Plant Cell, Development & Ultrastructure

Transcription

1 PCDU Lecture Plant Cell, Development & Ultrastructure Plant Cell Biology Labs Download at:

2 Cell Wall Structural Polysaccharides, Matrix Polysaccharides and up to 1000 Proteins are Required for Making Plant Cell Wall

3 Cell Wall Secondary Cell Wall in Sklerenchymatic Fiber Cells Middle Lamella Stained with Ruthenium Red Primary Cell Wall, Secondary Cell Wall and Middle Lamella are Structurally and Chemically Different

4 Cell Wall Cellulose Synthesis in Plant Protoplasts Fluorescence: Chlorophyll + Calcofluor White Sato et al min 12 hrs

5 Glycosyl Transferases Transfer of Carbohydrate Moieties from Donor Molecules to Acceptor Molecules IRX3 IRX5 IRX1 10 CesA Genes in Arabidopsis 18 CesA Genes in Populus nigra CesA Cellulose Synthase Xylem-mutants IRX= irregular xylem

6 Cellulose Synthase Sucrose Synthase Hydrogen bonds in and between cellulose polymers Cellulose Synthase (Rosette Terminal Complexes) 1,4-beta-Glucan Glucosyltransferase Sucrose Synthase Provides UDP-Glucose Uridine triphosphate

7 Cellulose Synthase (Rosette) outside Topology of the CS-monomer 36 Glucane Chains Produced per Rosette N-terminal dimerization domain (RING-finger, 2 Zn-bound) Cys3-His-Cys4 motif, modulated by redox-processes N inside DDD QVLRW C Goubet et al RING finger domain Saxena & Brown 2005

8 Cellulose Synthase (Rosette) 36 Glucane Chains Produced per Rosette Doblin et al. 2002

9 Cellulose Synthase (Rosette) - EM E-surface E-surface 25 nm P-surface

10 Cellulose Synthase (Rosette) Redox Regulation Kor Korrigan Mt Metallothionein MT Microtubules LTP Lipid Transfer Protein PM SuSy Plasma Membrane Bound Sucrose Synthase Doblin et al. 2002

11 Cell Wall Cellulose Synthesis Bashline et al. Ann. Bot. (2014)

12 Glycosyl Transferases Kor = Korrigan beta-1,4-glucanase Kob = Kobra GPI-anchored Scheible & Pauly 2004

13 Korrigan Function Sitosterol Cellodextrin beta-glucoside is Required for Chain Initiation. Korrigan, a β-1,4-glucanase, transfers the cellodextrin chain from the sitosterol glycoside to the CES subunits, which uses it as primer for the addition of further β-d-glucose molecules. Korrigan = celtic, meaning dwarf

14 Formation of Cell Wall Matrix Golgi ER AGPs = Arabinogalaktan proteins HRGPs = Hydroxyprolin-rich Glycoproteins PRPs = Prolin-rich-Proteins GRPs = Glycin-rich Proteins Buchanan et al. 2000

15 Matrix Polysaccharides Pectins = Heterogeneous Polygalacturonates ionic cross bridges 2-keto-3-deoxyoctonate

16 Matrix Polysaccharides Hemicelluloses (Xyloglucanes/ Arabinogalactanes)

17 Matrix Polysaccharides WS13/14 Ara Arabinoxyloglucane in the Solanaceae Ara Xyl Xyl Ara Xyl Xyl

18 Plastic Expansion of Cell Wall Expansin genes 38 in Arabidopis 80 in Rice XET = Xyloglucane endo/trans-glycosylase

19 Multinet Growth of Cell Wall C Stretching of the wall D Burk & Ye 2002 c: transversal orientation in recently deposited layer d: as the wall is stretched in longitudinal direction, cellulose fibrils get passively reoriented c: final state of fibril orientation in fully elongated cell Saxena & Brown 2005

20 Primary Cell Wall Cellulose Hemicellulose Pectin (Extensin) Buchanan et al Cell Wall-Preparation - Freeze-Etch Method

21 Cell Wall Proteins WAK = Wall-associated Kinases involved in development pathogen infection wounding GRP= Glycine-rich proteins PRP= Prolin-rich proteins HRGP= Hydroxyprolin-rich glycoproteins AGPs = Arabinogalactan Proteins & other Proteoglycanes COBRA GPI-anchored AGP Kohorn 2000

22 Arabinogalactan Proteins (AGPs) Majewska-Sawka & Nothnagel 2000 Glycoproteins: 90% protein/10% sugar residues Proteoglycanes: 10% Protein/ 90% sugar residues

23 Arabinogalactan Proteins (AGPs) Arabinogalactan Proteins Serve as Developmental Markers Coimbra et al Young pollen in the Arabidopsis archespore. Labelling of a specific subfraction of AGPs on the cell surface of the generative cell in the pollen with monoclonal antibody JIM8.

