Defining natural factors that stimulate and inhibit cellulose:xyloglucan hetero-transglucosylation.

Certain transglucanases can covalently graft cellulose and mixed-linkage ß-glucan (MLG) as donor substrates onto xyloglucan as acceptor substrate and thus exhibit cellulose:xyloglucan endotransglucosylase (CXE) and MLG:xyloglucan endotransglucosylase (MXE) activities in vivo and in vitro. However, missing information on factors that stimulate or inhibit these hetero-transglucosylation reactions limits our insight into their biological functions. To explore factors that influence hetero-transglucosylation, we studied Equisetum fluviatile hetero-trans-ß-glucanase (EfHTG), which exhibits both CXE and MXE activity, exceeding its xyloglucan:xyloglucan homo-transglucosylation (XET) activity. Enzyme assays employed radiolabelled and fluorescently labelled oligomeric acceptor substrates, and were conducted in vitro and in cell walls (in situ). With whole denatured Equisetum cell walls as donor substrate, exogenous EfHTG (extracted from Equisetum or produced in Pichia) exhibited all three activities (CXE, MXE, XET) in competition with each other. Acting on pure cellulose as donor substrate, the CXE action of Pichia-produced EfHTG was up to approximately 300% increased by addition of methanol-boiled Equisetum extracts; there was no similar effect when the same enzyme acted on soluble donors (MLG or xyloglucan). The methanol-stable factor is proposed to be expansin-like, a suggestion supported by observations of pH dependence. Screening numerous low-molecular-weight compounds for hetero-transglucanase inhibition showed that cellobiose was highly effective, inhibiting the abundant endogenous CXE and MXE (but not XET) action in Equisetum internodes. Furthermore, cellobiose retarded Equisetum stem elongation, potentially owing to its effect on hetero-transglucosylation reactions. This work provides insight and tools to further study the role of cellulose hetero-transglucosylation in planta by identifying factors that govern this reaction.

Hetero-trans-ß-Glucanase Produces Cellulose-Xyloglucan Covalent Bonds in the Cell Walls of Structural Plant Tissues and Is Stimulated by Expansin.

Current cell-wall models assume no covalent bonding between cellulose and hemicelluloses such as xyloglucan or mixed-linkage ß-d-glucan (MLG). However, Equisetum hetero-trans-ß-glucanase (HTG) grafts cellulose onto xyloglucan oligosaccharides (XGOs) - and, we now show, xyloglucan polysaccharide - in vitro, thus exhibiting CXE (cellulose:xyloglucan endotransglucosylase) activity. In addition, HTG also catalyzes MLG-to-XGO bonding (MXE activity). In this study, we explored the CXE action of HTG in native plant cell walls and tested whether expansin exposes cellulose to HTG by disrupting hydrogen bonds. To quantify and visualize CXE and MXE action, we assayed the sequential release of HTG products from cell walls pre-labeled with substrate mimics. We demonstrated covalent cellulose-xyloglucan bonding in plant cell walls and showed that CXE and MXE action was up to 15% and 60% of total transglucanase action, respectively, and peaked in aging, strengthening tissues: CXE in xylem and cells bordering intercellular canals and MXE in sclerenchyma. Recombinant bacterial expansin (EXLX1) strongly augmented CXE activity in vitro. CXE and MXE action in living Equisetum structural tissues potentially strengthens stems, while expansin might augment the HTG-catalyzed CXE reaction, thereby allowing efficient CXE action in muro. Our methods will enable surveys for comparable reactions throughout the plant kingdom. Furthermore, engineering similar hetero-polymer formation into angiosperm crop plants may improve certain agronomic traits such as lodging tolerance.

Bonds broken and formed during the mixed-linkage glucan : xyloglucan endotransglucosylase reaction catalysed by Equisetum hetero-trans-ß-glucanase.

