Saccharide analysis of onion outer epidermal walls

Abstract

Background: Epidermal cell walls have special structural and biological roles in the life of the plant. Typically they are multi-ply structures encrusted with waxes and cutin which protect the plant from dehydration and pathogen attack. These characteristics may also reduce chemical and enzymatic deconstruction of the wall for sugar analysis and conversion to biofuels. We have assessed the saccharide composition of the outer epidermal wall of onion scales with different analytical methods. This wall is a particularly useful model for cell wall imaging and mechanics.

Results: Epidermal walls were depolymerized by acidic methanolysis combined with 2M trifluoracetic acid hydrolysis and the resultant sugars were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Total sugar yields based on wall dry weight were low (53%). Removal of waxes with chloroform increased the sugar yields to 73% and enzymatic digestion did not improve these yields. Analysis by gas chromatography/mass spectrometry (GC/MS) of per-O-trimethylsilyl (TMS) derivatives of the sugar methyl glycosides produced by acidic methanolysis gave a high yield for galacturonic acid (GalA) but glucose (Glc) was severely reduced. In a complementary fashion, GC/MS analysis of methyl alditols produced by permethylation gave substantial yields for glucose and other neutral sugars, but GalA was severely reduced. Analysis of the walls by ¹³C solid-state NMR confirmed and extended these results and revealed 15% lipid content after chloroform extraction (potentially cutin and unextractable waxes).

Conclusions: Although exact values vary with the analytical method, our best estimate is that polysaccharide in the outer epidermal wall of onion scales is comprised of homogalacturonan (~ 50%), cellulose (~ 20%), galactan (~ 10%), xyloglucan (~ 10%) and smaller amounts of other polysaccharides. Low yields of specific monosaccharides by some methods may be exaggerated in epidermal walls impregnated with waxes and cutin and call for cautious interpretation of the results.

Keywords: Cuticle; Gas chromatography/mass spectrometry; High-performance anion-exchange chromatography; Methanolysis/trifluoracetic acid hydrolysis; Monosaccharide analysis; Onion epidermal wall; Pulsed amperometric detection; Solid-state NMR.

Fig. 1

Representative HPAEC-PAD chromatograms from saccharide analysis of onion epidermal peels with monosaccharides of interest labeled. Samples were separated on a CarboPac PA20 analytical column on a Dionex ICS5000 as detailed in the methods. a Soluble fraction after met/TFA hydrolysis; b insoluble fraction of met/TFA hydrolysis further analyzed with 2-step sulfuric acid hydrolysis, neutralized, and separated on the column. GalA galacturonic acid, GlcA glucuronic acid

Fig. 2

GC/MS chromatograms of chloroform pretreated onion epidermal peel using two different derivatization methods. a TMS derivatives of the monosaccharide methyl glycosides separated on an Equity-1 fused silica capillary column; b methyl alditol derivatives separated on an Equity-1 fused silica capillary column; c same sample as b but run on a SP2330 column to separate arabinose and xylose derivatives

Fig. 3

Solid-state NMR analysis of onion primary cell walls. a Spectral deconvolution of 1D ¹³C conventional CP spectrum that mainly detects rigid components. The experimental data is in black and the fitted line is in orange. Underlying Lorentzians are individual fitted components. b Spectral deconvolution of quantitative MultiCP spectrum that detects all molecules. c Expanded region of 52–95 ppm region of MultiCP spectrum. d Polymer composition from integrated ssNMR MultiCP spectra. Spectral deconvolution yields both polysaccharide to lipid ratio and sugar composition analysis. i (interior cellulose), s (surface cellulose), GA (GalA/GlcA), Gal (galactose), A (arabinose), R (rhamnose), GAMe (GalA/GlcA methylated), AcMe (acetylated methyl group), Lipid CO (carbon/oxygen), and XyG (xyloglucan). Following number refers to the associated ¹³C site number