24 Fasciclin-Like AGPs (FLAs) fascilin-like domain Hyp-rich domain ER-signal peptide unique sequence GPI-anchor domain/ CAAX-box Fascilin is an adhesion molecule originally found in the connective tissue of bundled muscle fibres (fascicles) Connection to the membrane may be terminated by enzymatic cleavage of the GPI-anchor by phospholipase C/D About 30% do not possess a GPIanchor. 21 FLA genes in Arabidopsis Johnson et al. 2003

25 GPI-Anchored AGPs 22Hyp for O-linked sugars 86 AA 3Hyp for O-linked sugars 10 AA 9Hyp for O-linked sugars 10 AA 23 AA GPI A-O-A-O-T-A-T-O-O-O-A-T-O-O-O-V A-O-A-O-S-O-T-T-T-V-T-P-O-O-V A-O-G-O-A-O-T-R-S-O-L-P-S-O-A A-O-A-O-T-O-T-A-T-O A-O-A-O-S-O-T-T-T-V-T-P A-T-S-O-O-T-A-A-O-A-O Typical AGP backbone sequences O = hydroxyprolin

26 Fasciclin-Like AGPs (FLAs) Fasciclin-Like AGPs in Secretory Tissues Transmission trackt, cross section TTS-protein monomer signal peptide Hydroxy-prolin rich domain Cystein-rich domain TTS-protein oligomer Wu et al. 2001

27 AGPs - Yariv-Reagent Azo-bond Yariv-Reagent is used: as inhibitor of AGP function in live tissues as cytochemical stain in situ as chemical agent to precipitate AGPs out of solution

28 Effects of Yariv Reagent on Cell Growth Root active Yariv reagent Hypocotyl Phytohormones enhance the Yariv-effect Parke et al. 2003

29 Effects of Yariv Reagent on Roots Treatment with Yariv Reagent for 24h 1µM inactive Yariv 1µM active Yariv 5µM active Yariv

30 Effects of Yariv Reagent on Roots Cortical Microtubule Become Disordered by Yariv Reagent control Yariv

31 Yariv Reagent-Induced AGP Clusters Yariv Reagent-Induced Clusters of AGPs in Unfixed Plant Cells

32 Arabinogalactan Proteins Cytoskeleton-Dependent Relocation of AGPs A B C Sadar et al Heterologous expression of GFP-LeAGP-1 in tobacco BY2-cells (A) control (B) plasmolyzed (C) plasmolyzed after pretreatment with 30 µm APM Le = Lycopersicon esculentum

33 Lipid Raft-Associated AGPs AGP Function is Dependent on Cytoskeletal Integrity Sadar et al Le = Lycopersicon esculentum

34 Arabinogalactan Proteins Models for AGP Unvolvement in Cellular Signaling and Cell Adhesion A B C A) signaling by the release of a carbohydrate epitope (small squares) from an AGP, which binds to a plasma membrane receptor capable of initiating an intercellular signaling cascade B) signaling by an AGP directly or indirectly after binding ligand molecules (diamonds) for presentation to an appropriate plasma membrane receptor C) intercellular signaling by an AGP from one cell interacting with a plasma membrane receptor on an adjacent cell Showalter 2001

35 Extensins WS13/14 Hydroxyproline-Rich Glycoproteins (HRGPs) Extensin

36 Extensins Inhibit Cell Growth HO Hydroxy-Prolin (B) Tomato extensin (Extensive glycosylation) Isodityrosine (C) By the appearance of covalently linked Extensins in the primary wall, elongation growth stops

37 Microtubules in Secondary Cell Wall Formation Reorientation of microfibrils does not occur in the absence of MTs S3 MTs S2 S1 MTs Burk & Ye 2002 PW ML

38 Secondary Cell Wall Thickenings Coalign with Microtubules IRX = irregular xylem

39 Secondary Cell Wall Thickenings Coalign with Microtubules Wheat germ agglutinin (WGA) is a carbohydratebinding protein of approx. 36 kda, it binds to N- acetylglucosaminyl sugar residues. Oda & Hasezawa 2006 Texas Red WGA (pink)/ Tubulin-GFP (green) At this stage cell wall thickenings begin to appear MT-bands split and widen as wall thickenings increase

40 Secondary Cell Wall Thickenings Coalign with Microtubules glancing section Hepler & Newcomb 1964 cross section

41 Lignin Peroxidative Formation of Lignol Radicals 2x Peroxidase unpaired electron Monologinol Radicals Undergo Uncatalyzed Coupling to form Lignin Polymer

42 Lignification of Secondary Cell Walls Covalent Bonds Between Lignin and Structural Polysaccharides