 hour of induction with IPTG, lane 2. 1 hour after induction of IPTG and lane 3. 2 hours after induction. (C) Western blot showing the expression screening performed with the same protein sample as Figure 31B and labelled with Anti-PttMAP20 antibody, raised against the full length protein including the TPX2 domain found common between the PttMAP20 and its orthologs. The purified proteins were then labelled with [3H]DCPA and the competition experiments were performed with the DCB to understand the comparative binding properties of PttMAP20 and its orthologs. As seen in Figure 32, the affinity of DCB is indeed different between the proteins, with the protein from the monocot rice with the lowest affinity. Figure 32: Comparative Labelling of recombinant PttMAP20 and its orthologs by [3H]DCPA. Inhibition of the binding of [3H]DCPA to PttMAP20 and its orthologs by competition with DCB. The experiments were performed using 0.75 µM [3H]DCPA, 1 µg of PttMAP20 and its orthologs with increasing 3 concentrations of DCB as indicated in the plot. The amount of bound [ H]DCPA was determined by measuring the radioactivity in the PttMAP20 bands excised from SDS-PAGE gels (liquid scintillation; see Materials and Methods for experimental details). 58 4.3.4. In situ and in vivo localization 4.3.4.1. Poplar immunolocalization, in situ Immunolocalization of PttMAP20 protein in the wood forming tissues was then carried out by indirect immunofluorescence experiments. Strong signals corresponding to the anti-PttMAP20 antibodies were detected in all cell types (Figure 33A) while no background labeling was detected when using the pre-immune serum instead of primary antibodies (Figure 33C). The strongest signals were observed in developing xylem cells (fibers, vessel elements and ray parenchyma cells) at the early stages of secondary cell wall thickening (SW) whereas weaker signals were seen in the cambium (CA) and in the radial expansion zone (RE) (Figure 33A). The signals were located inside the protoplasts, which is also evident in the transmission electron microscopy (TEM) micrographs revealing the labeled PttMAP20 along the plasma membrane (Figure 33D and E). Also the microtubules were visualized in the same tissues using a monoclonal antibody DM1A, which is specific for the AALEK epitope at the C-terminus of mammalian α-tubulins (Blose et al., 1984). The specificity of the antibody was verified by western blot analysis of hybrid aspen xylem and phloem protein extracts that revealed a single protein band of 49 kDa, corresponding to the predicted size of aspen α-tubulin (data not shown). As expected, signals specific for α-tubulin were detected in all stem cells except for the sieve tube cells (indicated by an arrow in Figure 33B). The labelling of poplar MT gave a continuous signal typical for microtubular networks (Figure 33F). In developing wood cells, the α-tubulin signals were moderate in the cambium (CA), lower in the radial expansion zone (RE) and strongest during secondary wall deposition (SW) (Figure 33B). Although the signals specific for both α-tubulin and PttMAP20 coincided in the same cell types in developing wood, there were differences in the intensity and the patterning of the labeling. The α-tubulin signals were clearly more distinct than the PttMAP20 signals in the cambium, where microtubules have a major role in cell division and primary wall deposition. Furthermore, in contrast to the relatively even and continuous labeling of α-tubulin, the PttMAP20 signals appeared more sporadic, forming discontinuous patches of labeling (Figure 33, compare panels A and B). The patches observed in TEM experiments were about 100 nm long and spaced about 1 μm from one another. These observations suggest that PttMAP20 has a spatially restricted role specifically during secondary cell wall thickening. 59 B A F D C 1µm E F 0.1µm Figure 33: Immunolocalization of PttMAP20 and microtubules in the wood forming tissues of hybrid aspen using confocal microscopy (A-C and F) and transmission electron microscopy (TEM) (D and E). Panes A, D and E - Affinity purified polyclonal anti-PttMAP20 antibodies; B - Monoclonal anti-α-tubulin antibodies; C - Pre-immune serum – a negative control for the MAP20 antibodies. Transverse sections through the wood-forming tissues show developing and mature phloem (dPH and mPH), vascular cambium (CA), radial expansion zone (RE) and the secondary wall deposition zone (SW). The arrow in B points to the sieve tube cell. D - A region in the secondary wall forming xylem ray cell showing the distribution of the label (arrows) along the plasma membrane. E - A magnified image of panel D showing details of the label distribution. F - Typical microtubular networks in aspen parenchyma cells as labelled by monoclonal anti-α-tubulin antibodies. 60 4.3.4.2. Transient expression in tobacco, in vivo To corroborate the binding of PttMAP20 to microtubules in vivo, we examined the subcellular localization of PttMAP20 by imaging of PttMAP20-YFP fusion protein transiently expressed in tobacco leaves (Figure 34). Confocal microscopy observations of the YFP signal clearly show that the fusion protein localizes to interphase microtubules in leaf epidermal pavement cells and that the net-like structure is labeled uniformly throughout its length (Figure 34A). Interestingly, more detailed analysis of the YFP signals revealed some cells in which the protein occurred in patches of dot-like structures (Figure 34B), resembling those observed in the poplar tissues (see above). B A Figure 34: Confocal microscopy images of tobacco leaf epidermal cells transiently expressing PttMAP20 fused to YFP. The images were recorded 3-5 days after the agroinfiltration. The scale bar is 20 μm for all micrographs. (A) Decoration of the tobacco microtubules by the PttMAP20-YFP fusion proteins in vivo. (B) Images of rarely occurring cells in which the signals from the PttMAP20-YFP fusion proteins appear a patches of dotted signals. Characterization of the TPX2 domain of PttMAP20 The same technique (as explained in 4.3.6.) was used to visualize the location of truncated PttMAP20 protein. The truncated or deletion series were constructed based on the TPX2 domain found in the middle of the protein analysed with SMART analysis (http://smart.embl-heidelberg.de/), Figure 35A. The deletions series were cloned in the expression cassette in frame with C-terminus GFP fusion protein (Figure 35B). The transfected tobacco leaves were then visualized in confocal microscopy (Figure 35C). 