Equisetum arvense as a silica fertilizer.

The aim was to use the agricultural weed and silica (Si) hyperaccumulator Equisetum arvense as Si fertilizer in plant cultivation. We investigated (1) the Si uptake in various Equisetum species, (2) where Si accumulates in the Equisetum plant, (3) processing methods to release as much Si as possible from dried, ground E. arvense plants and (4) which treatment yields gives the highest uptake of Si in young wheat plants cultivated in soil containing ground E. arvense. The results showed that E. arvense containes 22% Si and was among the best Si accumulators. Equisetum arvense accumulates Si as both soluble and firmly bound fractions. Amorphous silica (SiO2) accumulates in the outer cell walls of epidermis of the entire plant. Regarding the processing method, a longer treatment time, greater concentration of Equisetum, boiling, and the addition of sodium bicarbonate increased the Si availability in ground, dried E. arvense. The addition of untreated, ground, dried E. arvense to the soil, corresponding to 160 kg Si ha-1, increased the available Si in the soil and the Si uptake in wheat plants by five-fold, compared with the control. Boiling the ground E. arvense increased the Si uptake by 10 times, and the of sodium bicarbonate increased the availability and uptake by 40 times, compared with the control. In conclusion, dried, ground E. arvense can be used as a Si fertilizer as is, after boiling for a slightly better effect, or with sodium bicarbonate (up to a similar amount as the ground material) for best effect.

Equisetum praealtum and E. hyemale have abundant Rubisco with a high catalytic turnover rate and low CO2 affinity.

The kinetic properties of Rubisco, a key enzyme for photosynthesis, have been examined in numerous plant species. However, this information on some plant groups, such as ferns, is scarce. This study examined Rubisco carboxylase activity and leaf Rubisco levels in seven ferns, including four Equisetum plants (E. arvense, E. hyemale, E. praealtum, and E. variegatum), considered living fossils. The turnover rates of Rubisco carboxylation (kcatc) in E. praealtum and E. hyemale were comparable to those in the C4 plants maize (Zea mays) and sorghum (Sorghum bicolor), whose kcatc values are high. Rubisco CO2 affinity, estimated from the percentage of Rubisco carboxylase activity under CO2 unsaturated conditions in kcatc in these Equisetum plants, was low and also comparable to that in maize and sorghum. In contrast, kcatc and CO2 affinities of Rubisco in other ferns, including E. arvense and E. variegatum were comparable with those in C3 plants. The N allocation to Rubisco in the ferns examined was comparable to that in the C3 plants. These results indicate that E. praealtum and E. hyemale have abundant Rubisco with high kcatc and low CO2 affinity, whereas the carboxylase activity and abundance of Rubisco in other ferns were similar to those in C3 plants. Herein, the Rubisco properties of E. praealtum and E. hyemale were discussed regarding their evolution and physiological implications.

Apocarotenoids from Equisetum debile Roxb. ex Vaucher regulate the lipid metabolism via the activation of the AMPK/ACC/SREBP-1c signaling pathway.

Sixteen undescribed apocarotenoids (1-16), along with 22 known analogues, were isolated from the aerial parts of Equisetum debile. Their structures, including absolute configurations, were elucidated by NMR, HRESIMS, X-ray diffraction analysis, the modified Mosher's method and the quantum-chemical calculation of electronic circular dichroism (ECD) spectra. Compounds 1-9, 11-12 are the first example of C16-apocarotenoids appeared in nature. The plausible biosynthetic pathway of 1-16 was proposed. Moreover, the isolates were evaluated for their lipid-lowering activity, and the results showed that 13, 14, 15, 22, 31, 32 and 33 could remarkably decrease the levels of both TC and TG in FFA induced HepG2 cells at 20 µM. The oil red staining assay further demonstrated the lipid-lowering effects of 13, 14 and 15. The western blot results indicated that compounds 13, 14 and 15 could regulate the lipid metabolism via the activation of the AMPK/ACC/SREBP-1c signaling pathway. A preliminary structure-activity relationship (SAR) study of the isolates indicated that the apocarotenoids with 6/5 ring system displayed more potent lipid-lowering effects.

Subcritical water extraction of Equisetum arvense biomass withdraws cell wall fractions that trigger plant immune responses and disease resistance.

