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.

Rapid and accurate determination of protein content in North Atlantic seaweed by NIR and FTIR spectroscopies.

Seaweed is considered a potentially sustainable source of protein for human consumption, and rapid, accurate methods for determining seaweed protein contents are needed. Seaweeds contain substances which interfere with common protein estimation methods however. The present study compares the Lowry and BCA protein assays and protein determination by N-ratios to more novel spectroscopic methods. Linear regression of the height or the integrated area under the Amide II band of diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) was used to predict seaweed protein with good prediction performance. Partial least squares regression (PLSR) was performed on both DRIFTS and near-infrared (NIR) spectra, with even higher prediction accuracy. Spectroscopy performed similar to or better than the calculated N-ratio of 4.14 for protein prediction. These spectral prediction methods require minimal sample preparation and chemical use, and are easy to perform, making them environmentally sustainable and economically viable for rapid estimation of seaweed protein.

Adaptive strategies of Scots pine under shade: Increase in lignin synthesis and ecotypic variation in defense-related gene expression.

Shade is a stressful condition for plants characterized by low Red:Far-Red (R:FR) ratio. The northern latitudes in Sweden daily receive more hours of FR-enriched light (twilight) or shade-like conditions compared to southern forests during the growing season. Scots pine (Pinus sylvestris L.) is a shade-intolerant species. Yet, it is well adapted to this latitudinal variation in light, which is evident by a northward increase in FR requirement to maintain growth. Shade adversely affects plant growth; it makes the plant weak and, therefore, susceptible to pathogen attack. Lignin is involved in plant protection against pathogen invasion mainly by forming a physical barrier. We studied lignin synthesis and expression of defense-related genes (growth-defense trade-offs) under a low R:FR (shade) ratio in Scots pine. A higher number of immunity/defense-related genes were up-regulated in response to shade in northern populations compared to southern ones, which can be viewed as a local adaptation to light quality for optimal growth and survival. Light quality regulates lignin metabolism; light stimulates lignin synthesis, while shade causes a decrease in lignin synthesis in most angiosperms. In contrast, Scots pine shows an increase in lignin synthesis supported by the higher expression of a few key genes in the lignin biosynthetic pathway, a novel finding reported by our study. These findings can be applied to future breeding strategies in forestry to produce disease-resilient trees.

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.

Enhanced lignin synthesis and ecotypic variation in defense-related gene expression in response to shade in Norway spruce.

During the growth season, northern forests in Sweden daily receive more hours of far-red (FR)-enriched light or twilight (shade) as compared to southern forests. Norway spruce (shade-tolerant) are adapted to latitudinal variation in twilight characterized by a northward increase in FR requirement to maintain growth. Shade is a stressful condition that affects plant growth and increases plant's susceptibility to pathogen attack. Lignin plays a central role in plant defense and its metabolism is regulated by light wavelength composition (light quality). In the current work, we studied regulation of lignin synthesis and defense-related genes (growth-defense trade-offs) in response to shade in Norway spruce. In most angiosperms, light promotes lignin synthesis, whereas shade decreases lignin production leading to weaker stem, which may make plants more disease susceptible. In contrast, enhanced lignin synthesis was detected in response to shade in Norway spruce. We detected a higher number of immunity/defense-related genes up-regulated in northern populations as compared to south ones in response to shade. Enhanced lignin synthesis coupled with higher defense-related gene expression can be interpreted as an adaptive strategy for better survival in northern populations. Findings will contribute to ensuring deployment of well-adapted genetic material and identifying tree families with enhanced disease resistance.

Polyoxoniobates as molecular building blocks in thin films.

Niobium oxide thin films have been prepared by spin-coating aqueous solutions of tetramethylammonium salts of the isostructural polyoxometalate clusters [Nb10O28]6-, [TiNb9O28]7- and [Ti2Nb8O28]8- onto silicon wafers, and annealing them. The [Nb10O28]6- cluster yields films of Nb2O5 in the orthorhombic and monoclinic crystal phases when annealed at 800 °C and 1000 °C, respectively, whereas the [TiNb9O28]7- and [Ti2Nb8O28]8- clusters yield the monoclinic crystal phases of Ti2Nb12O29 and TiNb2O7 (titanium-niobium oxides) in different ratios. We also demonstrate a protocol for depositing successive layers of metal oxide films. Finally, we explore factors affecting the roughness of the films.

The chromatin-modifying protein HUB2 is involved in the regulation of lignin composition in xylem vessels.

