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  • University of Alberta

    -Galactosidases and Fasciclin-like Arabinogalactan Proteins in Flax (Linum usitatissimum)

    Phloem Fibre Development

    by

    Neil Robert Hobson

    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

    Plant Biology

    Department of Biological Sciences

    Neil Robert Hobson

    Fall 2013

    Edmonton, Alberta

    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of

    this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only.

    Where the thesis is converted to, or otherwise made available in digital form, the University of

    Alberta will advise potential users of the thesis of these terms.

    The author reserves all other publication and other rights in association with the copyright in the

    thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof

    may be printed or otherwise reproduced in any material form whatsoever without the author's prior

    written permission.

  • Abstract

    The phloem fibres of flax (Linum usitatissimum) have specialized, cellulose-rich

    secondary walls of the gelatinous type (G-type), which give the fibres remarkable strength. G-

    type walls are also found in tension wood of many trees. -galactosidases (BGAL) and fasciclin-

    like arabinogalactan (FLA) genes are expressed during development of G-type walls in flax, but

    the functions of these genes and their possible interactions are not well understood. The recent

    assembly of the flax genome, to which I contributed, afforded an opportunity to characterize the

    BGAL and FLA gene families of flax. Each of these families comprises 43 predicted genes.

    Comparison of the BGAL family structure between species revealed the expansion and contraction

    of distinct sub-families, perhaps correlated with specialization of cell wall composition of flax

    tissues during its evolution and domestication. Phylogenetic analyses of the FLA gene family

    revealed a sub-family and an amino acid motif unique to the Rosids that is represented by many

    FLA genes known to be expressed in G-type walls. Transcript expression profiling of the BGAL

    and FLA families in 12 different flax tissues identified multiple genes from each family that were

    highly expressed in developing fibres. Transgenic analyses of selected, fibre-enriched genes

    (LuBGAL1 and LuFLA1) were conducted, first by generating promoter-reporter gene fusions.

    Within stems, the upstream regions of each of these genes directed reporter gene expression

    preferentially to developing phloem fibres. Because the RNAi loss-of-funtion phenotype of

    LuBGAL1 had been previously shown to reduce fibre strength and crystalinity, I extended this

    functional analysis by producing and characterizing lines overexpressing LuBGAL1 transcripts. I

    was unable to obtain evidence that LuBGAL1 overexpression affected the composition or

    mechanical properties of phloem fibres, suggesting that LuBGAL1 may be necessary but not

    sufficient for G-type wall development. I also characterized transgenic lines bearing an RNAi

    construct targeted towards LuFLA35 (which is closely related to LuFLA1 and whose transcripts are

    likewise highly enriched in fibres). LuFLA35-RNAi lines were slightly diminished in tensile

    strength in flax stems, providing the first evidence that FLAs are functionally required for G-type

    fibre development in flax.

  • Acknowledgement

    Thanks to my family, for all their support. A special thanks goes to Mike, for putting up with my

    shenanigans, and letting me participate in more studies and publications than I probably warranted.

    To dear departed Mary, who's moved on to a better place. Or, at least a new job. She was an

    invaluable help while working as our lab technician, and taught me everything I know about flax

    tissue culture. Hopefully, cattle research is treating her well. To Melissa, whose research projects

    I acquired upon her graduation, and with whom we've continued to collaborate. To David, for

    sharing the burden of experimenting with Fluidigm. And by sharing, I mean doing most of the

    work while I ran off to China for a summer. Thanks to all my labmates, past and present. Id also

    like to thank Dr. Kaushik Ghose and Dr. Bourlaye Fofana from the Crops and Livestock Research

    Centre in Charlottetown, PE, Canada, for generously aiding in the heterologous expression of

    LuBGAL1, and validating our results. Lastly, to the double negative, the sentence fragment, and

    the comma splice. To contractions, contradictions, and colloquialisms. I placed you where I

    could, and where I couldn't, you were missed.

  • Table of Contents

    Chapter 1 Literature Review ......................................................................................................... 1

    Flax .............................................................................................................................................. 1

    Flax Phloem Fibres ...................................................................................................................... 4

    Cell Walls..................................................................................................................................... 7

    Cellulose .................................................................................................................................. 7

    Pectin and Hemicellulose ........................................................................................................ 8

    Lignin .................................................................................................................................... 10

    Cell Wall Proteins .................................................................................................................. 11

    -Galactosidases ........................................................................................................................ 11

    Fasciclin-like Arabinogalactan Proteins ..................................................................................... 13

    Current Study ............................................................................................................................. 14

    References .................................................................................................................................. 16

    Chapter 2 - LuFLA1PRO and LuBGAL1PRO promote gene expression in the phloem fibres of flax

    (Linum usitatissimum) .................................................................................................................... 23

    Introduction ................................................................................................................................ 23

    Materials and Methods ............................................................................................................... 25

    Fosmid Library Construction and Screening ......................................................................... 25

    Plasmid Construction ............................................................................................................. 26

    Tissue Culture ........................................................................................................................ 26

    GUS Histochemistry .............................................................................................................. 27

    Bioinformatics ....................................................................................................................... 28

    MUG Assay ........................................................................................................................... 28

    Results ........................................................................................................................................ 29

    LuFLA1 .................................................................................................................................. 29

    LuBGAL1 ............................................................................................................................... 31

    MUG assay ............................................................................................................................ 33

  • Discussion .................................................................................................................................. 34

    Conclusion ................................................................................................................................. 37

    Figures and Tables ..................................................................................................................... 39

    References .................................................................................................................................. 45

    Chapter 3 - Whereupon we examine the glycosyl hydrolase 35 family of flax .............................. 51

    Introduction ................................................................................................................................ 51

    Materials and Methods ............................................................................................................... 52

    Gene Discovery ................