Plant cell walls are complex structures mainly made up of carbohydrate and phenolic polymers. In addition to their structural roles, cell walls function as external barriers against pathogens and are also reservoirs of glycan structures that can be perceived by plant receptors, activating Pattern-Triggered Immunity (PTI). Since these PTI-active glycans are usually released upon plant cell wall degradation, they are classified as Damage Associated Molecular Patterns (DAMPs). Identification of DAMPs imply their extraction from plant cell walls by using multistep methodologies and hazardous chemicals. Subcritical water extraction (SWE) has been shown to be an environmentally sustainable alternative and a simplified methodology for the generation of glycan-enriched fractions from different cell wall sources, since it only involves the use of water. Starting from Equisetum arvense cell walls, we have explored two different SWE sequential extractions (isothermal at 160 ºC and using a ramp of temperature from 100 to 160 ºC) to obtain glycans-enriched fractions, and we have compared them with those generated with a standard chemical-based wall extraction. We obtained SWE fractions enriched in pectins that triggered PTI hallmarks in Arabidopsis thaliana such as calcium influxes, reactive oxygen species production, phosphorylation of mitogen activated protein kinases and overexpression of immune-related genes. Notably, application of selected SWE fractions to pepper plants enhanced their disease resistance against the fungal pathogen Sclerotinia sclerotiorum. These data support the potential of SWE technology in extracting PTI-active fractions from plant cell wall biomass containing DAMPs and the use of SWE fractions in sustainable crop production.

Advances in Cell Wall Matrix Research with a Focus on Mixed-Linkage Glucan.

Mixed ß(1,3;1,4)-linkage glucan (MLG) is commonly found in the monocot lineage, at particularly high levels in the Poaceae family, but also in the evolutionally distant genus, Equisetum. MLG has several properties that make it unique from other plant cell wall polysaccharides. It consists of ß1,4-linked polymers of glucose interspersed with ß1,3-linkages, but the presence of ß1,3-linkages provides quite different physical properties compared to its closest form of the cell wall component, cellulose. The mechanisms of MLG biosynthesis have been investigated to understand whether single or multiple enzymes are required to build mixed linkages in the glucan chain. Currently, MLG synthesis by a single enzyme is supported by mutagenesis analyses of cellulose synthase-like F6, the major MLG synthase, but further investigation is needed to gather mechanistic insights. Because of transient accumulation of MLG in elongating cells and vegetative tissues, several hypotheses have been proposed to explain the role of MLG in the plant cell wall. Studies have been carried out to identify gene expression regulators during development and light cycles as well as enzymes involved in MLG organization in the cell wall. A role of MLG as a storage molecule in grains is evident, but the role of MLG in vegetative tissues is still not well understood. Characterization of a cell wall component is difficult due to the complex heterogeneity of the plant cell wall. However, as detailed in this review, recent exciting research has made significant impacts in the understanding of MLG biology in plants.

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.

Three highly acidic Equisetum XTHs differ from hetero-trans-ß-glucanase in donor substrate specificity and are predominantly xyloglucan homo-transglucosylases.

Transglycanases are enzymes that remodel the primary cell wall in plants, potentially loosening and/or strengthening it. Xyloglucan endotransglucosylase (XET; EC 2.4.1.207), ubiquitous in land plants, is a homo-transglucanase activity (donor, xyloglucan; acceptor, xyloglucan) exhibited by XTH (xyloglucan endotransglucosylase/hydrolase) proteins. By contrast, hetero-trans-ß-glucanase (HTG) is the only known enzyme that is preferentially a hetero-transglucanase. Its two main hetero-transglucanase activities are MLG : xyloglucan endotransglucosylase (MXE) and cellulose : xyloglucan endotransglucosylase (CXE). HTG is highly acidic and found only in the evolutionarily isolated genus of fern-allies, Equisetum. We now report genes for three new highly acidic HTG-related XTHs in E. fluviatile (EfXTH-A, EfXTH-H and EfXTH-I). We expressed them heterologously in Pichia and tested the encoded proteins' enzymic activities to determine whether their acidity and/or their Equisetum-specific sequences might confer high hetero-transglucanase activity. Untransformed Pichia was found to secrete MLG-degrading enzyme(s), which had to be removed for reliable MXE assays. All three acidic EfXTHs exhibited very predominantly XET activity, although low but measurable hetero-transglucanase activities (MXE and CXE) were also detected in EfXTH-H and EfXTH-I. We conclude that the extremely high hetero-transglucanase activities of Equisetum HTG are not emulated by similarly acidic Equisetum XTHs that share up to 55.5% sequence identity with HTG.

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.

