Derek T. A. Lamport

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.

Periplasmic arabinogalactan glycoproteins act as a calcium capacitor that regulates plant growth and development

Arabinogalactan glycoproteins (AGPs) are implicated in virtually all aspects of plant growth and development, yet their precise role remains unknown. Classical AGPs cover the plasma membrane and are highly glycosylated by numerous acidic arabinogalactan polysaccharides O-linked to hydroxyproline. Their heterogeneity and complexity hindered a structural approach until the recent determination of a highly conserved repetitive consensus structure for a 15-sugar residue arabinogalactan subunit with paired glucuronic carboxyls. Based on NMR data and molecular dynamics simulations, we identify these carboxyls as potential intramolecular Ca(2+)-binding sites. Using rapid ultrafiltration assays and mass spectrometry, we verified that AGPs bind Ca(2+) tightly (K(d) ~ 6.5 μM) and stoichiometrically at pH 5. Ca(2+) binding is reversible in a pH-dependent manner. As typical AGPs contain c. 30 Ca(2+)-binding subunits and are bulk components of the periplasm, they represent a Ca(2+) capacitor discharged at low pH by stretch-activated plasma membrane H(+)-ATPases, hence a substantial source of cytosolic Ca(2+). We propose that these Ca(2+) waves prime the calcium oscillator, a signal generator essential to the global Ca(2+) signalling pathway of green plants.

Back to the future with the AGP–Ca2+ flux capacitor

BACKGROUNDnArabinogalactan proteins (AGPs) are ubiquitous in green plants. AGPs comprise a widely varied group of hydroxyproline (Hyp)-rich cell surface glycoproteins (HRGPs). However, the more narrowly defined classical AGPs massively predominate and cover the plasma membrane. Extensive glycosylation by pendant polysaccharides O-linked to numerous Hyp residues like beads of a necklace creates a unique ionic compartment essential to a wide range of physiological processes including germination, cell extension and fertilization. The vital clue to a precise molecular function remained elusive until the recent isolation of small Hyp-arabinogalactan polysaccharide subunits; their structural elucidation by nuclear magentic resonance imaging, molecular simulations and direct experiment identified a 15-residue consensus subunit as a β-1,3-linked galactose trisaccharide with two short branched sidechains each with a single glucuronic acid residue that binds Ca(2+) when paired with its adjacent sidechain.nnnSCOPEnAGPs bind Ca(2+) (Kd ∼ 6 μm) at the plasma membrane (PM) at pH ∼5·5 but release it when auxin-dependent PM H(+)-ATPase generates a low periplasmic pH that dissociates AGP-Ca(2+) carboxylates (pka ∼3); the consequential large increase in free Ca(2+) drives entry into the cytosol via Ca(2+) channels that may be voltage gated. AGPs are thus arguably the primary source of cytosolic oscillatory Ca(2+) waves. This differs markedly from animals, in which cytosolic Ca(2+) originates mostly from internal stores such as the sarcoplasmic reticulum. In contrast, we propose that external dynamic Ca(2+) storage by a periplasmic AGP capacitor co-ordinates plant growth, typically involving exocytosis of AGPs and recycled Ca(2+), hence an AGP-Ca(2+) oscillator.nnnCONCLUSIONSnThe novel concept of dynamic Ca(2+) recycling by an AGP-Ca(2+) oscillator solves the long-standing problem of a molecular-level function for classical AGPs and thus integrates three fields: AGPs, Ca(2+) signalling and auxin. This accounts for the involvement of AGPs in plant morphogenesis, including tropic and nastic movements.

Structural Proteins of the Primary Cell Wall: Extraction, Purification, and Analysis

Structural proteins of the primary cell wall present unusual but interesting problems for structural biologists in particular and plant biologists in general. As structure is the key to function; then the biochemical isolation of these glycoproteins for further study is paramount. Here, we detail the classical method for isolating soluble extensin monomers by elution of monomeric precursors to network extensin from tissue cultures. We also outline an additional approach involving genetic engineering that can potentially yield the complete genomic range of extensins and other hydroxyproline-rich glycoprotein (HRGPs) currently underutilized for biotechnology.

Pollen tube growth and guidance: Occam's razor sharpened on a molecular arabinogalactan glycoprotein Rosetta Stone

Occams Razor suggests a new model of pollen tube tip growth based on a novel Hechtian oscillator that integrates a periplasmic arabinogalactan glycoprotein-calcium (AGP-Ca2+ ) capacitor with tip-localized AGPs as the source of tip-focussed cytosolic Ca2+ oscillations: Hechtian adhesion between the plasma membrane and the cell wall of the growing tip acts as a piconewton force transducer that couples the internal stress of a rapidly growing wall to the plasma membrane. Such Hechtian transduction opens stretch-activated Ca2+ channels and activates H+ -ATPase proton pump efflux that dissociates periplasmic AGP-Ca2+ resulting in a Ca2+ influx that activates exocytosis of wall precursors. Thus, a highly simplified pectic primary cell wall regulates its own synthesis by a Hechtian growth oscillator that regulates overall tip growth. By analogy with the three cryptic inscriptions of the classical Rosetta Stone, the Hechtian Hypothesis translates classical AGP function as a Ca2+ capacitor, pollen tube guide and wall plasticizer into a simple but widely applicable model of tip growth. Even wider ramifications of the Hechtian oscillator may implicate AGPs in osmosensing or gravisensing and other tropisms, leading us yet further towards the Holy Grail of plant growth.

Pollen tube growth and guidance: Occam's razor sharpened on a molecular AGP Rosetta Stone

Occam’s Razor suggests a new model of pollen tube tip growth based on a novel Hechtian oscillator that integrates: (1) a periplasmic AGP-Ca2+ calcium capacitor with tip-localised arabinogalactan glycoproteins (AGPs); (2) tip-focussed cytosolic Ca2+oscillations; (3) Hechtian strands evidence of adhesion between the plasma membrane and the cell wall of the growing tip. Thus Hechtian adhesion, as a piconewton force transducer, couples the internal stress of a rapidly growing wall to the plasma membrane. Such Hechtian transduction via stretch-activated Ca2+ channels and H+-ATPase proton efflux dissociating periplasmic AGP-Ca2+, creates a Ca2+ influx that activates exocytosis of wall precursors. In effect a highly simplified primary cell wall regulates its own synthesis and a Hechtian growth oscillator regulates overall tip growth. By analogy with the Rosetta Stone that translates trilingual inscriptions as a single identical proclamation, the Hechtian Hypothesis translates classical AGPs and their roles as a Ca2+ capacitor, pollen tube guide and wall plasticiser into a simple but widely applicable model of tip growth. Even wider ramifications of the Hechtian oscillator may implicate AGPs in osmosensing or gravisensing and other tropisms, leading us yet further towards the Holy Grail of plant growth.