Profiling the main cell wall polysaccharides of tobacco leaves using high-throughput and fractionation techniques

Abstract

Nicotiana species are used to study agriculturally and industrially relevant processes, but limited screening methods are available for this species. A tobacco leaf cell wall preparation was fractionated using both chemical and enzymatic methods; the fractions obtained were subsequently analysed using rapid high-throughput wall profiling tools. The results confirmed previous data showing that mature tobacco leaf cell walls are predominantly composed of pectic homogalacturonans with lesser amounts of hemicellulosic arabinoxyloglucan and glucuronoxylan polymers. This confirmation provided proof that the profiling methods could generate good-quality results and paves the way for high-throughput screening of tobacco mutants where a range of biological processes, where the cell wall profile is important, are studied. A novel enzymatic oligosaccharide fingerprinting method was optimized to rapidly analyse the structure of XXGG-rich arabinoxyloglucans characteristic of Solanaceae species such as tobacco. Digestion profiles using two available xyloglucanase-specific endoglucanases: Trichoderma reseei EGII and Paenibacillus sp. xyloglucanase were compared showing that the latter enzyme has a higher specificity toward tobacco arabinoxyloglucans, and is better-suited for screening. This methodology would be suitable for species, such as tomato (Solanum lycopersicum) or potato (Solanum tuberosum), with similar XXGG-type motifs in their xyloglucan structure.