PIRIN2 (PRN2) was earlier reported to suppress syringyl (S)-type lignin accumulation of xylem vessels of Arabidopsis thaliana. In the present study, we report yeast two-hybrid results supporting the interaction of PRN2 with HISTONE MONOUBIQUITINATION2 (HUB2) in Arabidopsis. HUB2 has been previously implicated in several plant developmental processes, but not in lignification. Interaction between PRN2 and HUB2 was verified by ß-galactosidase enzymatic and co-immunoprecipitation assays. HUB2 promoted the deposition of S-type lignin in the secondary cell walls of both stem and hypocotyl tissues, as analysed by pyrolysis-GC/MS. Chemical fingerprinting of individual xylem vessel cell walls by Raman and Fourier transform infrared microspectroscopy supported the function of HUB2 in lignin deposition. These results, together with a genetic analysis of the hub2 prn2 double mutant, support the antagonistic function of PRN2 and HUB2 in deposition of S-type lignin. Transcriptome analyses indicated the opposite regulation of the S-type lignin biosynthetic gene FERULATE-5-HYDROXYLASE1 by PRN2 and HUB2 as the underlying mechanism. PRN2 and HUB2 promoter activities co-localized in cells neighbouring the xylem vessel elements, suggesting that the S-type lignin-promoting function of HUB2 is antagonized by PRN2 for the benefit of the guaiacyl (G)-type lignin enrichment of the neighbouring xylem vessel elements.

Screening Suitability of Northern Hemisphere Algal Strains for Heterotrophic Cultivation and Fatty Acid Methyl Ester Production.

Rapid rises in atmospheric CO2 levels derived from fossil fuel combustion are imposing urgent needs for renewable substitutes. One environmentally friendly alternative is biodiesel produced from suitable microalgal fatty acids. Algal strains normally grow photoautotrophically, but this is problematic in Northern areas because of the light limitations for much of the year. Mixotrophic and particularly heterotrophic strains could be valuable, especially if they can be cultivated in municipal wastewater with contents of nutrients such as nitrogen and phosphorous that should be reduced before release into receiving water. Thus, the aim of this study was to screen for microalgal strains suitable for heterotrophic cultivation with a cheap carbon source (glycerol) for biodiesel production in Nordic, and other high-latitude, countries. One of the examined strains, a Desmodesmus sp. strain designated 2-6, accumulated biomass at similar rates in heterotrophic conditions with 40 mM glycerol as in autotrophic conditions. Furthermore, in heterotrophic conditions it produced more fatty acids, and ca. 50% more C18:1 fatty acids, as well as showing a significant decrease in C18:3 fatty acids, all of which are highly desirable features for biodiesel production.

SYNERGISTIC ON AUXIN AND CYTOKININ 1 positively regulates growth and attenuates soil pathogen resistance.

Plants as non-mobile organisms constantly integrate varying environmental signals to flexibly adapt their growth and development. Local fluctuations in water and nutrient availability, sudden changes in temperature or other abiotic and biotic stresses can trigger changes in the growth of plant organs. Multiple mutually interconnected hormonal signaling cascades act as essential endogenous translators of these exogenous signals in the adaptive responses of plants. Although the molecular backbones of hormone transduction pathways have been identified, the mechanisms underlying their interactions are largely unknown. Here, using genome wide transcriptome profiling we identify an auxin and cytokinin cross-talk component; SYNERGISTIC ON AUXIN AND CYTOKININ 1 (SYAC1), whose expression in roots is strictly dependent on both of these hormonal pathways. We show that SYAC1 is a regulator of secretory pathway, whose enhanced activity interferes with deposition of cell wall components and can fine-tune organ growth and sensitivity to soil pathogens.

Arabidopsis XTH4 and XTH9 Contribute to Wood Cell Expansion and Secondary Wall Formation.

Xyloglucan is the major hemicellulose of dicotyledon primary cell walls, affecting the load-bearing framework with the participation of xyloglucan endo-transglycosylase/hydrolases (XTHs). We used loss- and gain-of function approaches to study functions of XTH4 and XTH9 abundantly expressed in cambial regions during secondary growth of Arabidopsis (Arabidopsis thaliana). In secondarily thickened hypocotyls, these enzymes had positive effects on vessel element expansion and fiber intrusive growth. They also stimulated secondary wall thickening but reduced secondary xylem production. Cell wall analyses of inflorescence stems revealed changes in lignin, cellulose, and matrix sugar composition indicating an overall increase in secondary versus primary walls in mutants, indicative of higher xylem production compared with the wild type (since secondary walls were thinner). Intriguingly, the number of secondary cell wall layers compared with the wild type was increased in xth9 and reduced in xth4, whereas the double mutant xth4x9 displayed an intermediate number of layers. These changes correlated with specific Raman signals from the walls, indicating changes in lignin and cellulose. Secondary walls were affected also in the interfascicular fibers, where neither XTH4 nor XTH9 was expressed, indicating that these effects were indirect. Transcripts involved in secondary wall biosynthesis and cell wall integrity sensing, including THESEUS1 and WALL ASSOCIATED KINASE2, were highly induced in the mutants, indicating that deficiency in XTH4 and XTH9 triggers cell wall integrity signaling, which, we propose, stimulates xylem cell production and modulates secondary wall thickening. Prominent effects of XTH4 and XTH9 on secondary xylem support the hypothesis that altered xyloglucan affects wood properties both directly and via cell wall integrity sensing.