10 20 30 40 50 60 MEKAHTKSAL KKLVKASSQS ASWSNAARGM AKDDLKDPLY DKSKVAPKPL AKENTKPQEF 70 80 90 100 110 120 KLHTGQRALK RAMFNYSVAT KIYMNEQQKR QIERIQKIIE EEEVRMMRKE MVPRAQLMPY 130 140 150 160 170 FDRPFFPQRS SRPLTVPREP SFHMVNSKCW SCIPEDELYY YFEHAHPHDH AWKPVN Figure 35A: PttMAP20 sequence showing the TPX2 domain, marked in red, identified using SMART analysis. 61 1 61 123 176 PttMAP20-FL (1-176) 1 PttMAP20-N (1-61) 2 PttMAP20-TPX2 (61-123) 3 PttMAP20-C (116-176) 4 5 PttMAP20-N+TPX2 (1-123) PttMAP20-C+TPX2 (61-176) Figure 35B: Cartoon describing the serial deletions of PttMAP20 constructs (1 -5) visualized in tobacco transient expression system. FL, Full-length; N, N-terminus; C, C-terminus. N-terminus in blue; TPX2 domain in grey; C-terminus in green; YFP tag in yellow. 1 2 3 4 5 Figure 35C: Confocal microscopy images of tobacco leaf epidermal cells transiently expressing deletion series of PttMAP20 fused to YFP (explained in Figure 34). The images were recorded 3-5 days after the agroinfiltration. The results suggest that the signal indicating binding to tubulin, as seen before with the full length protein YFP labelling in tobacco transient expression (Figure 34A), is found only with the construct 4 (Figure 35C) containing both the N-terminal and the TPX2 domain of PttMAP20. A signal of much lower intensity was seen with the construct 1 (Figure 35C), containing only the N-terminus of PttMAP20 and construct 2, containing only the TPX2 domain of PttMAP20. Construct 3 (Figure 35C) with only the C-terminal domain of PttMAP20, doesn’t show any labelling. The construct 5 (Figure 35C), containing the TPX2 and the C-terminal domain of PttMAP20 shows weak but patched signals similar to those noted for the intact PttMAP20 in Poplar tissues (Figures 35 D and E) and in some cells of tobacco (Figure 34B). 62 4.4. PHENOTYPIC CHARACTERIZATION 4.4.1. Overexpression in Arabidopsis Stable Arabidopsis plants expressing PttMAP20 under the CaMV 35S promoter were generated and the overexpression was confirmed by real time PCR (Figure 36A). Inspection of the visual phenotypes of several independent over expressing (OE) lines revealed left-handed helical growth of rosettes leaves (Figure 36B) and stunted root growth (Figure 36C), two phenotypes that have been previously observed with microtubule destabilization (Smertenko et al., 2004; Valentine et al., 2004; Ishida et al., 2007). A B WT C WT PttMAP20-OE PttMAP20-OE Figure 36. Preliminary characterization of transgenic lines of Arabidopsis overexpressing PttMAP20 (A) RT-PCR analysis of wild type (WT) and transgenic (PttMAP20-OE) plants using primers specific for PttMAP20 and Actin. (B) Rosette leaves from 2 weeks old PttMAP20-OE and WT Arabidopsis plants. The helical growth typically associated with MAPs is apparent for plants overexpressing PttMAP20. (C) WT (left) and PttMAP20-OE Arabidopsis plants (right), the latter showing a stunted root phenotype. 63 64 5. DISCUSSION The economic importance of cellulose has imparted enormous scope for its research. High throughput techniques used in modern biotechnology such as genome sequencing and expression profiling have resulted in a huge amount of data. Such data is now available for many cellulose producing organisms. In general, it is known that there is a high score of unknown genes in these data and by characterizing these unknown genes the dark parts of cellulose science can be resolved. The discovery and characterization of PttMAP20 is one such example. The PttMAP20 gene was first discovered in a microarray experiment performed to expression profile the wood formation process in hybrid aspen. Bioinformatic analyses showed that PttMAP20 as a gene of unknown function had no hit in blast searches and it encoded a cytosolic coiled coil protein. At transcript level the expression pattern of PttMAP20 was found to follow the secondary cell wall specific CESA genes. This co-regulation suggests that PttMAP20 might be required directly or indirectly during cellulose biosynthesis and - more importantly - during the secondary cell wall formation where cellulose deposition is highly organized. Standard sequence comparisons revealed that PttMAP20 shares a small conserved domain with a large multidomain microtubule associated protein, TPX2, from Xenopus (Wittmann et al., 1998; Wittmann et al., 2000). The original TPX2 protein sequence contains two short domains predicted to adopt a coiled coil structure often involved in protein-protein interactions, one at the N-terminus (residues 171208) and one at the C-terminus (650-684). The C-terminal coiled coil domain is highly conserved in all TPX2-like proteins and it is thus called the TPX2 domain. Proteins containing a TPX2 domain have so far been identified in animals, fungi and plants but functional data concerning these proteins is scarce. Animal TPX2 is reported to interact with proteins using microtubules as a landing place to perform their function (Wittmann et al., 1998; Wittmann et al., 2000; Kufer et al., 2002; Brunet et al., 2004). Sequence alignments show that TPX2 proteins exhibit low or no similarity with each other beyond their TPX2 domains. This indicates that the TPX2 domain is a common nominator of many multi-domain proteins that overall do not have common evolutionary origins. However, a common origin is apparent for the TPX2 domains suggesting that these domains share a similar function. Sequence analyses also revealed genes encoding proteins with a TPX2 domain but no additional similarity to PttMAP20, hereby denoted MAP20-like genes (M20L) The phylogeny of the M20L genes clearly separates monocots and dicots and although several branches are not resolved, the dicot subtree does not contradict the species tree. The M20L phylogeny formed the