Sampling cambial region and mature xylem for non structural carbohydrates and starch analyses

This paper describes a procedure to analyze non-structural carbohydrates (NSC) and starch in xylem and cambial region extracted by the scraping method. An application on woody logs of 2-year-old Populus×canadensis Mönch 'I-214' during tree-ring formation is reported. Samples are freeze-dried in liquid nitrogen and, successively, the bark is removed. After drying, the breaks between bark and xylem take place in correspondence of differentiating xylem cells. The cambial region with the differentiating phloem and xylem is gently scraped with a razor blade from the inner side of the bark and the outermost side of the stem, respectively. Additionally the mature xylem is milled until obtaining a powder. The amount of the cambial region collected by scraping changes with respect to the width of the differentiating zone (differentiating phloem and xylem), with higher amount of dry matter obtained during the maximum rate of xylem formation and high stem water content. After extraction of the powder in an ethanol solution and centrifugation, the supernatant and the resulting pellet are used for assessing the contents of non structural carbohydrates (NSC) and starch, respectively. NSC are determined using the High-Performance Liquid Chromatography (HPLC) analysis and starch by colorimetric method. In both cambial region and xylem, the major NSC are glucose and fructose which represented together more than 80% of the total NSC during wood formation. However, the total NSC are higher in cambial region compared to xylem, especially when the xylem and phloem are differentiating. The scraping technique provides the possibility to sample cambial region during the year, allowing the investigation of metabolic changes during tree-ring formation

Equisetum species show uniform epicuticular wax structures but diverse composition patterns

In the Equisetopsida, different wax distribution and composition patterns in the plant organs indicate a close relationship between wax structure and chemistry and the assimilatory function of these organs. Diverging wax compound classes show the two subgenera of Equisetum to be well separated

Application of mild autohydrolysis to facilitate the dissolution of wood chips in direct-dissolution solvents

Wood is not fully soluble in current non-derivatising direct-dissolution solvents, contrary to the many reports in the literature quoting wood 'dissolution' in ionic liquids. Herein, we demonstrate that the application of autohydrolysis, as a green and economical wood pre-treatment method, allows for a massive increase in solubility compared to untreated wood. This is demonstrated by the application of two derivitising methods (phosphitylation and acetylation), followed by NMR analysis, in the cellulose-dissolving ionic liquids 1-allyl-3-methylimidazolium chloride ([amim]Cl) and 1,5-diazabicyclo[4.3.0]non-5-enium acetate ([DBNH][OAc]. In addition, the non-derivitising tetrabutylphosphonium acetate ([P-4444][OAc]) : DMSO-d6 electrolyte also allowed for dissolution of the autohydrolysed wood samples. By combination of different particle sizes and P-factors (autohydrolysis intensity), it has been clearly demonstrated that the solubility of even wood chips can be drastically increased by application of autohydrolysis. The physiochemical factors affecting wood solubility after autohydrolysis are also discussed.Peer reviewe

Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis

The involvement of two R2R3-MYB genes from Pinus taeda L., PtMYB1 and PtMYB8, in phenylpropanoid metabolism and secondary cell wall biogenesis was investigated in planta. These pine MYBs were constitutively overexpressed (OE) in Picea glauca (Moench) Voss, used as a heterologous conifer expression system. Morphological, histological, chemical (lignin and soluble phenols), and transcriptional analyses, i.e. microarray and reverse transcription quantitative PCR (RT-qPCR) were used for extensive phenotyping of MYB-overexpressing spruce plantlets. Upon germination of somatic embryos, root growth was reduced in both transgenics. Enhanced lignin deposition was also a common feature but ectopic secondary cell wall deposition was more strongly associated with PtMYB8-OE. Microarray and RT-qPCR data showed that overexpression of each MYB led to an overlapping up-regulation of many genes encoding phenylpropanoid enzymes involved in lignin monomer synthesis, while misregulation of several cell wall-related genes and other MYB transcription factors was specifically associated with PtMYB8-OE. Together, the results suggest that MYB1 and MYB8 may be part of a conserved transcriptional network involved in secondary cell wall deposition in conifers

Roles of silica and lignin in horsetail ( Equisetum hyemale

This research deals with detailed analyses of silica and lignin distribution in horsetail with special reference to mechanical strength. Scanning electron images of a cross-section of an internode showed silica deposited densely only around the outer epidermis. Detailed histochemical analyses of lignin showed no lignin deposition in the silica-rich outer internodes of horsetail, while a characteristic lignin deposition was noticed in the vascular bundle in inner side of internodes. To analyze the structure of horsetail from a mechanical viewpoint, we calculated the response of a model structure of horsetail to a mechanical force applied perpendicularly to the long axis by a finite element method. We found that silica distributed in the outer epidermis may play the major structural role, with lignin's role being limited ensuring that the vascular bundle keep waterproof. These results were in contrast to more modern tall trees like gymnosperms, for which lignin provides mechanical strength. Lignin has the advantage of sticking to cellulose, hemicellulose, and other materials. Such properties make it possible for plants containing lignin to branch. Branching of tree stems aids in competing for light and other atmospheric resources. This type of branching was impossible for ancient horsetails, which relied on the physical properties of silica. From the evolutional view points, over millennia in trees with high lignin content, true branching, and many chlorophyll-containing leaves developed. (C) 2012 American Institute of Physics. [https://doi.org/10.1063/1.3688253]ArticleJOURNAL OF APPLIED PHYSICS. 111(4):044703 (2012)journal articl

