Boron bridging of rhamnogalacturonan-II is promoted in vitro by cationic chaperones, including polyhistidine and wall glycoproteins

Abstract

Dimerization of rhamnogalacturonan-II (RG-II) via boron cross-links contributes to the assembly and biophysical properties of the cell wall. Pure RG-II is efficiently dimerized by boric acid (B(OH)3 ) in vitro only if nonbiological agents for example Pb(2+) are added. By contrast, newly synthesized RG-II domains dimerize very rapidly in vivo. We investigated biological agents that might enable this. We tested for three such agents: novel enzymes, borate-transferring ligands and cationic 'chaperones' that facilitate the close approach of two polyanionic RG-II molecules. Dimerization was monitored electrophoretically. Parsley shoot cell-wall enzymes did not affect RG-II dimerization in vitro. Borate-binding ligands (apiose, dehydroascorbic acid, alditols) and small organic cations (including polyamines) also lacked consistent effects. Polylysine bound permanently to RG-II, precluding electrophoretic analysis. However, another polycation, polyhistidine, strongly promoted RG-II dimerization by B(OH)3 without irreversible polyhistidine-RG-II complexation. Likewise, partially purified spinach extensins (histidine/lysine-rich cationic glycoproteins), strongly promoted RG-II dimerization by B(OH)3 in vitro. Thus certain polycations, including polyhistidine and wall glycoproteins, can chaperone RG-II, manoeuvring this polyanionic polysaccharide domain such that boron-bridging is favoured. These chaperones dissociate from RG-II after facilitating its dimerization, indicating that they act catalytically rather than stoichiometrically. We propose a natural role for extensin-RG-II interaction in steering cell-wall assembly.

Keywords: Boron; cell wall; cross-linking; dehydroascorbic acid; extension; pectic polysaccharides; polyhistidine; rhamnogalacturonan-II (RG-II).

Figure 1

Proposed two‐step dimerization of polyanionic rhamnogalacturonan‐II (RG‐II) and of chromotropic acid as a model. (a) The polyanion, RG‐II ⁽ⁿ⁻⁾, is proposed to undergo two reactions similar to those shown in (b). Owing to the strong negative charge on both RG‐II molecules, we propose that the second step will be favoured if the RG‐II ⁽ⁿ⁻⁾ is ionically complexed with a cationic ‘chaperone’. (b) The ionized form of chromotropic acid (CTA ²⁻) rapidly reacts with neutral trigonal boric acid (B(OH)₃), giving a (B:CTA)³⁻ complex containing a tetrahedral B⁻. This reacts slowly with a second CTA ²⁻, despite electrostatic repulsion, to give a relatively stable (B:(CTA)₂)⁵⁻ complex (Shao et al., 2000).

Figure 2

A parsley extract rich in numerous wall‐modifying enzyme activities fails to dimerize rhamnogalacturonan‐II (RG‐II). Monomeric RG‐II (a, 18 μM tritiated; b, 20 μM nonradioactive) was incubated in 1.2 mM B(OH)₃ at 20°C in the presence or absence of a wall‐enzyme‐rich extract from parsley shoots for 0–24 h, then frozen at −80°C. Later, (a) 2.4 kBq or (b) 0.8 μg of the RG‐II was subjected to polyacrylamide gel electrophoresis. In (b), the gel was silver‐stained and photographed. In (a), the zones corresponding to monomer and dimer were excised from the gel and separately assayed for radioactivity by scintillation counting. The ‘−0.1 h’ sample was taken just before addition of the parsley extract.

Figure 3

Low‐M _r putative boron carriers do not promote rhamnogalacturonan‐II (RG‐II) dimerization. Monomeric RG‐II (a, 18 μM tritiated; b, 20 μM nonradioactive) was incubated at 20°C for 24 h in the presence of the additives indicated at the top. DHA, dehydroascorbic acid. Later, (a) 2.4 kBq or (b) 0.8 μg of the RG‐II was subjected to polyacrylamide gel electrophoresis. In (a) the radioactive bands on the gel were detected by fluorography; in (b), the gel was silver‐stained and photographed.