basis for a search for signs of adaptive evolution (Yang, 1997; Bielawski and Yang, 2003). Only the extended TPX2 domain was studied since we could not derive a reliable multialignment over the full protein sequences. The hypothesis was that key properties in this conserved region could have changed, especially after the monocot/dicot split. However, no signs of positive selection were found, either over branches or on sequence sites. The extended TPX2 domain thus seems to have been under negative selection only. A recent study on gene family evolution dynamics (Wapinski et al., 2007) showed that duplication resistant characteristics are typical for genes related to essential growth processes, genes active in organelles and nucleus and genes essential for viability. It is possible that the proposed, but as yet undefined 65 role of MAP20 in cellulose biosynthesis would fulfil the requirements of such a process in plants. Our present data show that PttMAP20 binds to microtubules in vitro, in situ and in vivo and that its overexpression in Arabidopsis leads to a phenotype typical of microtubule associated proteins. The overexpression of PttMAP20 in Arabidopsis suggests that the hypocotyl morphology is affected and its twisted pattern suggests that the cellulose orientation is affected. An interesting future experiment would be to analyse the cellulose content in the plants presenting such phenotypes. DCB is an herbicide that inhibits cellulose synthesis leading to dwarfed plant phenotypes (Sabba and Vaughn, 1999 and references therein) and abnormal cell plate formation (Meyer and Herth, 1978). The herbicide has been shown to bind to small proteins of 12 – 18 kD, the amount of which seems to increase significantly at the onset of secondary cell wall synthesis (Delmer et al., 1987). It was suggested that the receptor protein for DCB might act as a regulator of cellulose synthesis, but its identity and the mode of action of DCB have remained unknown. Here we have shown that PttMAP20, which is also highly expressed during early xylogenesis and tightly co-regulated with the secondary cell wall associated CESA genes, is a target for DCB in poplar. This suggests that the microtubule associated protein PttMAP20 is also involved in cellulose biosynthesis. Labelling of PttMAP20 with DCB, or its functional analogs, in vivo might shed light on the possible connection of PttMAP20 and cellulose biosynthesizing machinery. Visualizing the CesA motion with GFP fusion studies as described by Paradez et al., 2007 can also be performed by overexpression of PttMAP20 in Arabidopsis plants, combined with DCB treatment. Classical hypotheses on the connection of cellulose biosynthesis and cortical microtubules suggest that microtubules form barriers that constrain the paths of cellulose synthase complexes on the plasma membrane thereby guiding the ordered deposition of the microfibrils (reviewed by Giddings and Staehelin, 1991). However, recent data suggest instead that organized microtubules may be required to establish rather than to maintain the pattern of cellulose microfibril deposition (Sugimoto et al., 2003; Roberts et al., 2004; Wasteneys, 2004), perhaps to correctly position CESA protein complexes in the plasma membrane (Gardiner et al., 2003b). In this respect, a model presented earlier by Baskin (2001) is interesting. According to him, the nascent microfibril could be incorporated into the cell wall by binding to a scaffold that is oriented around either already incorporated microfibrils and/or plasma membrane proteins. The role of cortical microtubules would thus be to assist in the deposition and orienting components of the scaffold at the plasma membrane. In the light of observations that cellulose deposition seems to be confined to discrete regions in the plasma membrane (Hogetsu, 1991; Roberts et al., 2004), it is interesting that PttMAP20 appears as patches of dot-like structures in immunolocalization experiments in poplar and in overexpression experiments with PttMAP20-YFP fusion proteins in tobacco. To observe such punctate patterns of cellular localization is not unusual among different microtubule associated proteins (Liu et al., 1995; Zhao et al., 2003; Preuss et al., 2004; Wang et al., 2007). As microtubules themselves are relatively uniform structures and offer few determinants for site specific association of other proteins along their length, it has been suggested that the punctate patterns of cellular localization arise when microtubule associated proteins occur as a part of larger molecular complexes. Examples of such proteins include e.g. the HaWLIM1 in sunflower (Briere et al., 2003), the MT plus end binding CLASP in Arabidopsis (Kirik et al., 2007) and the MP30-interacting protein, MPB2C in Zea mays (Kragler et al., 2003; Winter et al., 2007). In the case of cellulose 66 synthesis, the punctate localization of PttMAP20 could be explained if it bound, directly or indirectly, to the CESA complexes and/or ‘scaffoldins’ as suggested by (Baskin, 2001). In vivo imaging of a deletion series of PttMAP20 suggests that the protein sequence containing the TPX2 domain is required for the microtubule binding but that the intensity and punctuate pattern of its association is dependent on the Nterminus of the protein. It is possible that the N-terminus of MAP20 interacts with another protein thus forming a link between microtubuli, MAP20 and cellulose synthesis. The patches of punctuate signals corresponding to PttMAP20 may also be taken as an indication of that the protein is regulated between “on” and “off” binding to mirotubuli. Many MAPs are regulated by phosphorylation and sequence analyses indicate that PttMAP20 may also be a phosphoprotein, as it contains 1-6 putative phosphorylation sites. Interestingly, a putative targeting motif called as Tyrosine targeting motif (YYYF) is found in the C-terminus of PttMAP20 and such a targeting motifs are often phosphorylated. Therefore, future studies about the phosphorylation of PttMAP20 will be informative. If PttMAP20 will be proven to be a phosphoprotein, the kinase/s involved with the phosphorylation need to