1. Extensins Rich Wiemels

2. Assembly of cell wall proteins Proteins synthesized and hydroxylated in the ER Glyosylation occurs in the Golgi Golgi vesicles secrete monomers to the wall Monomers polymerize Cross-links form Ionic, covalent, H-bond, electrostatic etc. Elicitor stimulated cross-linking How does self assembly of the cell wall occur at the cell plate during cell division? Buchanan, Gruissem and Jones. (2000) Biochemistry & Molecular Biology of Plants

3. Plant Cell Wall Structural Proteins Characteristics: – Sequence information Motifs, palindromes, predictions – Physical properties Solubility, charge, structure, etc. – Post-translational modifications Hydroxylation (HRGPs) Glycosylation (amount, sugars involved, branching) Intramolecular cross-linking – Interaction with other molecules Ionic interactions, salt bridges Intermolecular interactions

4. HRGPs 3 types – Proline-rich proteins Lowly glycosylated, highly periodic Ara and Gal – Extensin Moderately glycosylated, less periodic Ara and Gal – Arabinogalactan proteins Highly glycosylated, least periodic Ara, Gal, Fuc, Rha, GlcNAc

5. Extensin Outline Phylogeny Motif comparisons Purification Cross-linking – Intramolecular IDT – Intermolecular Peroxidase, elicitors – di-IDT, pulcherosine Extensin pectate Formation of extensin scaffold (today’s paper) Ara on Hyp (contiguous only) Gal on Ser Buchanan, Gruissem and Jones. (2000) Biochemistry & Molecular Biology of Plants

6. Overall phylogeny of HRGPs Kieliszewski and Lamport 1994

7. Motifs The most common extensin motif is Ser-(Hyp) 4 Hydrophobic regions span intervals, very insoluble VYK –putative intermolecular crosslinking site (Schnabelrauch et al. 1996) Memelink et al. 1993 P hydroxylated to Hyp (O) PYYPPH and TPVYK

8. Comparing Motifs P1, P2 and P3 designated to differing extensin motifs Note YKYK in tomato P2, isodityrosine (IDT) motif (Tyr-X-Tyr-Lys) Kieliszewski and Lamport 1994

9. P1, P2, and P3 P1: Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys – No IDT motif P2: Ser-Hyp-Hyp-Hyp-Hyp-Val-Tyr-Lys-Tyr-Lys P3: Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Ser-Hyp- Hyp-Hyp-Hyp-Tyr-Tyr-Tyr-Lys – More Tyr cross-linking possibilities Smith et al. 1986

10. Purifying Extensin Monomers soluble until incorporated into wall, how to purify? Solubilize Hyp (Qi et al. 1995) – Digest homogalacturonan (EPG) – Digest cellulose, XyG (Cellulase) – HF at -73° to cleave furanosyl linkages, Ara removed – HF at 0° to cleave off all sugars – Ammonium carbonate to remove ionic interacting molecules

11. Purifying extensin, pectin cross-link RG-I still present RG-I remains in an insoluble fraction, suggesting covalent linkage to pectin Digests homogalacturonan Remove cellulose and XyG Remove furanosyl linkages (Ara) Remove ionically associated polymers Remove all sugars

12. Cross-links Peroxidase cross-linking – Needed for deposition, cross-links Tyr – Elicited defense mechanism IDT formation – Added insolubility and strength – Tyrosine tetramer (di-IDT) and trimer (pulcherosine) Extensin pectate – New model for wall assembly

13. Extensin Peroxidase (EP) Polymerizes extensin monomers (Schnabelrauch 1996) Oxidative cross-linking occurs at perception of stress or elicitors (Bradley et al., 1992) – Protects plant from pathogens, invaders Cross-linking occurs before transcription dependent response (Brisson et al. 1994) and after (Showalter, 1993). VYK possible cross-linking motif for EP in addition to Tyr motifs (Schnabelrauch et al., 1996)

