Boron bridging of rhamnogalacturonan-II in Rosa and arabidopsis cell cultures occurs mainly in the endo-membrane system and continues at a reduced rate after secretion

Abstract

Background and aims: Rhamnogalacturonan-II (RG-II) is a domain of primary cell-wall pectin. Pairs of RG-II domains are covalently cross-linked via borate diester bridges, necessary for normal cell growth. Interpreting the precise mechanism and roles of boron bridging is difficult because there are conflicting hypotheses as to whether bridging occurs mainly within the Golgi system, concurrently with secretion or within the cell wall. We therefore explored the kinetics of RG-II bridging.

Methods: Cell-suspension cultures of Rosa and arabidopsis were pulse-radiolabelled with [14C]glucose, then the boron bridging status of newly synthesized [14C]RG-II domains was tracked by polyacrylamide gel electrophoresis of endo-polygalacturonase digests.

Key results: Optimal culture ages for 14C-labelling were ~5 and ~1 d in Rosa and arabidopsis respectively. De-novo [14C]polysaccharide production occurred for the first ~90 min; thereafter the radiolabelled molecules were tracked as they 'aged' in the wall. Monomeric and (boron-bridged) dimeric [14C]RG-II domains appeared simultaneously, both being detectable within 4 min of [14C]glucose feeding, i.e. well before the secretion of newly synthesized [14C]polysaccharides into the apoplast at ~15-20 min. The [14C]dimer : [14C]monomer ratio of RG-II remained approximately constant from 4 to 120 min, indicating that boron bridging was occurring within the Golgi system during polysaccharide biosynthesis. However, [14C]dimers increased slightly over the following 15 h, indicating that limited boron bridging was continuing after secretion.

Conclusions: The results show where in the cell (and thus when in the 'career' of an RG-II domain) boron bridging occurs, helping to define the possible biological roles of RG-II dimerization and the probable localization of boron-donating glycoproteins or glycolipids.

Keywords: Arabidopsis thaliana; Rosa sp. (‘Paul’s Scarlet’); Boron bridges; borate diesters; cell-suspension cultures; cell-wall polysaccharides; pectin; polyacrylamide gel electrophoresis; radiolabelling; rhamnogalacturonan-II.

Fig. 1.

Uptake and metabolism of [¹⁴C]glucose by differently aged glycerol-grown cultures. Mini-cultures (100 µL in 6-mL tubes) of glycerol-grown rose and arabidopsis cells, sampled at 0 to 14 d after inoculation (always adjusted to 50 % settled cell volume and acclimated to the 6-mL vial for 4 h), were fed [6-¹⁴C]glucose (50 kBq; final concentration 0.25 mm) and incubated for a further 2 h. (A) Net uptake, estimated from ¹⁴C ‘remaining’ in the medium. (B) ¹⁴C-Incorporation into small intracellular metabolites. (C) ¹⁴C-Incorporation into non-pectic polymers (components not released from AIR by EPG). (D) ¹⁴C-Incorporation into pectin (components released from AIR by EPG). Error bars, where visible, show the range of two replicate mini-cultures.

Fig. 2.

¹⁴C-Labelling of RG-II by differently aged cultures. Pulse-labelling with [¹⁴C]glucose for 2 h was as in Fig. 1. Portions of each EPG digest (see Fig. 1D) corresponding to the products obtained from 1.0 mg of AIR were analysed by gel electrophoresis: culture ages as shown above each lane. Left, Rosa; right, arabidopsis. Autoradiograms of dried gels (greyscale; 4 weeks of exposure) are aligned below the corresponding silver-stained gels (shown in colour). M: non-radioactive markers (0.8 µg monomeric plus 0.8 µg dimeric RG-II). A repeat of this experiment with mini-cultures taken from independent standard cultures is shown in Fig. S1.

Fig. 3.

Net uptake and metabolism of [¹⁴C]glucose by Rosa and arabidopsis mini-cultures. Five-day Rosa and 1-d arabidopsis cultures were adjusted to 50 % (settled cell volume) SCV and dispensed as 100-µL ‘mini-cultures’ in 6-mL tubes. After 4 h acclimation in the new tubes, the mini-cultures were supplied with 50 kBq [6-¹⁴C]Glc (final concentration 250 µm). (A) Small portions of cell-free spent medium were collected at intervals and assayed for ¹⁴C. At other time-points, whole mini-cultures were sampled and assayed for (B) intracellular small (ethanol-soluble) metabolites and (C) non-pectic polymers (¹⁴C not released from de-esterified AIR by EPG). Data show the mean of two replicate mini-cultures ± range.

Fig. 4.

¹⁴C-Pectic fragments released by endo-polygalacturonase digestion. A sample of each EPG digest (corresponding to the products released from 0.125 mg of AIR; dried and re-dissolved in ethanol) was analysed by TLC in butan-1-ol/acetic acid/water (2 : 1 : 1; one ascent). (A) Rosa; (B) arabidopsis. Autoradiograms are shown alongside thymol-stained markers (M1–M4).

Fig. 5.

Quantification of ¹⁴C-labelling of pectic domains including monomeric and dimeric RG-II. Radiolabelling of (A) EPG-releasable GalA (quantified from TLC) and RG-I (from gel electrophoresis); (B) RG-II monomer and dimer (quantified from gel electrophoresis). After polyacrylamide gel electrophoresis (Fig. 6), the bands were cut out, acid-hydrolysed and assayed for ¹⁴C by scintillation-counting. Error bars show the range of the data; n = 2.

Fig. 6.

Time-course of ¹⁴C-labelling of monomeric and dimeric RG-II domains. Radiolabelling of mini-cultures with [¹⁴C]glucose for 0–1020 min was as in Fig. 3. Portions of each EPG digest (corresponding to the products obtained from 0.375 mg of AIR) were analysed by PAGE. Left, Rosa; right, arabidopsis. Autoradiograms (4 week of exposure) of the dried gels (shown in greyscale) are aligned below identical but silver-stained gels (shown in colour). M, non-radioactive markers (0.8 µg monomeric plus 0.8 µg dimeric RG-II). A repeat of this experiment with mini-cultures taken from independent standard cultures is shown in Fig. S4.