Quantitative and qualitative saccharide analysis of North Atlantic brown seaweed by gas chromatography/mass spectrometry and infrared spectroscopy.

Brown seaweeds contain a variety of saccharides which have potential industrial uses. The most abundant polysaccharide in brown seaweed is typically alginate, consisting of mannuronic (M) and guluronic acid (G). The ratio of these residues fundamentally determines the physicochemical properties of alginate. In the present study, gas chromatography/mass spectrometry (GC/MS) was used to give a detailed breakdown of the monosaccharide species in North Atlantic brown seaweeds. The anthrone method was used for determination of crystalline cellulose. The experimental data was used to calibrate multivariate prediction models for estimation of total carbohydrates, crystalline cellulose, total alginate and alginate M/G ratio directly in dried, brown seaweed using three types of infrared spectroscopy, using relative error (RE) as a measure of predictive accuracy. Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) performed well for the estimation of total alginate (RE = 0.12, R2 = 0.82), and attenuated total reflectance (ATR) showed good prediction of M/G ratio (RE = 0.14, R2 = 0.86). Both DRIFTS, ATR and near infrared (NIR) were unable to predict crystalline cellulose and only DRIFTS performed better in determining total carbohydrates. Multivariate spectral analysis is a promising method for easy and rapid characterization of alginate and M/G ratio in seaweed.

Comparative Analysis of Enzyme Production Patterns of Lignocellulose Degradation of Two White Rot Fungi: Obba rivulosa and Gelatoporia subvermispora.

The unique ability of basidiomycete white rot fungi to degrade all components of plant cell walls makes them indispensable organisms in the global carbon cycle. In this study, we analyzed the proteomes of two closely related white rot fungi, Obba rivulosa and Gelatoporia subvermispora, during eight-week cultivation on solid spruce wood. Plant cell wall degrading carbohydrate-active enzymes (CAZymes) represented approximately 5% of the total proteins in both species. A core set of orthologous plant cell wall degrading CAZymes was shared between these species on spruce suggesting a conserved plant biomass degradation approach in this clade of basidiomycete fungi. However, differences in time-dependent production of plant cell wall degrading enzymes may be due to differences among initial growth rates of these species on solid spruce wood. The obtained results provide insight into specific enzymes and enzyme sets that are produced during the degradation of solid spruce wood in these fungi. These findings expand the knowledge on enzyme production in nature-mimicking conditions and may contribute to the exploitation of white rot fungi and their enzymes for biotechnological applications.

Ethylene Signaling Is Required for Fully Functional Tension Wood in Hybrid Aspen.

Tension wood (TW) in hybrid aspen trees forms on the upper side of displaced stems to generate a strain that leads to uplifting of the stem. TW is characterized by increased cambial growth, reduced vessel frequency and diameter, and the presence of gelatinous, cellulose-rich (G-)fibers with its microfibrils oriented parallel to the fiber cell axis. Knowledge remains limited about the molecular regulators required for the development of this special xylem tissue with its characteristic morphological, anatomical, and chemical features. In this study, we use transgenic, ethylene-insensitive (ETI) hybrid aspen trees together with time-lapse imaging to show that functional ethylene signaling is required for full uplifting of inclined stems. X-ray diffraction and Raman microspectroscopy of TW in ETI trees indicate that, although G-fibers form, the cellulose microfibril angle in the G-fiber S-layer is decreased, and the chemical composition of S- and G-layers is altered than in wild-type TW. The characteristic asymmetric growth and reduction of vessel density is suppressed during TW formation in ETI trees. A genome-wide transcriptome profiling reveals ethylene-dependent genes in TW, related to cell division, cell wall composition, vessel differentiation, microtubule orientation, and hormone crosstalk. Our results demonstrate that ethylene regulates transcriptional responses related to the amount of G-fiber formation and their properties (chemistry and cellulose microfibril angle) during TW formation. The quantitative and qualitative changes in G-fibers are likely to contribute to uplifting of stems that are displaced from their original position.