Rough and tough. How does silicic acid protect horsetail from fungal infection?

Horsetail (Equisetum arvense) plants grew healthily for 10 weeks under both Si-deficient and Si-replete conditions. After 10 weeks, plants grown under Si-deficient conditions succumbed to fungal infection. We have used NanoSIMS and fluorescence microscopy to investigate silica deposition in the tissues of these plants. Horsetail grown under Si-deficient conditions did not deposit identifiable amounts of silica in their tissues. Plants grown under Si-replete conditions accumulated silica throughout their tissues and especially in the epidermis of the outer side of the leaf and the furrow region of the stem where it was continuous and often, as a double layer suggestive of a barrier function. We have previously shown, both in vivo (in horsetail and thale cress) and in vitro (using an undersaturated solution of Si(OH)4), that callose is a "catalyst" of plant silica deposition. Here we support this finding by comparing the deposition of silica to that of callose and by showing that they are co-localized. We propose the existence of a synergistic mechanical protection by callose and silica against pathogens in horsetail, whereby the induction of callose synthesis and deposition is the first, biochemical line of defence and callose-induced precipitation of silica is the second, adventitious mechanical barrier.

Identification and characterization of silicon efflux transporters in horsetail (Equisetum arvense).

Silicon (Si) is a beneficial element to plants, and its absorption via transporters leads to protective effects against biotic and abiotic stresses. In higher plants, two groups of root transporters for Si have been identified: influx transporters (Lsi1) and efflux transporters (Lsi2). Lsi1 transporters belong to the NIPIII aquaporins, and functional Lsi1s have been found in many plants species. Much less is known about Lsi2s that have been characterized in only a few species. Horsetail (Equisetum arvense), known among the highest Si accumulators in the plant kingdom, is a valuable model to study Si absorption and deposition. In this study, we first analyzed discrete Si deposition patterns in horsetail shoots, where ubiquitous silicification differs markedly from that of higher plants. Then, using the sequenced horsetail root transcriptome, two putative Si efflux transporter genes, EaLsi2-1 and EaLsi2-2, were identified. These genes share low sequence similarity with their homologues in higher plants. Further characterisation of EaLsi2-1 in transient expression assay using Nicotiana benthamiana epidermal cells confirmed transmembrane localization. In order to determine their functionality, the EaLsi2-1 was expressed in Xenopus oocytes, confirming that the translated protein was efficient for Si efflux. Both genes were equally expressed in roots and shoots, but interestingly, showed a much higher expression in the shoots than in the roots in contrast to Lsi2s found in other plants, a result consistent with the specific anatomy of horsetail and its rank as one of the highest Si accumulators among plant species.

Hetero-trans-ß-glucanase, an enzyme unique to Equisetum plants, functionalizes cellulose.

Cell walls are metabolically active components of plant cells. They contain diverse enzymes, including transglycanases (endotransglycosylases), enzymes that 'cut and paste' certain structural polysaccharide molecules and thus potentially remodel the wall during growth and development. Known transglycanase activities modify several cell-wall polysaccharides (xyloglucan, mannans, mixed-linkage ß-glucan and xylans); however, no transglycanases were known to act on cellulose, the principal polysaccharide of biomass. We now report the discovery and characterization of hetero-trans-ß-glucanase (HTG), a transglycanase that targets cellulose, in horsetails (Equisetum spp., an early-diverging genus of monilophytes). HTG is also remarkable in predominantly catalysing hetero-transglycosylation: its preferred donor substrates (cellulose or mixed-linkage ß-glucan) differ qualitatively from its acceptor substrate (xyloglucan). HTG thus generates stable cellulose-xyloglucan and mixed-linkage ß-glucan-xyloglucan covalent bonds, and may therefore strengthen ageing Equisetum tissues by inter-linking different structural polysaccharides of the cell wall. 3D modelling suggests that only three key amino acid substitutions (Trp → Pro, Gly → Ser and Arg → Leu) are responsible for the evolution of HTG's unique specificity from the better-known xyloglucan-acting homo-transglycanases (xyloglucan endotransglucosylase/hydrolases; XTH). Among land plants, HTG appears to be confined to Equisetum, but its target polysaccharides are widespread, potentially offering opportunities for enhancing crop mechanical properties, such as wind resistance. In addition, by linking cellulose to xyloglucan fragments previously tagged with compounds such as dyes or indicators, HTG may be useful biotechnologically for manufacturing stably functionalized celluloses, thereby potentially offering a commercially valuable 'green' technology for industrially manipulating biomass.

