1. Essentials of Glycobiology Lecture 29 May 18, 2004 Plant Glycans Marilynn Etzler Section of Molecular and Cellular Biology University of California Davis, CA 95616 e-mail: meetzler@ucdavis.edu

2. LECTURE OUTLINE Introduction Classes of glycans in plants Structure and biosynthesis of plant N-linked glycans Other types of plant glycans Plant cell wall Plant glycosylation mutants Molecular “farming”

3. PLANTSFUNGI ANIMALS Urochordates Yeasts Red algae Green algae Brown algae Vertebrates Chordates Mosses Liverworts Ferns Gymnosperms Angiosperms PROTOZOA EUKARYOTES Multicellular Unicellular Ancestral Prokaryotes Arthropods Insects Slime molds Sponges Coelenterates Mollusks Nematodes Echinoderms Adapted from Figure 1-38, Molecular Biology of the Cell, 3 rd ed., Garland Publishing, Inc.

5. Comparison of Classes of Plant and Animal Glycans From Lecture 2 by Dr. Varki Cell wall glycans Glycolipids Major Glycan Classes in Plant Cells

6. Major Classes of N-Glycans Found in Plants High Mannose 33 22 44 Complex Pauci-mannose Hybrid = Sialic acid = Man = Gal = GlcNAc = Fuc = Xylose

7. Sialic Acid Has Recently Been Found in Plants Evidence: Found in glycoproteins obtained from suspension-cultured cells from Arabidopsis thaliana, Nicotiana tabacum and Medicago sativa Bound to Sambucus nigra and Maackia amurensis lectins Did not bind to these lectins if pretreated with  2-3,6 sialidase Sialic acids were released chemically, derivatized with DMB and analyzed by reverse phase chromatography, yielding a prominent peak of Neu5Gc and a smaller peak of Neu5Ac. Similar results were obtained with sialic acids released enzymatically. Analyses of DMB-SA derivatives were confirmed by MALDI-TOF Reference: Shah, M.M., K. Fujiyama, C.R. Flynn, and L. Joshi (2003) Nature Biotechnology 21: 1470 – 1471.

8. Recognition and Processing of N-Glycans in the Plant Secretory Pathway Endoplasmic reticulum: Glucosidases I and II Calreticulin ER mannosidase  -mannosidase I Golgi:

9. Processing in Golgi (continued) GNT I  -Man II GNT II  2-XylT  3-FucT 33 22  3-GalT  4-FucT  4

10. J. Cell Science (2002) 115: 2423 Most of the volume of a typical plant cell is occupied by the vacuole(s)

11. Processing in Vacuole or Enroute to Vacuole:

12. Other Types of Plant Glycans Plant Glycolipids : Galactolipids – in chloroplast membranes Monogalactosyldiacylglycerol Digalactosyldiacylglycerol O-Diacylglycerol 66  Sphingolipids – in plasma membrane Glucosylceramide ceramide Glycosylphosphatidylinositol anchors: 44 66 22 NH2NH2 Protein - Ethanol amine - PO 4 Phosphatidylinositol 44 44 66 22 NH2NH2 Protein - Ethanol amine - PO 4 Phosphatidylinositol

13. Types of O-Linked Glycans Found in Plants GlcNAc Ser/Thr GalNAc Gal GlcNAcGal(Ara) 1-4 Hydroxyproline (Glycosylation of hydroxyproline is unique to plants and Chlorophycean algae) Arabinogalactan proteins (carbohydrate usually > 90% by weight) 33 33 33 33 33 33 ) ( (many variations) - Hyp

14. Cell Wall Glycans Cellulose [ Glc  4 Glc] n Pectins: Homogalacturanan [GalU  4 GalU  4] n Rhamnogalacturonan I [GalU  2 L-Rha  4] n Rhamnogalacturonan II Figure 3 from Phytochem. 57: 929 (2001)

15. Xyloglucan: 44 44 44 44 44 44 22 66 66 66 66 66 66 22 nn Hemicelluloses: Cell Wall Glycans (continued) Galactomannan

16. Plant Cell Wall Alberts, et al., Molecular Biology of the Cell, Fig. 19-75 Constitutes the extracellular matrix

17. cellulose galactans homogalacturonan rhamnogalacturonan I calcium rhamnogalacturonan II xyloglucan arabinans Plasma membrane Adapted from Figure 2, Trends in Plant Science 9: 203 (2004) Model of Plant CellWall

18. Plant Glycosyltransferases and Glycosidases Almost 800 glycosyltransferase and glycosidase-related genes have been found in the Arabidopsis genome. Comprises > 3.3% of its genes. By contrast, human genome has about 350 glycosyltransferase and glyco- sidase-related genes.

19. Arabidopsis thaliana as a Model Plant System SOME ADVANTAGES: Complete genome sequenced Diploid Easily transformed Relatively rapid life cycle Many mutants available Plants small and thus take up little space Arabidopsis seed Corn kernel

20. ARABIDOPSIS cgl MUTANT Identified by screening leaf extracts of EMS mutants with antiserum against complex glycans. DEFECT: Missing GNT I PHENOTYPE: No apparent effect on development and morphology of plants. No complex glycans. Accumulates Man 5 GlcNAc 2

21. ARABIDOPSIS mur 1 MUTANT DEFECT: Deficient in an isoform of GDP-D-mannose-4,6-dehydratase. PHENOTYPE: Plants are dwarfed and have fragile cell walls. Deficient in fucose. Identified by making acid hydrolysates of cell walls of EMS mutants and screening the alditol acetate derivatives by GLC. 33 22 L-Gal 44 44 44 44 44 44 22 66 66 66 66 66 66 22 nn

22. ARABIDOPSIS SPY MUTANTS DEFECT: Deficient in O-linked GlcNAc transferase activity. PHENOTYPE: A variety of alterations in growth and development. Originally identified in genetic screen for mutants with increased response to gibberellins from T- DNA mutants. Also from EMS mutants. Proposed to be involved in various aspects of regulation of plant development.