be explored. Furthermore, determination of the structure of the PttMAP20 with or without its binding partner/s could help to understand its function. Since PttMAP20 is the target of DCB in poplar and putative orthologs of the protein were identified in Arabidopsis and other plant species, it is conceivable that it is the inactivation of MAP20 by DCB that leads to the inhibition of cellulose synthesis. Interestingly, DCB treatment has previously been shown to cause changes in number of intact rosettes at the plasma membranes of algae and wheat (Herth, 1987; Rudolph et al., 1989) and accumulation of cellulose synthase subunits within localized regions at the plasma membrane of Arabidopsis hypocotyls (DeBolt et al., 2007a). DCB also causes cessation of the CESA mobility at the plasma membrane (DeBolt et al., 2007a). Phylogenetic analysis has showed that MAP20 proteins could be grouped into two clades, one for monocots and the other for dicots. Interestingly their putative transcription factors were found to be conserved within the monocot and dicot species as well. The trend seen in the differential sensitivity of Rice and the rest of dicot species, Arabidopsis, Cotton and Poplar, to DCB could be due to the differences in the MAP20 proteins of dicots and moncots. Differential sensitivity of DCB to crop species has been noticed previously in agronomic studies (Hardcastle and Wilkinson, 1971b) and it is interesting to note that a protein, PttMAP20, found to bind DCB has similar trend. We therefore hypothesize that PttMAP20 and its orthologs in other plants mediate a dynamic interaction between the cellulose synthase complexes and cortical microtubules and that this interaction, which is essential for successful initiation of cellulose synthesis, is disrupted by DCB. However, as DCB does not prevent the binding of PttMAP20 to microtubules, it may interfere with either direct or indirect binding of the protein to the CESA protein complexes. 67 68 6. CONCLUSION This work has shown that PttMAP20 shows a high transcript level during secondary cell wall formation and that it associates with the cytoskeletal network. The protein also binds to a cellulose biosynthesis inhibitor, DCB, suggesting relevance for cellulose biosynthesis. The characteristic features of PttMAP20 suggest a strong connection to cellulose biosynthesis in poplar secondary cell walls, known to have an organized cellulose orientation and cortical microtubuli. The work accomplished in this thesis has led to different hypotheses, described and illustrated in Figure 37 that will hopefully inspire more research efforts directed in solving the molecular function of PttMAP20 completely. Figure 37: A cartoon illustrating possible molecular functions of PttMAP20. A. PttMAP20 has been shown to bind microtubuli in vitro and in vivo. Hypothesis 1: Targeting of PttMAP20 and binding of it to microtubule might be regulated by phosphorylation by kinases that are specific for each phase of cellular development. B. PttMAP20 is found as patches in situ and also in vivo. Hypothesis 2: The patches might be formed by the change or microtubule filament surface which could be due to its contact with other organelles or its polymerizing and depolymerizing ends. C. Hypothesis 3: The patches could be an indication of a direct or indirect association of PttMAP20 to a protein complex example CesA complex. D. Hypothesis 4: The patches of MAP20 might be guiding the contact of the rosettes with microtubuli without itself having a direct binding to the rosette. E. PttMAP20 binds to DCB but DCB doesn’t affect the binding of PttMAP20 to microtubuli and microtubuli polymerization. Hypothesis 5: This is possible if hypothesis 3 is valid. PttMAP20 - when bound to DCB - might loose its contact with the CesA complex and thereby the complex will be derailed. 69 70 7. LIST OF ABBREVIATIONS Blast BMT BSA BY-2 cDNA CesA CFP CS Csl CSR DCB DCPA DNA DP EST FPLC GFP GH GPI GT HEPES HMM HVR IMAC IPTG kDa KOR M20L MAP mRNA MT Mw PBS PCR PLD Pt Ptt qPCR RACE RNA RNAi RT-PCR SDS-PAGE SuSy TATA TC TEM TPX2 UDP UTR WT YFP - basic local alignment tool bovine microtubule bovine serum albumin cultivar Bright Yellow - 2 of the tobacco complementary DNA cellulose synthase enzyme cyanine fluorescent protein cellulase synthase complex cellulose synthase-like class specific region 2,6-dichlorobenzonitrile 2,6-dichlorophenylazide deoxyribonucleic acid degree of polymerization expressed sequence tag Fast performance liquid chromatography green fluorescent protein glycosyl hydrolase glycosyl phosphatidylinositol glucosyltransferase 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid hidden Markov models hyper variable region immobilized metal affinity chromatography isopropyl-beta-D-thiogalactopyranoside 1000 Daltons KORRIGAN MAP20-like microtubule associated protein messenger ribonucleic acid microtubule molecular weight phosphate buffered saline Polymerase chain reaction phospholipase D Populus tremula Populus tremula x tremuloides quantitative PCR rapid amplification of cDNA ends ribonucleic acid RNA interference reverse transcriptase PCR sodium dodecyl sulphate polyacrylamide gel electrophoresis sucrose synthase TATA box, DNA sequence, cis-regulatory element terminal complex transmission electron microscopy targeting protein of kinesin-like protein uridine 5’-diphosphate’ untranslated region wild type yellow fluorescent protein 71 72 8. ACKNOWLEDGEMENTS My sincere gratitude to ‘Almighty’ for everything that I am blessed with. I would like to thank all the people responsible for the making of this thesis and without each and everyone involved, it would had been impossible to present this thesis. I am grateful to Prof. Tuula T. Teeri for providing an opportunity to pursue my PhD studies in Sweden. Thank you Tuula, for all that you have done during the academic tenure as a philosopher, guide and as a good friend. I am especially grateful for the tireless assistance and guidance in the making of this thesis and repetitive submission of several manuscripts even when you were sick. Tuula, I am also grateful for your guidance during the early times