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Extensins. Rich Wiemels. 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.

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extensins

Extensins

Rich Wiemels

assembly of cell wall proteins
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

plant cell wall structural proteins
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
hrgps
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
extensin outline
Extensin Outline
  • Phylogeny
  • Motif comparisons
  • Purification
  • Cross-linking
    • Intramolecular
      • IDT
    • Intermolecular
      • Peroxidase, elicitors
        • di-IDT, pulcherosine
      • Extensinpectate
  • 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

overall phylogeny of hrgps
Overall phylogeny of HRGPs

Kieliszewski and Lamport 1994

motifs
Motifs

PYYPPH and TPVYK

P hydroxylated to Hyp (O)

Memelink et al. 1993

The most common extensin motif is Ser-(Hyp)4

Hydrophobic regions span intervals, very insoluble

VYK –putative intermolecular crosslinking site (Schnabelrauch et al. 1996)

comparing motifs
Comparing Motifs

Kieliszewski and Lamport 1994

P1, P2 and P3 designated to differing extensin motifs

Note YKYK in tomato P2, isodityrosine (IDT) motif (Tyr-X-Tyr-Lys)

p1 p2 and p3
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

purifying extensin
Purifying Extensin
  • Monomers soluble until incorporated into wall, how to purify?
  • SolubilizeHyp (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
purifying extensin pectin cross link
Purifying extensin, pectin cross-link

Digests homogalacturonan

Remove cellulose and XyG

Remove furanosyl linkages (Ara)

Remove ionically associated polymers

RG-I still present

Remove all sugars

RG-I remains in an insoluble fraction, suggesting covalent linkage to pectin

cross links
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)
  • Extensinpectate
    • New model for wall assembly
extensin peroxidase ep
ExtensinPeroxidase (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)
various elicitors tested for cross linking stimulation of gvp1 an extensin from grapevine
Various elicitors tested for cross-linking stimulation of GvP1, an extensin from grapevine

Jackson et al. 2001

Elicitors cause cross-linking and increased insolubility of extensin

tyrosine linkages
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)

di idt and pulcherosine synthetic gene vs purified extensin
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

self assembly of the plant cell wall requires an extensin scaffold

Self-assembly of the plant cell wall requires an extensin scaffold

Maura C. Cannon1, Kimberly Terneus2, Qi Hall1, Li Tan2, Yumei Wang1, Benjamin L. Wegenhart2, Liwei Chen2, Derek T. A. Lamport3, Yuning Chen2, and Marcia J. Kieliszewski2

Department of Biochemistry and Molecular Biology, University of Massachusetts

Department of Chemistry and Biochemistry, Ohio University

Department of Biology and Environmental Science, University of Sussex, UK

the rsh mutant
The RSH mutant
  • -root, -shoot, -hypocotyl defective
  • Identified by Hall and Cannon 2002
  • Major developmental problems
    • Severely misshapen cells
    • Misplacement of cell plate
extensins in arabidopsis
Extensins in Arabidopsis
  • 20 homologous genes, but this one knockout has lethal phenotype!
r sh rsh phenotype
rsh/rshphenotype

Heart stage embryos

Root sections

C-F rsh/rshphenotype includes incomplete (floating, hanging) walls and wall stubs

purifying and identifying rsh
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 extensinarabinooligosaccharides
  • Deglycosylation by HF and sequencing confirmed identity as AtEXT3
protein sequence
Protein Sequence

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

cross linking assay
Cross-linking Assay
  • Pulcherosine is more prevalent than di-Idt as the cross-linking tyrosine derivative
calculating types of tyr derivatives
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?
explaining di idt absence
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)
c terminal significance
C-terminal significance
  • GFP fused to C-terminus did not rescue rsh/rshdouble mutant
  • N-terminal fused GFP yielded functional RSH
afm data
AFM data

(YK)20

RSH monomer

-Forms dendritic

structure

  • Segments calculated to be 127nm for polyproline II helical secondary structure
  • Molecules are overlapping, stretch longer than RSH molecule from staggered alignment
self assembly
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
conclusions a new paradigm for cell wall self assembly
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 extensinpectate

9.Extensin scaffolds template for orderly pectic matrix