Properties of an alkali-thermo stable xylanase from Geobacillus thermodenitrificans A333 and applicability in xylooligosaccharides generation

An extracellular thermo-alkali-stable and cellulase-free xylanase from Geobacillus thermodenitrificans A333 was purified to homogeneity by ion exchange and size exclusion chromatography. Its molecular mass was 44 kDa as estimated in native and denaturing conditions by gel filtration and SDS-PAGE analysis, respectively. The xylanase (GtXyn) exhibited maximum activity at 70 °C and pH 7.5. It was stable over broad ranges of temperature and pH retaining 88 % of activity at 60 °C and up to 97 % in the pH range 7.5–10.0 after 24 h. Moreover, the enzyme was active up to 3.0 M sodium chloride concentration, exhibiting at that value 70 % residual activity after 1 h. The presence of other metal ions did not affect the activity with the sole exceptions of K+ that showed a stimulating effect, and Fe2+, Co2+ and Hg2+, which inhibited the enzyme. The xylanase was activated by non-ionic surfactants and was stable in organic solvents remaining fully active over 24 h of incubation in 40 % ethanol at 25 °C. Furthermore, the enzyme was resistant to most of the neutral and alkaline proteases tested. The enzyme was active only on xylan, showing no marked preference towards xylans from different origins. The hydrolysis of beechwood xylan and agriculture-based biomass materials yielded xylooligosaccharides with a polymerization degree ranging from 2 to 6 units and xylobiose and xylotriose as main products. These properties indicate G. thermodenitrificans A333 xylanase as a promising candidate for several biotechnological applications, such as xylooligosaccharides preparation

Insights into the chemical composition of Equisetum hyemale by high resolution Raman imaging

Equisetaceae has been of research interest for decades, as it is one of the oldest living plant families, and also due to its high accumulation of silica up to 25% dry wt. Aspects of silica deposition, its association with other biomolecules, as well as the chemical composition of the outer strengthening tissue still remain unclear. These questions were addressed by using high resolution (<1 μm) Confocal Raman microscopy. Two-dimensional spectral maps were acquired on cross sections of Equisetum hyemale and Raman images calculated by integrating over the intensity of characteristic spectral regions. This enabled direct visualization of differences in chemical composition and extraction of average spectra from defined regions for detailed analyses, including principal component analysis (PCA) and basis analysis (partial least square fit based on model spectra). Accumulation of silica was imaged in the knobs and in a thin layer below the cuticula. In the spectrum extracted from the knob region as main contributions, a broad band below 500 cm−1 attributed to amorphous silica, and a band at 976 cm−1 assigned to silanol groups, were found. From this, we concluded that these protrusions were almost pure amorphous, hydrated silica. No silanol group vibration was detected in the silicified epidermal layer below and association with pectin and hemicelluloses indicated. Pectin and hemicelluloses (glucomannan) were found in high levels in the epidermal layer and in a clearly distinguished outer part of the hypodermal sterome fibers. The inner part of the two-layered cells revealed as almost pure cellulose, oriented parallel along the fiber

Comparative in situ analysis reveals the dynamic nature of sclerenchyma cell walls of the fern Asplenium rutifolium

Background and Aims: A key structural adaptation of vascular plants was the evolution of specialized vascular and mechanical tissues, innovations likely to have generated novel cell wall architectures. While collenchyma is a strengthening tissue typically found in growing organs of angiosperms, a similar tissue occurs in the petiole of the fern Asplenium rutifolium. Methods: The in situ cell wall (ultra)structure and composition of this tissue was investigated and characterized mechanically as well as structurally through nano-indentation and wide-angle X-ray diffraction, respectively. Key Results: Structurally the mechanical tissue resembles sclerenchyma, while its biomechanical properties and molecular composition both share more characteristics with angiosperm collenchyma. Cell wall thickening only occurs late during cell expansion or after cell expansion has ceased. Conclusions: If the term collenchyma is reserved for walls that thicken during expansive growth, the mechanical tissue in A. rutifolium represents sclerenchyma that mimics the properties of collenchyma and has the ability to modify its mechanical properties through sclerification. These results support the view that collenchyma does not occur in ferns and most probably evolved in angiosperms