when I was new to the Swedish system and its research curriculum. I feel obliged to thank Sweden in general for providing the infrastructure and the safety during my stay here for the PhD studies. I sincerely thank Dr. Stuart Denman and Dr. Kristina Blomqvist for their initial start-up supervisions. Special thanks to Dr. Henrik Aspeborg for being my Microarrays guru and providing enormous resource to the group. I would like to thank my previous co-supervisor Dr. Emma Master for her supervision of glycosyltransferases recombinant expression in Pichia. Emma, your work stratergy is an inspiration to me and belated thanks for the help for the critical reading of my licentiate thesis. I would like to thank Dr. Peter Nilsson, KTH, for his supervision of the work with the Microarrays technology. I would like to thank Dr. Dan Cullen, USDA, Madison, USA, for a nice collaboration and for the friendly accommodation in his lab during my visits. I would like to acknowledge Dr. Lars Arvestad, SCB, KTH, for being an excellent collaborator. Lars, I really enjoyed our fruitful discussions and team work and hope we will work together in the future as well. I would like to thank Prof. Vincent Bulone for a fruitful collaboration and many invaluable discussions during the later phase of the MAP20 project. Thanks Vincent, for the help with the critical reading of the manuscripts and the encouragements. I am grateful to Doc. Ines Ezcurra and Dr. Gea Guerriero for a fruitful collaboration for the plant biology part especially with the confocal sessions and the research discussions. Thank you Ines and Gea, I feel that your team brought additional light to the MAP20 project with your plant science expertise. I would like to thank Dr. Ewa Mellerowicz and Prof. Björn Sundberg from UPSC, Umeå for a fruitful collaboration for the plant science part of the MAP20 project and for providing my requirements during confocal work sessions in Umeå. The heartfelt gratitude extends to other collaborators and co-workers in my PhD projects; Christian Brown (GT, GH and MAP20 projects), Dr. Charlotta Filling (GT expression in Pichia) anders Winzéll (GT expression in Pichia), Doc. Harry Brummer (TmGH36 project and DCB part of MAP20 project), Potjamas Pansri (MAP20 crystallography), Doc. Christina Divne (MAP20 crystallography), Dr. Tan Tien-Chye (MAP20 crystallography) and Prof. Sophia Hober (Antibody development). 73 I would like to acknowledge the European Union (Project no. QLK5-CT200100443), the Wallenberg Foundation and the Swedish Research Council (Formas) for the funding of my PhD research. I would like to thank all my office roommates; Aihua Pei, Luminita Balau, Christian Brown, Houssine Sehaqui and Anders Winzéll for bringing a warm atmosphere in the office room. Chris, you are a wonderful work mate, I admire your ‘dearly critical’ quality. I enjoyed working with you very much. Andarsh, thank you for your Swedish language help. Special thanks to all my ex-wood colleagues; Dr. Martin Baumann, Dr. Soraya Djerbi, Dr. Åsa Kallas and Dr. Ulla Rudsander for all the memorable times at work, conferences and official trips. Also I would like to thank all other members in BIOMIME center especially the past and present administrators Alphonsa Lourdudoss, Lotta Rosenfeldt, Elzbieta Nilsson and Marita Johnsson for running a smooth adiministration. Alphonsa mam, thank you so much for your helps as a mentor and for being a great friend for all these years. Lotta, thank you for your care when I was sick. I would like to thank the past and the present members of the Biochemistry, the Microbiology and the Pyrosequencing groups for presenting a friendly coexistence in floor 2 during all these years. Special thanks to Sofia Andersson, Doc. Per Berglund, Magnus Eriksson, Linda Fransson, Dr. Malin Gustavsson anders Hamberg, Prof. Karl Hult, Marianne Larsen, Doc. Gunaratna Rajarao, Dr. Joke Rotticci-Mulder and Mohamad Takwa for all the lunch discussions, group activities and parties. Linda and Marianne, I cherish your amazing comfort talks. Friendly hugs to Jens, Ela, Fredrika, Cecilia, Karim, Per-Olof, Farid, Erik, Qi and Johanna for their cheerful chit-chats at leisure times. Special thanks to Fredrika Gullfot and Jens Eklöf for the Swedish translation of the thesis abstract. Special thanks to Doc. Gunaratna Rajarao, Jeyaseeli Fernando and Noel Gomez for proofreading the Tamil abstract. I would like to thank Hans Johansson and Vanitha Corera for the proofreading of the thesis. ‘Kiitti Kiitti’™ to Kaj Kauko for his assistance with the microscopy facility and specially for taking care of all my stupid computer problems. Thanks to Dr. Gustav Sundqvist for running the mass spectrometry facility smoothly as a friendly peer. I would like to thank all my teachers in SBOA School especially the Principal Mrs. Shirley Paul. I am very grateful to the American College, India, especially to my favourite tutors Dr. Karunyal Samuel, Dr. G. C. Abraham, Dr. P. Anbudurai, late Dr. Balakumar, Dr. Anbudoss Barnabas and others whom have contributed in every bit of my career development. I will love to live that college life again...I wish I could relive that part of life over and over again. I would like to acknowledge Prof. A. Ganapathi, BDU, India, who initiated my research career by selecting me as a research scholar in an Indo-Israel project. I would like to extend my sincere gratitude to my friends in India and in Sweden without whom I would have missed the fun and comfort that I am blessed with. Dear Andrews, Preethi & Amma; Kumar & family, Prasana, Sundar, Amma & Dushinthi; Sankar, Noel, Baskar and Selvin; Babuji annan & Rajini athachi; Ranjani akka & Sudhakar athan; Vimal, Ashwini, Vinoth and Leo; I am so happy to have you guys in my life and you guys cared and loved me the same way even when I am far away from you for all these years. I am really grateful to all my friends in Sweden; Alphonsa mam, Doss sir, Cecilia, Elango and Per; Tuula, Vincent, Petri and Vinski; Thulasi akka, Hans and Babu; Kathleen and Wald family; Guna mam, Vasu annan and Sugeerthy; Anna and 74 Torkel family; Siju and Shalini family; Anand