AN EXAMINATION OF LEAF MORPHOGENESIS IN THE MOSS, PHYSCOMITRELLA PATENS A Thesis Submitted to the Faculty of Graduate Studies and Research In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In Biology University of Regina By Elizabeth Io Barker Regina, Saskatchewan August, 2011 Copyright 2011: E.I. Barker UNIVERSITY OF REGINA FACULTY OF GRADUATE STUDIES AND RESEARCH SUPERVISORY AND EXAMINING COMMITTEE Elizabeth Barker, candidate for the degree of Doctor of Philosophy in Biology, has presented a thesis titled, An Examination of Leaf Morphogenesis In The Moss, Physcomitrella Patens, in an oral examination held on August 30, 2011. The following committee members have found the thesis acceptable in form and content, and that the candidate demonstrated satisfactory knowledge of the subject material. External Examiner: Dr. David Cove, University of Leeds Supervisor: Dr. Neil Ashton, Department of Biology Committee Member: Dr. Harold Weger, Department of Biology Committee Member: Dr. William Chapco, Department of Biology Committee Member: *Dr. Janis Dale, Department of Geology Chair of Defense: Dr. Philip Charrier. Department of History *Not present at defense ABSTRACT Physcomitrella patens is a simple model plant belonging to the bryophytes, which diverged from the tracheophytes approximately 500 million years ago. The leaves of the moss are similar in form to vascular plant leaves although leaves evolved independently in the bryophyte and tracheophyte lineages. Close examination of the morphology of Physcomitrella leaves and investigation of the morphogenetic processes that result in the leaf form and of the hormonal and genetic regulation of those processes will elucidate the evolutionary trajectory of moss leaves. Photomicroscopy and measurement of moss leaves were performed to provide detailed descriptions of leaves in strains of Physcomitrella that exhibited normal and aberrant morphology. Low concentrations of two major phytohormones, auxins and cytokinins, were applied to growing moss cultures to investigate their effects on leaf morphogenesis. A bioinformatic analysis of homologues of vascular plant genes that are involved in leaf development was performed to identify candidate genes that may have been co-opted for regulation of leaf morphogenesis during the early course of plant evolution. The transition from the basal leaf form to the adult form is gradual for all of the heteroblastic features of Physcomitrella leaves with the possible exception of the midrib. Low concentrations of exogenous auxin and cytokinin stimulate cell expansion and cell divisions respectively and auxin may be a key regulator of leaf heteroblasty. Homologues of almost all tracheophyte genes that are known to be involved in leaf morphogenesis are present in the Physcomitrella genome. However, few of the i Physcomitrella homologues exhibit both a high degree of sequence similarity to the vascular plant genes and high levels of expression in leaves. Of these, class I and class II homeodomain-leucine zipper genes are predicted to play key roles in leaf development in the moss. Other genes may have been co-opted to regulate the processes of cell division, cell expansion and adaxialization/abaxialization of the midrib in bryophyte leaves. Preliminary models of auxin-cytokinin activity and of genetic and hormonal regulation of leaf morphogenetic processes in Physcomitrella provide testable hypotheses for further investigation. ii ACKNOWLEDGEMENTS I would like to thank Dr. N. W. Ashton for introducing me to the fascinating plant, Physcomitrella patens, and for guiding this project. I would also like to thank the members of my committee for their interest, encouragement and support. Generous funding has been provided by the Natural Sciences and Engineering Research Council (CGS-M, PGS-D) and by the Faculty of Graduate Studies and Research in the form of enhancement awards and student teaching assistantships. iii DEDICATION This thesis is dedicated to my mother, my inspiration. iv TABLE OF CONTENTS ABSTRACT ........................................................................................................................ i ACKNOWLEDGEMENTS ............................................................................................ iii DEDICATION.................................................................................................................. iv LIST OF TABLES ............................................................................................................ x LIST OF FIGURES ......................................................................................................... xi LIST OF APPENDICES ............................................................................................... xiv LIST OF ABBREVIATIONS ........................................................................................ xv 1 INTRODUCTION.......................................................................................................... 1 1.1 Land plant leaves ................................................................................................................... 1 1.2 Leaf morphology .................................................................................................................... 1 1.2.1 Common and diverse features of leaf morphology ......................................................... 1 1.2.2 Leaf shape and size ......................................................................................................... 2 1.2.3 Simple and compound leaves .......................................................................................... 3 1.2.4 Leaf vasculature .............................................................................................................. 3 1.2.5 Stomata ........................................................................................................................... 5 1.2.6 Trichomes ....................................................................................................................... 5 1.2.7 Cuticle ............................................................................................................................. 6 1.2.8 Phyllotaxy ....................................................................................................................... 6 1.2.9 Leaf morphology in major land plant taxa ...................................................................... 7 1.2.10 Leaf nomenclature ........................................................................................................ 9 1.3 Plant development .................................................................................................................. 9 1.3.1 Apical growth ................................................................................................................ 10 1.3.3 Determinacy in leaves ................................................................................................... 12 1.3.4 Patterning in leaf development ..................................................................................... 13 1.3.5 Development of leaf vasculature ................................................................................... 14 1.3.6 Growth of leaves ........................................................................................................... 14 1.3.7 Leaf heteroblasty ........................................................................................................... 15 1.4 Hormones involved in leaf development ............................................................................. 18 1.4.1 The roles of auxins in plant development ..................................................................... 18 1.4.2. The role of auxin in acid growth .................................................................................. 19 v 1.4.3 The role of auxin in vascular patterning ....................................................................... 19 1.4.4 Cytokinins ..................................................................................................................... 21 1.4.5 Auxin-cytokinin interactions......................................................................................... 21 1.4.6 Gibberellins. abscisic acid, jasmonates, ethylene and brassinosteroids ........................ 21 1.5 Genes involved in leaf development .................................................................................... 22 1.5.1 Gene nomenclature ....................................................................................................... 22 1.5.2 Maintenance of the SAM and differentiation of leaf primordia.................................... 23 1.5.3 WUS and CLV ............................................................................................................... 23 1.5.4 KNOX and ARP ............................................................................................................. 24 1.5.5 Specification of adaxial and abaxial domains in leaves ................................................ 25 1.5.6 Regulation of development of leaf vasculature ............................................................. 26 1.5.7 Regulation of leaf size .................................................................................................. 27 1.5.8 Regulation of development of the leaf cuticle, stomata and trichomes ........................ 28 1.6 Plant evolution ....................................................................................................................