Fluorescent analysis for bioindication of ozone on unicellular models.

Unicellular model plant systems (vegetative microspores of horsetail Equisetum arvense and pollen of six plant species Corylus avellana, Dolichothele albescens Populus balsamifera, Salix caprea, Saintpaulia ionantha, Tulipa hybridum, on which autofluorescence and fluorescence after histochemical treatment studied, have been represented as bioindicators of ozone. It has found that low doses of ozone 0.005 or 0.008 µl/l did not affect or stimulate the autofluorescence of the samples with the ability to germinate in an artificial medium. In higher ozone concentrations (0.032 µl/l) either the decrease in the intensity of the emission or changing in the position of the maxima in the fluorescence spectrum (new 515-520 nm maximum characteristic for the green-and yellow area has appeared) were observed. In dose of 0.2 µl/l, higher than above the threshold of danger to human health, autofluorescence in all samples fell down to up to zero, and there was no the ability to germinate. In this case the formation of lipofuscin-like compounds fluoresced in blue with maxima from 440 to 485 nm was observed. Stress metabolites, known as neurotransmitters biogenic amines, were found in treated cells as determined on the characteristic fluorescence at 460-480 nm in the samples after a specific histochemical reactions for catecholamines (with glyoxylic acid) or for histamine (with o-phthalic aldehyde). Increased intensity of the emission under the treatment with ozone (total doses from 0.012 to 0.032 µl/l) was associated with an increase in the concentrations of catecholamines and histamine. The fluorescent analysis on undamaged cells-possible bioindicators of ozone can be useful in ecomonitoring for earlier warning about health hazardous concentrations of this compound in the air.

Comparative proteomic analysis of developing rhizomes of the ancient vascular plant Equisetum hyemale and different monocot species.

The rhizome is responsible for the invasiveness and competitiveness of many plants with great economic and agricultural impact worldwide. Besides its value as an invasive organ, the rhizome plays a role in the establishment and massive growth of forage, providing biomass for biofuel production. Despite these features, little is known about the molecular mechanisms that contribute to rhizome growth, development, and function in plants. In this work, we characterized the proteome of rhizome apical tips and elongation zones from different species using a GeLC-MS/MS (one-dimensional electrophoresis in combination with liquid chromatography coupled online with tandem mass spectrometry) spectral-counting proteomics strategy. Five rhizomatous grasses and an ancient species were compared to study the protein regulation in rhizomes. An average of 2200 rhizome proteins per species were confidently identified and quantified. Rhizome-characteristic proteins showed similar functional distributions across all species analyzed. The over-representation of proteins associated with central roles in cellular, metabolic, and developmental processes indicated accelerated metabolism in growing rhizomes. Moreover, 61 rhizome-characteristic proteins appeared to be regulated similarly among analyzed plants. In addition, 36 showed conserved regulation between rhizome apical tips and elongation zones across species. These proteins were preferentially expressed in rhizome tissues regardless of the species analyzed, making them interesting candidates for more detailed investigative studies about their roles in rhizome development.

Effective half-lives of ¹³7Cs in giant butterbur and field horsetail, and the distribution differences of potassium and ¹³7Cs in aboveground tissue parts.

Concentrations of (137)Cs and (40)K in different tissues of edible wild herbaceous plants, that is, leaf blade and petiole for giant butterbur (Petasites japonicas (Siebold et Zucc.) Maxim.), and leaf, stem and strobilus for fertile shoot of field horsetail (Equisetum arvense L.) were measured in 2012-2014 to clarify the effect in Japan from the Fukushima Daiichi Nuclear Power Plant accident. The concentrations of (137)Cs decreased with time with effective half-lives of ca. 450 d and 360 d for giant butterbur and field horsetail, respectively. The ANOVA test revealed that (40)K and (137)Cs distributions in leaf blade and petiole for giant butterbur and leaf and stem for field horsetail were different. Therefore, other plants, leaf and stem for Japanese knotweed (Fallopia japonica (Houtt.) Ronse Decr.) and Canada goldenrod (Solidago canadensis L.), and leaf blade and petiole for gingko (Ginkgo biloba L.) and Someiyoshino cherry (Cerasus × yedoensis (Matsum.) A.V.Vassil. 'Somei-yoshino') were collected from the same sampling field and their (137)Cs and (40)K concentrations were compared to those in the giant butterbur and field horsetail parts. For (137)Cs, concentrations in leaf blade and leaf parts were 1.1-6.0 times higher than those in petiole and stem parts for all six plants. On the other hand, (40)K concentrations in leaf blade and leaf parts were 0.40-0.97 of those observed in petiole and stem parts. Discrimination ratios of (40)K/(137)Cs of leaf blade to petiole or leaf to stem were then calculated and they ranged from 0.09 to 0.57. These results suggested that Cs and K did not behave similarly in these plants. Thus, to understand the radiocesium fate in plants, K measurement results should not be used as an analog for Cs behavior although Cs is known to have a similar chemical reactivity to that of K.