annan and Indrapreeth mam family, Ulla and Patrick family; Kannan annan and Nomchit, all for treating me with dinners, picnics and affection during my stay in Sweden. I would like to thank my buddies: Prasana, Jaya Seelan, Shriram, Ananth, Harish, Bragadeesh, Prakash, Madurai Prakash, Basil and many other Stockholm friends with whom I enjoyed organizing parties, cultural programs, trips and loads of fun. I hope we will be in touch for the rest of the life and my family will be very happy to host your visits. I would like to thank the International office of KTH and Indian embassy, Sweden for giving me chances to organize cultural programs. I would like to share my happiness with all family members: my siblings, Chandar annan, Jawahar annan, Ravi annan and Anjala akka; in-laws, Seelan athan, Parameshwari athachi, Jenita athachi and Jaya athachi; nieces and newphews, Dharshini, Anne, Jerin, Caroline, Adarsh, Ashwin and Alicia whom have made my sweet home into a paradise. Every spot of my life is soaked in your care and thank you all for the love, trust, prayers and supports during all these years. Appa, I am very proud to be your son and I know that I am giving that feeling to you only once in a while. Thank you dad for all that you have done for me. I know you will be the happiest person in the world seeing me with this degree. Love you´pa. Amma, I don’t have words or don’t know how I will thank you for your love, affection, care and everything you have given me in this life. You were waiting to see me become a Doctor and now you are not here to see it... Amma, please come back´ma... I don’t know how I will go home without you being there. Love you´ma. My personal aims during the PhD life could be poetically bench-marked with few representative verses from a two thousand year old Tamil literature (couplets), Thirukural. Italicized explanations are in English made by Dr. Rev. G. U. Pope. When I arrived to this beautiful snow world I tried to blend in as said in... ¯Ä¸ò§¾¡Î ´ð¼ ´Ø¸ø ÀÄ¸üÚõ ¸øÄ¡÷ «È¢Å¢Ä¡ ¾¡÷ Those who know not how to act agreeably to the world, though they have learnt many things, are still ignorant. ...aferwards I tried to get wiser as said in... ¦¾¡ð¼¨Éò àÚõ Á½ü§¸½¢ Á¡ó¾÷ìÌì ¸üÈ¨Éò àÚõ «È¢×. In sandy soil, when deep you delve, you reach the springs below; The more you learn, the freer streams of wisdom flow. ...stronger as said in... «ïÍÅ ¾ïº¡¨Á §À¨¾¨Á «ïÍÅÐ «ïºø «È¢Å¡÷ ¦¾¡Æ¢ø. Not to fear what ought to be feared, is folly; it is the work of the wise to fear what should be feared. ...more disciplined as said in... ´Øì¸õ Å¢ØôÀó ¾ÃÄ¡ý ´Øì¸õ ¯Â¢Ã¢Ûõ μõÀô ÀÎõ Propriety of conduct leads to eminence, it should therefore be preserved more carefully than life. ...more forgiving as said in... þýÉ¡¦ºö ¾¡¨Ã ´Úò¾ø «Å÷¿¡½ ¿ýÉÂï ¦ºöÐ Å¢¼ø The (proper) punishment to those who have done evil (to you), is to put them to shame by showing them kindness, in return and to forget both the evil and the good done on both sides. ..and so on. Now I am leaving Sweden with loads of gratitude and good memories as said in... ¿ýÈ¢ ÁÈôÀÐ ¿ýÈýÚ ¿ýÈøÄÐ «ý§È ÁÈôÀÐ ¿ýÚ It is not good to forget a benefit; it is good to forget an injury even in the very moment (in which it is inflicted) ¾ì §º¡ Á¢§¸ Š§ÅÃ¢Â¡!!! Ssvedanuku Nandree!!! 75 76 9. REFERENCES Albersheim, P., Darvill, A., Roberts, K., Staehelin, L.A. and Varner, J.E. (1997). 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Gene P. trichocarpa Gene Model Forward Primer (5'-3') Reverse Primer (5'-3') PtCesA1 Pt553321, eugene3.00002636 AATTGATCCCTTCTTGCCCA ATCCATACAATGACCACTCCAC PtCesA3-2 Pt555650, eugene3.00040363 CAGAGATTGACAATTATGACGAGC ACTTAAGATATCCTCAGTGACCG PtCesA9-2 Pt262611, gw1.XVIII.3152.1 AACCTGGAAGTGCAAGATGG GCCAGTATGATTAGCCTAGC Ubiquitin Pt794626, fgenesh4_pg.C _scaffold_13026000001 AGATGTGCTGTTCATGTTGTCC ACAGCCACTCCAAACAGTACC EF 1β Pt815174, estExt_fgenesh4 _pg.C_LG_I1178 TTAGGAGTATTGAGATGCCCG CAAAGATGGCTACACTACACAACC 95 Appendix Table 2: TPX2 domain containing Poplar proteins Gene model Protein sequence Pt195236 gw1.IV.325.1 LNTKNPKSATPVKDRHGFQSKLSENSNPNLSHLSPCSKPTNSPS TKSQKSASKNPTLNPNPAIFSPRKKIRERRFVVAKKNSKKETLNS NPTTVDCKCKERYGGSVKKCLCLAYETLRASQEEFFKNKNDVE EKDHLMDQNLDIEDREGSDAQYSCEIEKSGQMGSSKIKRRRNKL LEEARDSAPDNGKVKHLVEAFEKLFTLPNPKESDRTEEEEIKEN RKKAMQWALPGFQLPKTNVSSSSFCPSGFFLTSENLGLDTRISI SSSWDGSQGSNSSRSSNGGRRSRRNSAESGATTGGRRLKKK QIKITSQKPFKLRTEQRGRLKEEEFTKKIQEIMTEEEKLRIPIAQGL PWTTDEPECLIKPPVKENTRPIDLKLRSDIRAVERADFDHQVSEK MSLIEQYKMERERQQKLAEEEEVRRLRKELVPKAQPMPYFDRP FIPRRSMKHPTMANEAKLRRHKKIKFCQSWNDVSSYSYDQQ* 2. Pt75311 fgenesh1_pg.C_LG_VII000075 MAAESDDSSTATTATTTTSLTANATTSLKENTTTLMLVDEMYEFS APKFYDFVKGESDEDSRNAELWFDVTAAYAPSPFMPRIKTGRSF KVETLCDFSQADQLHKVAESSDSKPTDSSSDSNSQSEVMPLPE PAASSEMRKEETSSNEENNANLINVISAGDVTCEKEKKVGFACA ESNGRCTSSLQIENTDGKSSKNDAYCTPKQPMSSRNRGILTDSK KNQSARHIASLVKNPSSVKPKGQSQSSRVKGIKPSSVKKDPNVK NVAGTANLAQENQAIKKQKLDGGRSRQIVNAKPPQPLMHKSKL GLSSGNSNFCSSVPNKMQKVDRKVYVREQAAPAPFVSMAEMM KKFQSNTRELSLPHDGPASVIQRKPKLTLTRPKEPEFETAQRVR SVKIKSTAEIEEEMMAKIPKFKARPLNKKILEAPTLPALPRSTPHR PEFQEFHFVTAARANQNAESASVASTEVSCQSNQWKPHHLTEP KTPVLHTSLRRPAMVKSSLELEKEELEKIPKFKARPLNRKIFESK GEMGIFCHVKKQVTIPQEFHFATNERIPPASSVVDMFEKHMNKS NTCISSCWSSSQLSLRSEPTNENPIPRNTLPNPFHLHTEERGAE KERKFVMELMQKQMEEERAFHRANPYPYTTDYPVIPPRPEPKP CTKAEPFQLESLVRHEEEMQREMQERERKEKEEAQMRIFRAQP VLKEPIPLPEKVRKPLTQVQQFNLNADHRAVGRAEFDQKVKEKE MLYKRYREESETARMMEEEKALKQLRRTMVPHARPVPNFNRPF FPEKSSKETTNARSPNLRVLQRRERRKMMVNAASSATASGMR* 3. Pt85496 fgenesh1_pg.C_LG_XIII000413 MEKKPNGLAVKFNGVSHDRVHFAPKLSEGVIKAKEYVEKETAEE SEKQDVLGVKSTNFDADVSDEKDEKPEAQKSSDDRNSSSPSLK AGGVGNAHVRQTVPQPFALATDKRVGRNTFTNSNNAQSPATM KNSQQNSPSTARKPLQPDNKKRHDEEDSWSVASSYPVRFEQH LSLAMFSTAASVRTVKSVTVGTAPTFRSAERAAKRKEYYSKLEE KHRALEKERSQAEERTKEEQEAAIRQLRKNMAYKANPVPNFYY EPPPPKVERKKLPLTRPQSPKLNRRKSCSDAVQTSQEEVGKHC ARHRHSIGNHKDSTATSTAKAKVQISSQTANGIRKVTGRSKQER VTAEAVPEKTAEPTNADISVQS* 4. Pt355005 fgenesh1_pg.C_LG_VIII001919 5. Pt354840 No 1. fgenesh1_pg.C_LG_VIII001754 MGESLVAASSYEDKIGGTVASDPALQASVSFGRFENDSLSWDK WSSFSQNKYLEEVEKCATPGSVAEKRAYFEAHYKKIAARKAELL DQEKQIEHDLSRANNQNSGDLIVKTSQMDSDFDASNGQTSSEGI RPESKFDNEWDGGHIDKPTEDAAIDAHGQASTNKPYEDTAVDA HGQASSNDPYEDAAFSVHGQASLNEPYEDAAIDVQGQVPLNGR VKEEQDSELDTPVSAKLEEVALMKKEETGSQDMRELPKNLEKE MESILMIKEEKVKLDHRKESPKISPMSKVRDLAMAKKKPEPPITK RPQISSLKFSKPASTSSSLSASQSSIKKVNGSSLPRSKNTPVGGN KKVNPKSLHMSLSMDSPNSETVPLTTTRKSFIMEKMGDKDIVKR AFKTFQNNFSQLKSSAEERSIGAKQMPAKEIGVKVSTSMTPRKE NIGSFKSGGVDRRTAKLAPSSSVLKSDERAERRKEFSKKLEEKS KTEAESRRLGTKSKEEREAEIKKPRRSLNFKATPMPGFYRGQKA SKSPLDKVFAFGFPFLISVYMLVGCLFMDTKPDKIEKPIK* MGESIVAASSYEDKIGGTAASDPALQVSVSFGRFENDSLSWEK WSSFSQNKYLEEVEKCASPGSVAEKKAYFEAHYKKIAARKAELF DQEKQMEHESSMENNHNIGDLTGKNGQTDSSFDVSNGQTSAE GIWHESKLDNERDGGHVDEPYEDAAIDVHGQASLSGLYEDAAN DVQSQASSNGRVKEELENKLDSPESTKLEELALIKEEEKGYQDT RELPKNSEKEKESILMIKEEKVKFDHQRGSSKIIPLSKVRDIARAK KKPEPLVTKQPQISTPKVSKRVPTSSSLSASQSSTKKMNGSLLP 96 RSKNPPAGENKKVTSKSLHLSLTMDPSNSEPDPLITTRKSFIREK MGDKDIVKRAFKTFQNNFSQLKSSAEERAIREKQVPAKGTDVKV STSMTPRTENIVSLKSSRVDRKTAKLAPSSFVLKSDERAKRRKEL SMKMEEKSNAKPAESTHLRTKSKEEKEEEIIKQRHSSNFKPTPM PGFYRAQKASKSPLDKVCPVPESCHTLMK 6. 