14. Various elicitors tested for cross-linking stimulation of GvP1, an extensin from grapevine Elicitors cause cross-linking and increased insolubility of extensin Jackson et al. 2001

15. Tyrosine linkages Tyr-X-Tyr-Lys motif Isodityrosine (IDT) intramolecular linkages stabilizes extensin (P2, P3) and does not disrupt helical conformation (Epstein and Lamport, 1984)

16. Brady and Fry, 1998 di-IDT and pulcherosine: Intermolecular cross-linking

17. di-IDT and pulcherosine: synthetic gene vs. purified extensin di-IDT (tetromer) forms in vitro from synthetic P3 extensin (Held et al., 2004) – SPPPPYYYKSPPPPSP repeated 20x = (YK) 20 Very little di-IDT observed in RSH, an Arabidopsis extensin. Instead Tyr trimer, pulcherosine (Cannon et al. 2008) ? …and that brings us to today’s paper

18. Self-assembly of the plant cell wall requires an extensin scaffold Maura C. Cannon 1, Kimberly Terneus 2, Qi Hall 1, Li Tan 2, Yumei Wang 1, Benjamin L. Wegenhart 2, Liwei Chen 2, Derek T. A. Lamport 3, Yuning Chen 2, and Marcia J. Kieliszewski 2 1.Department of Biochemistry and Molecular Biology, University of Massachusetts 2.Department of Chemistry and Biochemistry, Ohio University 3.Department of Biology and Environmental Science, University of Sussex, UK

19. The RSH mutant -root, -shoot, -hypocotyl defective Identified by Hall and Cannon 2002 Major developmental problems – Severely misshapen cells – Misplacement of cell plate

20. Extensins in Arabidopsis 20 homologous genes, but this one knockout has lethal phenotype!

21. rsh/rsh phenotype Heart stage embryos Root sections C-F rsh/rsh phenotype includes incomplete (floating, hanging) walls and wall stubs

22. Purifying and Identifying RSH Lys-rich positively charged extensin monomers salt eluted – Superose-6 gel filtration yielded monomer – Cation-exchange chromatography and alkaline hydrolysis yielded extensin arabinooligosaccharides Deglycosylation by HF and sequencing confirmed identity as AtEXT3

23. Protein Sequence Highly periodic. 11 identical 28-residue repeats, monomer = 308 amino acids

24. Cross-linking Assay Pulcherosine is more prevalent than di-Idt as the cross-linking tyrosine derivative

25. Calculating Types of Tyr derivatives Very little di-Idt forms unlike (YK) 20 (Held et al. 2004) Why are pulcherosine and Idt the dominant Tyr cross-linking derivatives?

26. Explaining di-Idt absence Parallel alignment, as with (YK) 20 yields only di-Idt motifs (A) Staggering the alignment yields only pulcherosine motifs– Ser-(Hyp) 4 still aligned (B)

27. C-terminal significance GFP fused to C-terminus did not rescue rsh/rsh double mutant N-terminal fused GFP yielded functional RSH

28. AFM data Segments calculated to be 127nm for polyproline II helical secondary structure Molecules are overlapping, stretch longer than RSH molecule from staggered alignment RSH monomer -Forms dendritic structure (YK) 20

29. Self Assembly Monomers of extensin self assemble into dendritic scaffold – Consistent with other self-assembling amphiphiles at liquid interfaces (Rapaport 2006) – Able to form template for pectin New paradigm for cell wall assembly – Extensin needed at cell initiation, not cessation of growth

30. Conclusions: A new paradigm for cell wall self-assembly 1. Liquid-liquid interface promotes self-assembly of ordered amphiphilic arrays 2. Alternating hydrophilic/phobic modules induce self- recognition 3. Periodicity aligns monomers 4. C-terminus sequence may initiate end-on adhesion 5. Intermolecular Tyr cross-links stabilize 6. Pulcherosine cross-links favor staggered RSH alignment 7. Staggered alignment allows 2D growth 8. Extensin and pectin form extensin pectate 9.Extensin scaffolds template for orderly pectic matrix