Ethylene signaling induces gelatinous layers with typical features of tension wood in hybrid aspen.

The phytohormone ethylene impacts secondary stem growth in plants by stimulating cambial activity, xylem development and fiber over vessel formation. We report the effect of ethylene on secondary cell wall formation and the molecular connection between ethylene signaling and wood formation. We applied exogenous ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to wild-type and ethylene-insensitive hybrid aspen trees (Populus tremula × tremuloides) and studied secondary cell wall anatomy, chemistry and ultrastructure. We furthermore analyzed the transcriptome (RNA Seq) after ACC application to wild-type and ethylene-insensitive trees. We demonstrate that ACC and ethylene induce gelatinous layers (G-layers) and alter the fiber cell wall cellulose microfibril angle. G-layers are tertiary wall layers rich in cellulose, typically found in tension wood of aspen trees. A vast majority of transcripts affected by ACC are downstream of ethylene perception and include a large number of transcription factors (TFs). Motif-analyses reveal potential connections between ethylene TFs (Ethylene Response Factors (ERFs), ETHYLENE INSENSITIVE 3/ETHYLENE INSENSITIVE3-LIKE1 (EIN3/EIL1)) and wood formation. G-layer formation upon ethylene application suggests that the increase in ethylene biosynthesis observed during tension wood formation is important for its formation. Ethylene-regulated TFs of the ERF and EIN3/EIL1 type could transmit the ethylene signal.

A genome-wide screen for ethylene-induced ethylene response factors (ERFs) in hybrid aspen stem identifies ERF genes that modify stem growth and wood properties.

Ethylene Response Factors (ERFs) are a large family of transcription factors that mediate responses to ethylene. Ethylene affects many aspects of wood development and is involved in tension wood formation. Thus ERFs could be key players connecting ethylene action to wood development. We identified 170 gene models encoding ERFs in the Populus trichocarpa genome. The transcriptional responses of ERF genes to ethylene treatments were determined in stem tissues of hybrid aspen (Populus tremula × tremuloides) by qPCR. Selected ethylene-responsive ERFs were overexpressed in wood-forming tissues and characterized for growth and wood chemotypes by FT-IR. Fifty ERFs in Populus showed more than five-fold increased transcript accumulation in response to ethylene treatments. Twenty-six ERFs were selected for further analyses. A majority of these were induced during tension wood formation. Overexpression of ERFs 18, 21, 30, 85 and 139 in wood-forming tissues of hybrid aspen modified the wood chemotype. Moreover, overexpression of ERF139 caused a dwarf-phenotype with altered wood development, and overexpression of ERF18, 34 and 35 slightly increased stem diameter. We identified ethylene-induced ERFs that respond to tension wood formation, and modify wood formation when overexpressed. This provides support for their role in ethylene-mediated regulation of wood development.

Non-cell-autonomous postmortem lignification of tracheary elements in Zinnia elegans.

Postmortem lignification of xylem tracheary elements (TEs) has been debated for decades. Here, we provide evidence in Zinnia elegans TE cell cultures, using pharmacological inhibitors and in intact Z. elegans plants using Fourier transform infrared microspectroscopy, that TE lignification occurs postmortem (i.e., after TE programmed cell death). In situ RT-PCR verified expression of the lignin monomer biosynthetic cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase in not only the lignifying TEs but also in the unlignified non-TE cells of Z. elegans TE cell cultures and in living, parenchymatic xylem cells that surround TEs in stems. These cells were also shown to have the capacity to synthesize and transport lignin monomers and reactive oxygen species to the cell walls of dead TEs. Differential gene expression analysis in Z. elegans TE cell cultures and concomitant functional analysis in Arabidopsis thaliana resulted in identification of several genes that were expressed in the non-TE cells and that affected lignin chemistry on the basis of pyrolysis-gas chromatography/mass spectrometry analysis. These data suggest that living, parenchymatic xylem cells contribute to TE lignification in a non-cell-autonomous manner, thus enabling the postmortem lignification of TEs.