7. 8. 9. 10. 11. Pt564607 eugene3.00081202 Pt564928 eugene3.00081523 MCFKISFYTQNSESGNSRIHPVNKRYSPFLANCKPASSMVSDFK LSIFLCSEKEPRVKKFESPSSRSKRVEPIAHLSTNRTKQNASSINP DTRPSASTFSFKSDERAERRKEFYMKLEEKWHAKEAEMNQIQA KTQEKTEAEIKQFRKSLNFKATPMPSFYHVAVPPASNGNKVQTV Q* MGIEVTDICMDKESDSVIVYSNGVSHDQTHETVPHDHGVLESYE PINGVPELHSSEESTEAKEYEVKECTTEVSVEVTELSHAEKSKE GQHVVCSNFEDGLKVKKVKAVNRKSKDIGQQKSSIKRVSKPASA AIARTKHTVPQPFALATEKRASSGIRPSGPEPDITNGVNKSFKAN NVLRQNPMKQNQPLSVSRKPLQPNNKKHPDEEDNCSVTSSTTA SARPIMSKATAVAAPVFRCTERAEKRKEFYSKLEEKYQALEAEK TQSEARTKEEKEAAIKQLRKSLTFKANPMPSFYHEGPPPKVELK KLPPTRAKSPKLGRRKSCNNRVNSSQPDKVKRDFNDEKNQSQ DSSREDTSNPVSQHSVLKGHAISKFEDETQQAEEINE* Pt566133 eugene3.00100691 MGIEVTDVCMDKEPNCVIVYSNGVSHDPTHETVPDDHGVLESY EPINGVPELHSSEESTEAKEYVVKECTTEVSVEVTELSHAEKSKE DQTVVCSNFEDGLKVEKVKALNRKSKDIGQKKSSTKHASKPAPA GLARTKHTVPQPFALATEKRASLGMRPSGEPDITNGLNKSFKAN NALRPNPIKQNQPLSVSRKPLQPNNKKHPDEEDNCSVTSSYPVS SILTSARPAKSKPAAVAAPVFRCNERAEKRKEFYSKLEEKHLALE AEKTQSEARTKEEKEAAIKQLRKSLMFKASPMPSFYHEGPPPKV ELKKLPPTRAKSPKLGRRKSCSNGVNSSQPDRVKGACGDGNN QSQGIFREDTSNPVSQHSIPKGHVICKFEDETREMEGIDELIPLE VSGQSFAGIGLQS* Pt578210 eugene3.00180434 MDSDYHLFPDDGLETVHQNGVHEQSAAAREDGVVSNNLSGSM GNTFEVDDCTNDNLSTREVEGELKEGEAKVKDADNSEKARSQK GSGKGGNAKPSNPKNVSATQVKGKDGRDAVARTAVSNGSVAV NSQLKQSLKSNSFNERQGQASKQSGKSDAVLSAGLVEKAKPLK KGPVVKAEGETESTSSPTAEDAKSRKFGTLPNYGFSFKCDERA EKRKEFYTKLEEKIHAKEVEKSTLQAKSKETQEAEIKLFRKSLAFK ATPMPSFYQEPAPLKVELKKIPTTRAKSPKLGRKKSPSPADSEG NNSQSNRSGRLSLDEKISSKIPIRGLSPAHPKKPQRKSLPKLPSE KINLYANDEKGKLPKASNEENTTLSDQTNEGVSANQEQEAVSKN EASEFLPPKEEVVVQEEAATLMKGPIALAV* estExt_fgenesh4_pg.C_440200 Pt594654 eugene3.00640242 MEKAHTKSALKKLVKASSQSAPWSNAARGMAKDDLKDPLYDKS KVAPKPFAKENTKPQEFKLHTGQRALKRAMFNYSVATKIYMNEQ QKRQIERIQKIIEEEEVRTMRKEMVPRAQLMPYFDRPFFPQRSS RPLTVPREPSFHMVNSKCWSCIPEDELYYYFEHAHPHDHAWKP VK* MAAESEDSSTATTMTNATTLMMFVDEAYEFSAPKFYDFVKGES DEDSRNAELWFDVTASYAPSPFMPRIKTGRSFKVETLCDFSQAD QFHKVAESSDSKASDSSSQSEVMPPPAEAAAPIGTGKEEKTSD EDNKENNANLVNVISAGEVTCEKEKKVGFACAEGNERSTSSLQT ENADGKESSKNEAYCTPKPPMSSRNRGPLTDSKKNHSARHIAS LVRNPSLLKPKSQSQSSQVKGIKPASVKKDRNVKNVAGTTNLAQ ENQAIKKQKLEGGRSRQILNAKPPQPLTHKSKLGLSSGSSNLCS SVANKMKKEERKVYVREQAAPGPFVSTAEMMNKFQSNTRGLS MPRFNNSISHDGPASVIQRKPKLTLTRPKEPEFETAQRVRSVKIK SSAEIEEEMMAKIPKFKARPLNKKILEAATLPALPRSTPQPPEFLE FHLETAARANQNAESTSVASTEVSHQSNLWKPHHLTEPKTPVLH TSLRARPARVKSSLELEKEEIEKFPKFKARPLNKKIFESKGAMGIF CHAKKQVTVPQEFHFATNERIPPQAAVADMFDKLSLRSEPILDN PIPRNTKPNPFHLHTEERGAEKERKFWMELVQKQMEEERARVP RANPYPYTTDYPVVPPRPEPKPCTKPEPFQLESLVRHEEEMQR EMEERERKEKEEAQMRIFRAQPVLKEDPIPVPEKARKPLTQVQQ FNLHADQRAVERAEFDHKVKEKEMLYKRYREESETAKMMEEEK 97 ALKQLRRTMVPHARPVPNFNHPFCPQKSSKEATKAKSPNLRVL QRRERRKMMMVNAASSAAASGMR* Pt595399 eugene3.00700040 MGRELQRADMEKKPNGLAAKFHGVSHDKVHISPKLSKAVIEAKE YVEKETAEKSEKQDVLGVKSTNFDADPSDGKDEKPGAQKLSDD KNSSSPSQKIGVNGNKHAHRAHQTVPQQFALATDKHVGGNSST NSNKTQSPVSMKNSQQNSPSTARKPLHPDNKKHHDEEDSWSV TSSTSASVRTVKSVTVGTAPTFRSSERAAKRKEYYSKLEEKHRA LEKERSQAEARTKEEQEAAIKQLRKSMLYKANPVPSFYHEPPPP QVELKKLPLTRPQSPKLNRRKSCSDAVRTSQEEVGKHCARHRH SIGSHKDSTGANTAKAKVQISSQTANGIRKVKDRSKQDHVATKA DPEKIAGPTNADISVQS* 13. Pt653406 grail3.0030003201 SRANRLAPTGANSKESNINGSKTLTKQTSSTSKSSSQQAASVKS SSLTEAAKCPPPQVSESAADQNSKPETTTFSSKEEDDTHSTTSS ATLSGRRSSGSGFSFRLEERAEKRKEFFSKLEEKIHAKEIEQTNL QAKSKESQEAEIKKLRKSLTFKAAPMPCFYKEPPPKVELKKIPTT RAKSPKLGRRKSSTTSMNNSLEDVGSSFSPRASHSPHLNQESS NPTKGAQRNGNVDNGASKTPIRKSQPKHQSRQITANGMEGKTV KSKAKLPGAESQTQKANVEKVEVNENNSMKVPVCENGIETMPE NNTPQNNGPVLSSSNPEIMLPHVTVGG* 14. Pt415809 gw1.VI.182.1 KVKDADNSENAKSQKGPGKRGTAKPSHLKNASATQVKKGKDG RDAEVQLTVSNGSVAVNSQLKQHLKSKSFNERQGQASKQSGTS DAGPPEGIVEKMKLKPLKKGPVDKAEADTDSTSSPTVEDAKPRK VGALPNYGFSFKCDERAEKRKEFYSKLEEKIHAKEVEKTTLQAK SKETHEAEIKMLRKSLGFKATPMPSFYQEPTPPKVELKKIPTTRA KSPKLGRRKSSSPADTEGNNSQSYRPGRLSLDEKVSSNIPIKGL SPAHPKKPQRKSLPKLPSEKTKL* 15. Pt658207 grail3.0036016201 MPEEKMPNKVNNHPNQEAAEMENVALPNNKRQMSSLSNSLSQ SRASKLPKSSAKLSSSTRLNATPNSKKSAGELVGEKRATSKSIHV SINFASRLQDTNKSYVRVSKDRSATPENPTRGSVHGVSKLLPLIF RHSQDRRSKSELNKSVSGKITPGEISQTLSSDCSKSSSAKGSKS RPPLISSPFSFRSEERVAKRKEFFQKLGEKNNAKEDTEKKHLHA RPKEKAEHDLKKLRQSAVFRGKPSDDLHRGLHSPYNSMKKIPLT RPQSPKLGRKSTPNAVREASLQLHQRPSVNAETSKPFIQKSNHS STCPVNLLPKKKALENASPNILW* 16. Pt658783 grail3.0006023901 MRSPINGSQFQKILNNVSKTTAKTQNRGEGETPQRAKSEKQSS RATTPTRRTLHRAKNEENSESGNLRLHPVNRSERASRVNKFES PPSRSKKVEPMSHLRANRNKQIVNSIKPDTMPCAAAFSFKSDER AERRKEFYMKLEEKLHAKEAEMNQIQAKTQEQKKAEIKKFRERL NFKAAPMPSFYRVAVSPGSDGNK 12. 17. 18. Pt667581 grail3.0004025702 Pt668651 grail3.0059011801 RNRCSFPHISVTSSPRLNQANRRVPTGVNSKESNINCSKTLTRQ SSSAGKSCSQQATSVKSSSLNEAAKGHPPQASESAAHQNSKPE TTTLSSKEDDDTHSTTSSATPSGRRSSGSGFSFRLEERAEKRKE FFSKIEEKIHAKEIEQTNLQEKSKENQEAEIKQLRKSLTFKATPMP SFYKEPPPKAELKKIPTTRAISPKLGRRKSSTTLTNNSLEDSGSS FSPRASHSPRLNQESSNPTKGIQRNGNKDNGASKTPIRKSQPKL QSHQIMANGLEGKTVKSKAKPPGAENQTQKAGVGKVEENENNS KKIPLCDNGIQTMPENNTPQNDGLVLSSSNPEFMLPQVTVGG* MGLRRLKKKKQLKVTSQKPFKLRTEQRGRQKEEEFTKKIQEIMM EEERLRIPVAQGLPWTTDEPECLIKPPVKENTKPVDLKLHSDIRA VERADFDHQVSEKMSLIEQYKMERERQRKLAEEEEIRRLRKELV PKAQPMPYFDRPFIPRRSIKHPTVPREPRFHMPQHKKIKCCLSW SNV 98 19. Pt691617 estExt_fgenesh1_pg_v1.C_LG_I2234 MGDTTCVMQPFSYAAGISNDAKEGNPIHALGQSISFGRFMSDSL SWEKWSSFSHNRYVEEAEKFSRPGSVAQKKAFFEAHYRNLAAR KAAALLEQANAEANNVQEPENEGGIHDKTTQDSLTVATNSQEA GDREEVHVQQVNCEASFVADDNTRTSNVDMERFESSNVEEVE PSAENEILVENCVKNETLNQIVKVDNKEEVKEMELSVSKQMEKP LLKDFMSCKDDAASMSKKKPAVSSSKSSIYDKASKLPSTPAKPA PSVRAKKENTATPISKKSALESVERRKPTPKSTHKSMNFTPARE