Profiling the main cell wall polysaccharides of grapevine leaves using high-throughput and fractionation methods

Abstract

Vitis species include Vitis vinifera, the domesticated grapevine, used for wine and grape agricultural production and considered the world's most important fruit crop. A cell wall preparation, isolated from fully expanded photosynthetically active leaves, was fractionated via chemical and enzymatic reagents; and the various extracts obtained were assayed using high-throughput cell wall profiling tools according to a previously optimized and validated workflow. The bulk of the homogalacturonan-rich pectin present was efficiently extracted using CDTA treatment, whereas over half of the grapevine leaf cell wall consisted of vascular veins, comprised of xylans and cellulose. The main hemicellulose component was found to be xyloglucan and an enzymatic oligosaccharide fingerprinting approach was used to analyze the grapevine leaf xyloglucan fraction. When Paenibacillus sp. xyloglucanase was applied the main subunits released were XXFG and XLFG; whereas the less-specific Trichoderma reesei EGII was also able to release the XXXG motif as well as other oligomers likely of mannan and xylan origin. This latter enzyme would thus be useful to screen for xyloglucan, xylan and mannan-linked cell wall alterations in laboratory and field grapevine populations. This methodology is well-suited for high-throughput cell wall profiling of grapevine mutant and transgenic plants for investigating the range of biological processes, specifically plant disease studies and plant-pathogen interactions, where the cell wall plays a crucial role.

Keywords: AIR; CBM; Cell wall polysaccharides; CoMPP; EPG; ESI-MS; Endopolygalacturonase (EPG); FT-IR; Fourier transform-infrared spectroscopy; Grapevine profiling; HG; HPAEC-PAD; Paenibacillus sp xyloglucan specific endoglucanase 5; Pectin-rich; PspXEG5; RG; TrEGII; Tricshoderma reesei endoglucanase II; XEG; XyG; Xyloglucan; Xyloglucan-specific endoglucanase (XEG); alcohol insoluble residue; carbohydrate binding module; comprehensive microarray polymer profiling; electrospray ionization-mass spectrometry; endopolygalacturonase; high performance anion exchange chromatography-pulse amperometric detection; homogalacturonan; mAb; monoclonal antibody; rhamnogalacturonan; xyloglucan; xyloglucan-specific endoglucanase.