Mixed-linkage glucan∶xyloglucan endotransglucosylase (MXE) is one of the three activities of the recently characterised hetero-trans-ß-glucanase (HTG), which among land plants is known only from Equisetum species. The biochemical details of the MXE reaction were incompletely understood - details that would promote understanding of MXE's role in vivo and enable its full technological exploitation. We investigated HTG's site of attack on one of its donor substrates, mixed-linkage (1→3),(1→4)-ß-d-glucan (MLG), with radioactive oligosaccharides of xyloglucan as the acceptor substrate. Comparing three different MLG preparations, we showed that the enzyme favours those with a high content of cellotetraose blocks. The reaction products were analysed by enzymic digestion, thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC) and gel-permeation chromatography (GPC). Equisetum HTG consistently cleaved the MLG at the third consecutive ß-(1→4)-bond following (towards the reducing terminus) a ß-(1→3)-bond. It then formed a ß-(1→4)-bond between the MLG and the non-reducing terminal glucose residue of the xyloglucan oligosaccharide, consistent with its xyloglucan endotransglucosylase/hydrolase subfamily membership. Using size-homogeneous barley MLG as the donor substrate, we showed that HTG does not favour any particular region of the MLG chain relative to the polysaccharide's reducing and non-reducing termini; rather, it selects its target cellotetraosyl unit stochastically along the MLG molecule. This work improves our understanding of how enzymes can exhibit promiscuous substrate specificities and provides the foundations to explore strategies for engineering novel substrate specificities into transglycanases.

Purification, cDNA cloning, and characterization of LysM-containing plant chitinase from horsetail (Equisetum arvense).

Chitinase-A (EaChiA), molecular mass 36 kDa, was purified from the vegetative stems of a horsetail (Equisetum arvense) using a series of column chromatography. The N-terminal amino acid sequence of EaChiA was similar to the lysin motif (LysM). A cDNA encoding EaChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1320 nucleotides and encoded an open reading frame of 361 amino acid residues. The deduced amino acid sequence indicated that EaChiA is composed of a N-terminal LysM domain and a C-terminal plant class IIIb chitinase catalytic domain, belonging to the glycoside hydrolase family 18, linked by proline-rich regions. EaChiA has strong chitin-binding activity, however, no antifungal activity. This is the first report of a chitinase from Equisetopsida, a class of fern plants, and the second report of a LysM-containing chitinase from a plant.

Mixed-linkage glucan:xyloglucan endotransglucosylase (MXE) re-models hemicelluloses in Equisetum shoots but not in barley shoots or Equisetum callus.

Among land-plant hemicelluloses, xyloglucan is ubiquitous, whereas mixed-linkage (1→3),(1→4)-ß-D-glucan (MLG) is confined to the Poales (e.g. cereals) and Equisetales (horsetails). The enzyme MLG:xyloglucan endotransglucosylase (MXE) grafts MLG to xyloglucan. In Equisetum, MXE often exceeds extractable xyloglucan endotransglucosylase (XET) activity; curiously, cereals lack extractable MXE. We investigated whether barley possesses inextractable MXE. Grafting of endogenous MLG or xyloglucan onto exogenous [(3)H]xyloglucan oligosaccharides in vivo indicated MXE and XET action, respectively. Extractable MXE and XET activities were assayed in vitro. MXE and XET actions were both detectable in living Equisetum fluviatile shoots, the MXE : XET ratio increasing with age. However, only XET action was observed in barley coleoptiles, leaves and roots (which all contained MLG) and in E. fluviatile intercalary meristems and callus (which lacked MLG). In E. fluviatile, extractable MXE activity was high in mature shoots, but extremely low in callus and young shoots; in E. arvense strobili, it was undetectable. Barley possesses neither extractable nor inextractable MXE, despite containing both of its substrates and high XET activity. As the Poales are xyloglucan-poor, the role of their abundant endotransglucosylases remains enigmatic. The distribution of MXE action and activity within Equisetum suggests a strengthening role in ageing tissues.