FNRITSSIIRKIDNSRVGSHSKSSKDCPTPSRTPMMMVSIAESKH PLATPQSEKRRAKTPLHPSTSGSKTVRSKWHFLPKDCSMFMTS SRNRSQSPSASIPFSFRTEERAARRKEKLEEKFNAYQAQKVQLQ VTLKEKAETELKRLRQSLCFKARPLPDFYKQRVAPNNQMEKVPL THSESPEPGRKMTPSKIRSASQLPQWSSLKNSGSKDAMQKKSD NPRSLASRLKASPHENTSPNIQHE* 99 Appendix Table 3: Microtubule associated and binding proteins reported in Pfam. Accession ID Description PF00225 PF02991 PF00414 PF00418 PF03271 PF03607 PF03999 PF05672 PF06886 PF08154 PF00022 PF00334 PF00784 PF00956 PF00994 PF01031 PF01221 PF03028 PF03311 PF03378 PF04402 PF05804 PF05937 PF06098 PF06705 PF07202 PF07544 PF07781 PF00307 PF00091 PF03953 PF00004 Kinesin MAP1_LC3 MAP1B_neuraxin Tubulin-binding EB1 DCX MAP65_ASE1 MAP7 TPX2 NLE Actin NDK MyTH4 NAP MoCF_biosynth Dynamin_M Dynein_light Dynein_heavy Cornichon CAS_CSE1 DUF541 KAP EB1_binding Radial_spoke_3 SF-assemblin Tcp10_C CSE2 Reovirus_Mu2 CH Tubulin Tubulin_C AAA Kinesin motor domain Microtubule associated protein 1A/1B, light chain 3 Neuraxin and MAP1B repeat Tau and MAP protein, tubulin-binding repeat EB1-like C-terminal motif Doublecortin Microtubule associated protein (MAP65/ASE1 family) MAP7 (E-MAP-115) family Targeting protein for Xklp2 (TPX2) NLE (NUC135) domain Actin Nucleoside diphosphate kinase MyTH4 domain Nucleosome assembly protein (NAP) Probable molybdopterin binding domain Dynamin central region Dynein light chain type 1 Dynein heavy chain Cornichon protein CAS/CSE protein, C-terminus Protein of unknown function (DUF541) Kinesin-associated protein (KAP) EB-1 Binding Domain Radial spoke protein 3 SF-assemblin/beta giardin T-complex protein 10 C-terminus RNA polymerase II transcription mediator Reovirus minor core protein Mu-2 Calponin homology (CH) domain Tubulin/FtsZ family, GTPase domain Tubulin/FtsZ family, C-terminal domain ATPase family associated with various cellular activities (AAA) PF00018 PF00036 PF00041 PF00069 PF00400 PF00433 PF00435 PF00566 PF00595 PF00612 PF00622 SH3_1 efhand fn3 Pkinase WD40 Pkinase_C Spectrin TBC PDZ IQ SPRY SH3 domain EF hand Fibronectin type III domain Protein kinase domain WD domain, G-beta repeat Protein kinase C terminal domain Spectrin repeat TBC domain PDZ domain (Also known as DHR or GLGF) IQ calmodulin-binding motif SPRY domain 100 PF00627 PF00642 PF00692 PF00838 PF01302 PF01472 PF01509 PF01669 PF02149 PF02187 PF02971 PF02985 PF03451 PF04961 PF05091 UBA zf-CCCH dUTPase TCTP CAP_GLY PUA TruB_N Myelin_MBP KA1 GAS2 FTCD HEAT HELP FTCD_C eIF-3_zeta UBA/TS-N domain Zinc finger C-x8-C-x5-C-x3-H type (and similar) dUTPase Translationally controlled tumour protein CAP-Gly domain PUA domain TruB family pseudouridylate synthase (N terminal domain) Myelin basic protein Kinase associated domain 1 Growth-Arrest-Specific Protein 2 Domain Formiminotransferase domain HEAT repeat HELP motif Formiminotransferase-cyclodeaminase Eukaryotic translation initiation factor 3 subunit 7 (eIF-3) PF05217 PF05622 PF06740 PF07058 PF07145 PF07837 PF08068 PF08239 PF08377 PF08926 PF08953 PF08954 PF09041 PF09336 STOP HOOK DUF1213 Myosin_HC-like PAM2 FTCD_N DKCLD SH3_3 MAP2_projctn DUF1908 DUF1899 DUF1900 Aurora-A_bind Vps4_C STOP protein HOOK protein Protein of unknown function (DUF1213) Myosin II heavy chain-like Ataxin-2 C-terminal region Formiminotransferase domain, N-terminal subdomain DKCLD (NUC011) domain Bacterial SH3 domain MAP2/Tau projection domain Domain of unknown function (DUF1908) Domain of unknown function (DUF1899) Domain of unknown function (DUF1900) Aurora-A binding Vps4 C terminal oligomerization domain 101 Appendix Table 4: ESTs corresponding to PttMAP20 identified in different Populus species. Species / hybrid Populus tremula x tremuloides EST ID /s A001P11U, A010P57U, Tissue type Cambial region A016P72.5pR, A016P72.3pR, A068P39U and A089P68U Populus trichocarpa G122P48Y Developing tension wood M129E12 Female catkins xylem.est.98 Developing xylem WS0196.B21_I23, Xylem WS0161.B21_L22, WS0199.B21_B20, WS0165.B21_L15, PX0019.B21.1_J10 Populus alba x tremula WS0116.B21_M24 Cambium and phloem WS01227.B21.1_D04 Leaves and green stems PtaJXT0021G4G0414, Developing tension wood PtaJXT0028H5H0515 and PtaJXT0021H2H0216 PtaJXO0010F12C0105 Young opposite side xylem Populus trichocarpa x deltoides pni266-2 Shoot tips, leaves and wood Populus trichocarpa x nigra WS02028.B21_G07 Insect fed bark 102 Appendix Table 5: ESTs homologous to PttMAP20 in different plants as identified using tblastn. Species / hybrid EST ID / s Tissue type Aquilegia formosa x Aquilegia DR915799, DT750924, Mixed shoot and floral apical meristem, pubescens DT747806, DR951414 flower buds, leaves and roots Brassica napus CD830242, CN734834 Seeds Eragrostis tef DN482641 Young leaves Glycine max BQ612497, BM522504 Roots BI972132 Etiolated hypocotyls Hordeum vulgare CX630106 Roots Malus x domestica CV083029 Shoot internodes Medicago truncatula DW019348 Flowers, early seeds, late seeds and stems Picea engelmannii x Picea CO211859 Bark (with phloem and cambium attached) sitchensis DR550456 Cones Picea glauca DR563898 Phloem Pinus taeda BQ198662 Primary xylem DR742411 Roots plus added copper DR051360 Roots plus added boron Vitis pseudoreticulata DT661589 Infected young leaves Vitis vinifera CF213900 Stems Triticum aestivum BJ280729 Roots BJ257649 Spike at heading date BM074169 Seedling and silk Zea mays 103 Appendix Figure 1: Sequence alignment of MAP20 proteins made with the MUSCLE alignment tool. The TPX2 domain is marked with a black bar and the extended TPX2 domain is indicated using dots. Notes above the TPX2 domain indicate where residues are lost relative to the Pfam domain model. Colors indicate similarity to consensus sequence. 104 Arabidopsis TPX2 genes expression levels 3000 Log (2) scale 2500 2000 1500 1000 500 0 Callus Cell Suspension Seedling Inflorescence Rosette Roots Tissue types Appendix Figure 2: Expression profile of TPX2 genes found in Arabidopsis. 105 AT3g01015 At5g15510 At5g44270.1 At1g03780.2 At2g35880 At4g32330.1 At3g26050 Atg70950.1 At3g23090.1 At1g54460 At3g04630.1 At1g54460.1 106

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Download Discovery and Characterization of a Novel Microtubule