Summary• Mixed-linkage (1→3,1→4)-β-D-glucan (MLG) is a hemicellulose reputedly confined to certain Poales. Here, the taxonomic distribution of MLG, and xyloglucan, especially in early-diverging pteridophytes, has been re-investigated.• Polysaccharides were digested with lichenase and xyloglucan endoglucanase (XEG), which specifically hydrolyse MLG and xyloglucan, respectively. The oligosaccharides produced were analysed by thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC) and alkaline peeling.• Lichenase yielded oligo-β-glucans from all Equisetum species tested (Equisetum arvense, Equisetum fluviatile, Equisetum scirpoides, Equisetum sylvaticum and Equisetum × trachyodon). The major product was the tetrasaccharide β-glucosyl-(1→4)-β-glucosyl-(1→4)-β-glucosyl-(1→3)-glucose (G4G4G3G), which was converted to cellotriose by alkali, confirming its structure. Minor products included G3G, G4G3G and a nonasaccharide. By contrast, poalean MLGs yielded G4G3G > G4G4G3G > nonasaccharide > dodecasaccharide. No other pteridophytes tested contained MLG, including Psilotum and eusporangiate ferns. No MLG was found in lycopodiophytes, bryophytes, Chara or Nitella. XEG digestion showed that Equisetum xyloglucan has unusual repeat units.• Equisetum, an exceedingly isolated genus whose closest living relatives diverged > 380 million years ago, has evolved MLG independently of the Poales. Equisetum and poalean MLGs share basic structural motifs but also exhibit clear-cut differences. Equisetum MLG is firmly wall-bound, and may tether neighbouring microfibrils. It is also suggested that MLG acts as a template for silica deposition, characteristic of grasses and horsetails.

SummaryMixed-linkage (1 fi 3,1 fi 4)-b-D-glucan (MLG), a hemicellulose long thought to be confined to certain Poales, was recently also found in Equisetum; xyloglucan occurs in all land plants. We now report that Equisetum possesses MLG:xyloglucan endotransglucosylase (MXE), which is a unique enzyme that grafts MLG to xyloglucan oligosaccharides (e.g. the heptasaccharide XXXGol). MXE occurs in all Equisetum species tested (Equisetum arvense, Equisetum fluviatile, Equisetum hyemale, Equisetum scirpoides, Equisetum telmateia and Equisetum variegatum), sometimes exceeding xyloglucan endotransglucosylase (XET) activity. Charophytic algae, especially Coleochaete, also possess MXE, which may therefore have been a primordial feature of plant cell walls. However, MXE was negligible in XET-rich extracts from grasses, dicotyledons, ferns, Selaginella and bryophytes. This and the following four additional observations indicate that MXE activity is not the result of a conventional xyloglucan endotransglucosylase/hydrolase (XTH): (i) XET, but not MXE, activity correlates with the reaction rate on water-soluble cellulose acetate, hydroxyethylcellulose and carboxymethylcellulose, (ii) MXE and XET activities peak in old and young Equisetum stems, respectively, (iii) MXE has a higher affinity for XXXGol (K m $ 4 lM) than any known XTH, (iv) MXE and XET activities differ in their oligosaccharide acceptor-substrate preferences. High-molecular-weight (M r ) xyloglucan strongly competes with [ 3 H]XXXGol as the acceptor-substrate of MXE, whereas MLG oligosaccharides are poor acceptor-substrates. Thus, MLGto-xyloglucan grafting appears to be the favoured activity of MXE. In conclusion, Equisetum has evolved MLG plus MXE, potentially a unique cell wall remodelling mechanism. The prominence of MXE in mature stems suggests a strengthening/repairing role. We propose that cereals, which possess MLG but lack MXE, might be engineered to express this Equisetum enzyme, thereby enhancing the crop mechanical properties.