Three 2-oxoglutarate-dependent dioxygenase activities of Equisetum arvense L. forming flavone and flavonol from (2S)-naringenin.

Equisetum arvense L. (Equisetaceae-horsetail) accumulates various flavones and flavonols in infertile shoot. Enzyme assays conducted with crude extracts of the green tissue revealed chalcone synthase activity and also three further activities assigned to flavonoid biosynthesis and identified as flavone synthase I, flavanone 3ß-hydroxylase and flavonol synthase. The latter three activities were characterized as soluble, 2-oxoglutarate-dependent dioxygenases by their typical cofactor requirements and peculiar inhibition. Notably, this is the first report of flavone synthase I which had been considered to be restricted solely to species of the Apiaceae from a distant plant taxon.

Exploring Equisetum arvense L., Equisetum ramosissimum L. and Equisetum telmateia L. as sources of natural antioxidants.

The antioxidant and scavenging activities of above ground parts of Equisetum arvense L., Equisetum ramosissimum L. and Equisetum telmateia L. phosphate buffer (pH 7) extracts were investigated. Activities of antioxidant enzymes (superoxide dismutase, catalase, guaiacol peroxidase and glutathione peroxidase), quantities of reduced glutathione, malonyldialdehyde, superoxide and hydroxyl radicals and flavonoid, soluble protein, chlorophyll a, b and carotenoid contents were determined. The total antioxidant capacity was determined by ferric reducing antioxidant power (FRAP) assay. The Equisetum telmateia extract demonstrated scavenging and antioxidant properties better than Equisetum ramosissimum and Equisetum arvense. The ESR signal of DMPO-OH radical adducts in the presence of Equisetum telmateia phosphate buffer (pH 7) extract was reduced by 98.9% indicating that Equisetum telmateia could be a useful source of antioxidants with huge scavenging ability.

Mixed-linkage beta-glucan : xyloglucan endotransglucosylase, a novel wall-remodelling enzyme from Equisetum (horsetails) and charophytic algae.

Mixed-linkage (1-->3,1-->4)-beta-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) approximately 4 microM) 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, MLG-to-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.

Diverse chalcone synthase superfamily enzymes from the most primitive vascular plant, Psilotum nudum.

Psilotum nudum Griseb is a pteridophyte and belongs to the single family (Psilotaceae) of the division, Psilophyta. Being the only living species of a once populated division, P. nudum is the most primitive vascular plant. Chalcone synthase (CHS; EC 2.3.1.74) superfamily enzymes are responsible for biosyntheses of diverse secondary metabolites, including flavonoids and stilbenes. Using a reverse transcription-polymerase chain reaction strategy, four CHS-superfamily enzymes (PnJ, PnI, PnL and PnP) were cloned from P. nudum, and heterologously expressed in Escherichia coli. These four enzymes of 396-406 amino acids showed sequence identity of > 50% among themselves and to other higher-plant CHS-superfamily enzymes. PnJ and PnP preferred p-coumaroyl-CoA and isovaleryl-CoA respectively, as starter CoA and catalyzed CHS-type ring formation, indicating that they are CHS and phlorisovalerophenone synthase, respectively. On the other hand, PnI and PnL preferred cinnamoyl-CoA as starter CoA and catalyzed stilbene synthase-type cyclization and thus were determined to be pinosylvin synthases (EC 2.3.1.146). In addition, PnE, which uniquely contains a glutamine in place of otherwise strictly conserved histidine, had no apparent in vitro catalytic activity. Phylogenetic analysis indicated that these P. nudum clones form a separate cluster together with Equisetum arvense CHS. This cluster of pteridophytes is located next to the cluster formed by pine (gymnosperm) enzymes, in agreement with their evolutionary relationships. Psilotum nudum represents a plant with the most diverse CHS-superfamily enzymes and this ability to diverge may have provided a survival edge during evolution.