Bioatividade de extratos aquosos de plantas medicinais em sementes de feijão-fava / Behavioral and physiological health of lima bean seeds subjected to aqueous extract of medicinal plants

O objetivo do presente estudo foi avaliar a bioatividade de extratos aquosos de plantas medicinais em sementes de Phaseolus lunatus L. (feijão-fava) via comportamento fisiológico e fitossanitário. Foram utilizadas sementes de feijão-fava da variedade Anduzinha tratadas com seis extratos aquosos de plantas medicinais a 5% (Ocimun gratissimum, Plectranthus neachilus, Vernonia condensata, Cymbopogom citratus, Equisetum sp., e Piper aduncum L.), juntamente com a testemunha (água destilada). A bioatividade foi determinada pelo comportamento fisiológico e sanitário avaliados por meio dos testes de germinação, primeira contagem de germinação, índice de velocidade de germinação, comprimento de raiz na primeira e última contagem, e teste de sanidade. Realizou-se a análise de variância e teste Tukey a 5% de probabilidade. O extrato aquoso de cavalinha (Equisetum sp.) promoveu a melhor qualidade fisiológica das sementes de feijão-fava. Houve maior incidência de fungos nas sementes de feijão-fava que receberam o extrato de boldinho (Plectranthus neachilus).

The aim of this study was to evaluate the bioactivitt of medicinal lants aqueous extracts in Phaseolus lunatus L. (lima bean) s via through physiological and behavioral health. We the llima bean seeds of the variety Anduzinha ins of medicinal lants aqueous extracs ato 5% (Ocimun gratissimum, Plectranthus neachilus, Vernonia condensata, Cymbopogom citratus, Equisetum sp. and Piper aduncum L.) together with the control (distilled water). The bioactivity was determined by physiological and sanitary qutyies reviews through germination, first counting of germination, speed of germination, root length in the first and last counting, and sanity check. We carried out the analysis of variance and the Tukey test at 5% probability. The aqueous extract of Equisetum sp. promoted beseed physiological quality of the lima bean seed. There was a higher incidence of fungs of lima bean sract that received the extract of Plectranthus neacilus).

Abundance of mixed linkage glucan in mature tissues and secondary cell walls of grasses.

(1,3; 1,4)-ß-D-glucan, also known as mixed linkage glucan (MLG), is a polysaccharide that in flowering plants is unique to the cell walls of grasses and other related members of Poales. MLG is highly abundant in endosperm cell walls, where it is considered a storage carbohydrate. In vegetative tissues, MLG transiently accumulates in the primary cell walls of young, elongating organs. In evolutionary distant species such as Equisetum, MLG accumulates predominantly in old tissues in the stems. Similarly, we have recently shown that rice accumulates a large amount of MLG in mature stems, which prompted us to re-evaluate the hypothesis that MLG is solely related to growth in grass vegetative tissues. Here, we summarize data that confirms the presence of MLG in secondary cell walls and mature tissues in rice and other grasses. Along with these results, we discuss additional evidence indicating a broader role for MLG than previously considered.

Discovery of a multigene family of aquaporin silicon transporters in the primitive plant Equisetum arvense.

Plants benefit greatly from silicon (Si) absorption provided that they contain Si transporters. The latter have recently been identified in the roots of some higher plants known to accumulate high concentrations of Si, and all share a high level of sequence identity. In this study, we searched for transporters in the primitive vascular plant Equisetum arvense (horsetail), which is a valuable but neglected model plant for the study of Si absorption, as it has one of the highest Si concentrations in the plant kingdom. Our initial attempts to identify Si transporters based on sequence homology with transporters from higher plants proved unsuccessful, suggesting a divergent structure or property in horsetail transporters. Subsequently, through sequencing of the horsetail root transcriptome and a search using amino acid sequences conserved in plant aquaporins, we were able to identify a multigene family of aquaporin Si transporters. Comparison of known functional domains and phylogenetic analysis of sequences revealed that the horsetail proteins belong to a different group than higher-plant Si transporters. In particular, the newly identified proteins contain a STAR pore as opposed to the GSGR pore common to all previously identified Si transporters. In order to determine its functionality, the proteins were heterologously expressed in both Xenopus oocytes and Arabidopsis, and the results showed that the horsetail proteins are extremely efficient a transporting Si. These findings offer new insights into the elusive properties of Si and its absorption by plants.

