Poster: Cell Walls

Abs # 1253: Synthetic genes for elucidating cross-linking amino acids in p1 type extensins

Presenter:	Lloyd, Elizabeth A, libbyanne@hotmail.com
Authors	Lloyd, Elizabeth A (A)   Diezman, Jill M (A)   Leykam, Joseph F (B)   Kieliszewski, Marcia J (A)
Affiliations:	(A): Ohio University (B): Michigan State University

P1 extensin is a hydroxyproline-rich glycoprotein (HRGP) that is an integral part of the primary plant cell wall. It consists mainly of two repetitive motifs: SPPPPTPVYK and SPPPPVKPYHPTPVYK. Earlier work demonstrated the requirement of a Val-Tyr-Lys motif for in vitro intermolecular cross-linking of P1 by extensin peroxidase. Tyrosine and lysine contain reactive groups, which are proposed to play a role in the cross-linking of extensin. Here we have tested the role of Val-Tyr-Lys motif in cross-linking by designing synthetic genes. These genes encode for the P1 motifs and variants that differ in their cross-linking motifs with substitutions of Tyr to Phe and Lys to Leu substitution. The genes were expressed in Nicotiana tabacum BY2 suspension cultured cells as enhanced green fluorescent protein (EGFP) fusion proteins and targeted through the ER and Golgi to the cell wall with a tobacco extensin signal sequence. The transgene products were isolated from cell culture medium by a combination of hydrophobic interaction and reverse phase C4 column chromatography. The fusion proteins underwent hydroxylation of proline and extensive arabanosylation of Hyp residues similar to endogenous P1. The VYK construct [SPPPPTPVYK]₆ cross-linked in vitro by tomato extensin peroxidase, while the VFL construct [SPPPPTPVFL]₆ showed no cross-linking activity.