Cross-Linking of Soluble Extensin in Isolated Cell Walls

Abstract

The extension component of primary cell walls has generally been considered to be an intrinsily insoluble cell wall glycoprotein. Recent data have established that cell wail extensin is in fact secreted in a soluble monomeric form which slowly becomes insolubilized in the cell wail probably through the oxidative formation of Isodityrosine cross-liks.We now show that isolated cell walls from aerated root slices of Dawc carota have the capacity to insolubllize extensin through the formation of isodityrosine. This in vitro cross-linking is specific for the extension glycoproten, as other wall proteins are not cross-linked by the isolated wall system. Although extensin can be cross-linked in solution by peroxidase and H202, dityrosine and not isodityrosine is the phenolic crosslink formed. Wall-catalyzed cross-linking of soluble extension is inhibited by L-ascorbate, and both the initial rate and total extent of cross4iking are inhibited by acidic pH in the physiological range (pH 4 to 6). We suggest several mechanisms by which add might inhibit cross-nkn and propose that cytoplasmic factors ascorbatee and/or hydrogen ions) may regulate the solubility of extensin in vivo.Cell walls of dicotyledonous plants contain an insoluble glycoprotein component which is distinguished by its content of 4-hydroxyproline and has been given the name extensin (for review, see 7, 19). Despite suggestions that this integral wall polymer is important in growth control (4,17,25), disease resistance (10), and morphogenesis (1), the role of extensin in cell wall structure and function remains speculative and thus controversial (12,19).The biochemical characterization of extensin has proved especially difficult. For two decades it was generally believed that wall extensin is an intrinsically insoluble HRGP3 which could only be solubilized by proteolysis (17,18). Recently, we have shown, using aerated carrot root slices, that insoluble wall extensin is secreted into the cell wall as a soluble monomeric HRGP (5) which has been well characterized (28,29). This soluble extensin4 has a polycationic rod-like structure resembling bacterial agglutinins isolated from potato (20) extensin (5, 27). This insolubilization is inhibited in vivo by free radical scavengers and antioxidants (especially ascorbic acid), and is accompanied by the derivatization of tyrosine to IDT (5), a diphenyl ether-linked bityrosine recently characterized from plant cell wall hydrolysates and proposed to be responsible for covalently cross-linking extensin in the wall (5, 13).Understanding the manner in which extensin rods are assembled into insoluble wall extensin should provide new insight into the molelcular organization of plant cell walls. Because of the complexities associated with biochemical studies using in vivo systems, we have developed an in vitro system to study extensin cross-linking. Here we report the results of our studies. MATERIALS AND METHODSTissue Preparation. Large tap roots of Daucus carota were obtained from local merchants and stored at 4C...

Rapid Deposition of Extensin during the Elicitation of Grapevine Callus Cultures Is Specifically Catalyzed by a 40-Kilodalton Peroxidase

“…The deposition of extensin is thought to be catalyzed by class III peroxidases (Cooper and Varner, 1984). This appears to be true in grapevine callus cultures, where the elicitor-induced deposition of GvP1 extensin was inhibited by the peroxidase inhibitors azide and ascorbate.…”

“…Peroxidase and peroxide are believed to be required for the catalysis of extensin deposition (Cooper and Varner, 1984). In the tomato system, at least two peroxidases have been demonstrated to have a high capacity for the oxidative cross-linking of extensin, whereas other tomato peroxidases (Brownleader et al, 1995) and several others derived from different species (Schnabelrauch et al, 1996) did not.…”

Identification and Molecular Characterization of Novel Peroxidase with Structural Protein-like Properties

“…Ser-Hyp-Hyp-Hyp-Hyp for extensins, and Pro-Pro-Val-Tyr-Lys for PRPs) (2)(3)(4). Their insolubilization mechanisms have been also well examined, confirming that insolubilization of both structural proteins, which involves H 2 O 2 -mediated oxidative cross-linking to cell walls, is catalyzed by coexisting peroxidases (5)(6)(7).…”

Solubilization of Covalently Bound Extensin from Capsicum Cell Walls

“…This insolubility hindered the study ofextensin until the isolation ofsalt-soluble extensin precursors (monomers ofMr about 9 x 104) from cell suspension cultures (21) and wounded carrot disks (4, 23), which has enabled progress on extensin's structure and properties to be made in recent years (see review, ref. 26 the wall network is unknown, but the isolation ofthe phenolic amino acid isodityrosine (an oxidatively coupled dimer of tyrosine units linked by a diphenylether bridge) from cell wall hydrolysates (8) led to the suggestion that isodityrosine could form inter-polypeptide cross-links between extensin molecules (2, 9).Studies on isolated carrot root disks (4,5) showed that newly synthesized extensin monomers were secreted from the cytoplasm and were slowly insolubilized in the walls. Insolubilization was accompanied by isodityrosine formation, but it was not proven that the isodityrosine formed was responsible for insolubilization.…”

“…Studies on isolated carrot root disks (4,5) showed that newly synthesized extensin monomers were secreted from the cytoplasm and were slowly insolubilized in the walls. Insolubilization was accompanied by isodityrosine formation, but it was not proven that the isodityrosine formed was responsible for insolubilization.…”