Figure 4

Low‐M _r organic cations and free apiose do not promote rhamnogalacturonan‐II (RG‐II) dimerization. Monomeric RG‐II (a, 18 μM tritiated; b, 20 μM nonradioactive) was incubated at 20°C for 24 h in the presence of the additives indicated at the top. Later, (a) 2.4 kBq or (b) 0.8 μg of the RG‐II was subjected to polyacrylamide gel electrophoresis. In (a) the radioactive bands on the gel were detected by fluorography; in (b), the gel was silver‐stained and photographed.

Figure 5

Polylysine and polyhistidine have different effects on rhamnogalacturonan‐II (RG‐II). Monomeric RG‐II (0.1 mg ml⁻¹; 20 μM) was incubated at 20°C for 24 h with a dilution series of (a) polylysine and (b) polyhistidine, with and without the other additives indicated. Later, 0.8 μg of the RG‐II was subjected to polyacrylamide gel electrophoresis, and the gel was silver‐stained and photographed.

Figure 6

Partial characterization of a spinach extensin preparation. (a) Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) of (glyco)proteins eluted from the cells of a live 6‐d‐old spinach suspension‐culture with water or 0.1 M CaCl₂, and comparable markers. M, commercial protein marker‐mixture. Lanes: 1, potato lectin; 2, de‐arabinofuranosylated potato lectin; 3, water‐eluate of spinach cells; 4, 0.1 M CaCl₂‐eluate of spinach cells; ‐, blank lane. Stains: left panel, Coomassie Blue (showing total proteins); right panel, periodate/Schiff's reagent (showing glycoproteins). E, spinach extensin. (b) High‐voltage paper electrophoresis (at pH 2.0) of the alkali hydrolysis products of [pentosyl‐³H]extensin. Nonradioactive markers were run in parallel and detected by staining (horizontal lines). (c) SDS‐PAGE of total cellular proteins extracted with warm phenol/acetic acid/water from suspension‐cultured spinach cells at various times (0.3–11 d after subculture). Stain: Coomassie Blue. E, spinach extensin. Each loading is the equivalent of 0.8 mg DW of cells.

Figure 7

An extensin preparation promotes rhamnogalacturonan‐II (RG‐II) dimerization. Monomeric RG‐II (0.1 mg ml⁻¹; 20 μM) was incubated at 20°C in the presence of various combinations of 1.2 mM boric acid, 0.5 mM Pb²⁺ and crude spinach extensin. In (a), the incubation time was varied. In (b), the extensin concentration was varied, incubation time being held constant at 4 h. In (c), the extensin, when present, was at a final concentration of 0.3 mg ml⁻¹, and the incubation time was 3 h; the extensin used for the sample in lane 2 had been pretreated at 100°C for 30 min before being mixed with RG‐II; that for lane 3 was an unboiled control; and in lane 1 the reaction was conducted in the presence of nonboiled extensin plus 0.2 M trifluoroacetic acid (TFA). In each case, 0.8 μg of the RG‐II was subjected to polyacrylamide gel electrophoresis, and the gel was then silver‐stained and photographed.

Figure 8

Cation‐exchange fractions of crude extensin promote rhamnogalacturonan‐II (RG‐II) dimerization. (a) Fractionation of 3 mg crude spinach extensin on sulphopropyl‐Sephadex (SP‐Sephadex) by elution with a pH gradient. Column bed volume, 5 ml; fraction volume, 2 ml. Bars indicate the pH of the eluent being applied to the column while the indicated fractions were being collected. (b) Monomeric RG‐II (0.1 mg ml⁻¹; 20 μM) was incubated at 20°C for 4 h with the (dialysed and dried) extensin fractions and/or other additives as indicated. Approximate concentrations of the extensin fractions present in the reaction mixtures were estimated from A ₂₈₀ (each concentration is the equivalent of the whole pool diluted into 2 ml of reaction mixture). After the incubation, 0.8 μg of the RG‐II was subjected to polyacrylamide gel electrophoresis, and the gel was then silver‐stained and photographed.