Evolution of mixed-linkage (1 -> 3, 1 -> 4)-ß-D-glucan (MLG) and xyloglucan in Equisetum (horsetails) and other monilophytes.

BACKGROUND AND AIMS: Horsetails (Equisetopsida) diverged from other extant eusporangiate monilophytes in the Upper Palaeozoic. They are the only monilophytes known to contain the hemicellulose mixed-linkage (1 → 3, 1 → 4)-ß-d-glucan (MLG), whereas all land plants possess xyloglucan. It has been reported that changes in cell-wall chemistry often accompanied major evolutionary steps. We explored changes in hemicelluloses occurring during Equisetum evolution. METHODS: Hemicellulose from numerous monilophytes was treated with lichenase and xyloglucan endoglucanase. Lichenase digests MLG to di-, tri- and tetrasaccharide repeat-units, resolvable by thin-layer chromatography. KEY RESULTS: Among monilophytes, MLG was confined to horsetails. Our analyses support a basal trichotomy of extant horsetails: MLG was more abundant in subgenus Equisetum than in subgenus Hippochaete, and uniquely the sister group E. bogotense yielded almost solely the tetrasaccharide repeat-unit (G4G4G3G). Other species also gave the disaccharide, whereas the trisaccharide was consistently very scarce. Tetrasaccharide : disaccharide ratios varied interspecifically, but with no consistent difference between subgenera. Xyloglucan was scarce in Psilotum and subgenus Equisetum, but abundant in subgenus Hippochaete and in the eusporangiate ferns Marattia and Angiopteris; leptosporangiate ferns varied widely. All monilophytes shared a core pattern of xyloglucan repeat-units, major XEG products co-chromatographing on thin-layer chromatography with non-fucosylated hepta-, octa- and nonasaccharides and fucose-containing nona- and decasaccharides. CONCLUSIONS: G4G4G3G is the ancestral repeat-unit of horsetail MLG. Horsetail evolution was accompanied by quantitative and qualitative modification of MLG; variation within subgenus Hippochaete suggests that the structure and biosynthesis of MLG is evolutionarily plastic. Xyloglucan quantity correlates negatively with abundance of other hemicelluloses; but qualitatively, all monilophyte xyloglucans conform to a core pattern of repeat-unit sizes.

Cell wall polysaccharides from fern leaves: evidence for a mannan-rich Type III cell wall in Adiantum raddianum.

Primary cell walls from plants are composites of cellulose tethered by cross-linking glycans and embedded in a matrix of pectins. Cell wall composition varies between plant species, reflecting in some instances the evolutionary distance between them. In this work the monosaccharide compositions of isolated primary cell walls of nine fern species and one lycophyte were characterized and compared with those from Equisetum and an angiosperm dicot. The relatively high abundance of mannose in these plants suggests that mannans may constitute the major cross-linking glycan in the primary walls of pteridophytes and lycophytes. Pectin-related polysaccharides contained mostly rhamnose and uronic acids, indicating the presence of rhamnogalacturonan I highly substituted with galactose and arabinose. Structural and fine-structural analyses of the hemicellulose fraction of leaves of Adiantum raddianum confirmed this hypothesis. Linkage analysis showed that the mannan contains mostly 4-Man with very little 4,6-Man, indicating a low percentage of branching with galactose. Treatment of the mannan-rich fractions with endo-ß-mannanase produced characteristic mannan oligosaccharides. Minor amounts of xyloglucan and xylans were also detected. These data and those of others suggest that all vascular plants contain xyloglucans, arabinoxylans, and (gluco)mannans, but in different proportions that define cell wall types. Whereas xyloglucan and pectin-rich walls define Type I walls of dicots and many monocots, arabinoxylans and lower proportion of pectin define the Type II walls of commelinoid monocots. The mannan-rich primary walls with low pectins of many ferns and a lycopod indicate a fundamentally different wall type among land plants, the Type III wall.