Marcia J. Kieliszewski

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

Cytokinesis partitions the cell by a cleavage furrow in animals but by a new cross wall in plants. How this new wall assembles at the molecular level and connects with the mother cell wall remains unclear. A lethal Arabidopsis embryogenesis mutant designated root-, shoot-, hypocotyl-defective (rsh) provides some clues: RSH encodes extensin AtEXT3, a structural glycoprotein located in the nascent cross wall or “cell plate” and also in mature cell walls. Here we report that electron micrographs of rsh mutant cells lacking RSH extensin correspond to a wall phenotype typified by incomplete cross wall assembly. Biochemical characterization of the purified RSH glycoprotein isolated from wild-type Arabidopsis cell cultures confirmed its identity as AtEXT3: a (hydroxy)proline-rich glyco protein comprising 11 identical amphiphilic peptide repeats with a 28-residue periodicity: SOOOOKKHYVYKSOOOOVKHYSOOOVYH (O = Hyp), each repeat containing a hydrophobic isodityrosine cross-link motif (YVY, underlined). Atomic force microscopy of RSH glycoprotein imaged its propensity for self-assembly into a dendritic scaffold. Extensin peroxidase catalyzed in vitro formation of insoluble RSH gels with concomitant tyrosine cross-linking, hence this likelihood in muro. We conclude that self-assembling amphiphiles of lysine-rich RSH extensin form positively charged scaffolds in the cell plate. These react with negatively charged pectin to create an extensin pectate coacervate that may template further orderly deposition of the new cross wall at cytokinesis.

Role of the Extensin Superfamily in Primary Cell Wall Architecture

Nearly two centuries of progress have established the major components of the plant cell wall, a composite that includes interpenetrating networks of cellulose (Payen, 1838; Schulze, 1891), microfibrils (Frey-Wyssling et al., 1948; Preston et al., 1948), pectin (Braconnot, 1825) and lignin (Payen, 1838). However, only over the last five decades has a relatively minor hydroxyproline-rich structural glycoprotein component emerged with essential roles in building and maintaining the growing primary cell wall. Here we highlight unique advances of each decade from the initial discovery of hydroxyproline (Hyp) in cell walls to the current definition of extensins as self-assembling amphiphiles that generate scaffolding networks, and where acid-base interaction - extensin pectate - may template assembly of the pectic matrix. Subsequent polymerization toughens up the wall as networks resisting both microbial and mechanical stress. At each stage we explore hypotheses arising from synthesis of emerging data with focus on structure. This review celebrates the 50th birthday of extensin.

KDEL-tailed cysteine endopeptidases involved in programmed cell death, intercalation of new cells, and dismantling of extensin scaffolds.

KDEL-tailed cysteine endopeptidases are a group of papain-type peptidases found in senescing tissue undergoing programmed cell death (PCD). Their genes have so far been cloned and analyzed in 12 angiosperms. They are synthesized as proenzymes with a C-terminal KDEL endoplasmatic reticulum retention signal, which is removed with the prosequence to activate enzyme activity. We previously identified three genes for KDEL-tailed cysteine endopeptidases (AtCEP1, AtCEP2, AtCEP3) in Arabidopsis thaliana. Transgenic plants of A. thaliana expressing β-glucuronidase (GUS) under the control of the promoters for the three genes were produced and analyzed histochemically. GUS activity was promoter- and tissue-specific GUS activity during seedling, flower, and root development, especially in tissues that collapse during final stages of PCD, and in the course of lateral root formation. KDEL-tailed cysteine endopeptidases are unique in being able to digest the extensins that form the basic scaffold for cell wall formation. The broad substrate specificity is due to the structure of the active site cleft of the KDEL-tailed cysteine endopeptidase that accepts a wide variety of amino acids, including proline and glycosylated hydroxyproline of the hydroxyproline rich glycoproteins of the cell wall.