PIRIN2 suppresses S-type lignin accumulation in a noncell-autonomous manner in Arabidopsis xylem elements.

PIRIN (PRN) genes encode cupin domain-containing proteins that function as transcriptional co-regulators in humans but that are poorly described in plants. A previous study in xylogenic cell cultures of Zinnia elegans suggested a role for a PRN protein in lignification. This study aimed to identify the function of Arabidopsis (Arabidopsis thaliana) PRN proteins in lignification of xylem tissues. Chemical composition of the secondary cell walls was analysed in Arabidopsis stems and/or hypocotyls by pyrolysis-gas chromatography/mass spectrometry, 2D-nuclear magnetic resonance and phenolic profiling. Secondary cell walls of individual xylem elements were chemotyped by Fourier transform infrared and Raman microspectroscopy. Arabidopsis PRN2 suppressed accumulation of S-type lignin in Arabidopsis stems and hypocotyls. PRN2 promoter activity and PRN2:GFP fusion protein were localised specifically in cells next to the vessel elements, suggesting a role for PRN2 in noncell-autonomous lignification of xylem vessels. Accordingly, PRN2 modulated lignin chemistry in the secondary cell walls of the neighbouring vessel elements. These results indicate that PRN2 suppresses S-type lignin accumulation in the neighbourhood of xylem vessels to bestow G-type enriched lignin composition on the secondary cell walls of the vessel elements. Gene expression analyses suggested that PRN2 function is mediated by regulation of the expression of the lignin-biosynthetic genes.

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.

Statistical Methods for Rapid Quantification of Proteins, Lipids, and Carbohydrates in Nordic Microalgal Species Using ATR-FTIR Spectroscopy.

Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy is a simple, cheap, and fast method to collect chemical compositional information from microalgae. However, (semi)quantitative evaluation of the collected data can be daunting. In this work, ATR-FTIR spectroscopy was used to monitor changes of protein, lipid, and carbohydrate content in seven green microalgae grown under nitrogen starvation. Three statistical methods-univariate linear regression analysis (ULRA), orthogonal partial least squares (OPLS), and multivariate curve resolution-alternating least squares (MCR-ALS)-were compared in their ability to model and predict the concentration of these compounds in the biomass. OPLS was found superior, since it i) included all three compounds simultaneously; ii) explained variations in the data very well; iii) had excellent prediction accuracy for proteins and lipids, and acceptable for carbohydrates; and iv) was able to discriminate samples based on cultivation stage and type of storage compounds accumulated in the cells. ULRA models worked well for the determination of proteins and lipids, but carbohydrates could only be estimated if already determined protein contents were used for scaling. Results obtained by MCR-ALS were similar to ULRA, however, this method is considerably easier to perform and interpret than the more abstract statistical/chemometric methods. FTIR-spectroscopy-based models allow high-throughput, cost-effective, and rapid estimation of biomass composition of green microalgae.

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.

Isolation and characterization of a respirable particle fraction from residential house-dust.

Indoor air pollution has caused increasing concern in recent years. As we spend most of our lives indoors, it is crucial to understand the health effects caused by indoor air pollution. Household dust serve as good proxy for accessing indoor air pollution, especially smaller dust particles that can pass into the lungs are of interest. In this study we present an efficient method for the isolation of dust particles in the respirable size range. The respirable fraction was recovered from vacuum cleaner bags, separated by stepwise sieving, followed by characterization for size, morphology, surface area, organic content and elemental composition. The respirable fraction was obtained in a yield of 0.6% with a specific surface area of 2.5m2/g and a Mass Median Aerodynamic Diameter of 3.73 ± 0.15µm. Aluminum and zink were the dominating metals measured in the dust, whereas the major mineral components were found to be silicon dioxide and calcium carbonate. The fraction of organic matter in the dust was measured to be 69 ± 1%. The organic matrix contained bacterial and fungi and a presence of skin fragments. We present here an efficient and fast method for the isolation of dust particles in the respirable size range. That is of considerable value due to the need for large quantities of respirable particle fractions to conduct toxicological studies and risk assessment work.

Biochemical profiling of diabetes disease progression by multivariate vibrational microspectroscopy of the pancreas.

Despite the dramatic increase in the prevalence of diabetes, techniques for in situ studies of the underlying pancreatic biochemistry are lacking. Such methods would facilitate obtaining mechanistic understanding of diabetes pathophysiology and aid in prognostic and/or diagnostic assessments. In this report we demonstrate how a multivariate imaging approach (orthogonal projections to latent structures - discriminant analysis) can be applied to generate full vibrational microspectroscopic profiles of pancreatic tissues. These profiles enable extraction of known and previously unrecorded biochemical alterations in models of diabetes, and allow for classification of the investigated tissue with regards to tissue type, strain and stage of disease progression. Most significantly, the approach provided evidence for dramatic alterations of the pancreatic biochemistry at the initial onset of immune-infiltration in the Non Obese Diabetic model for type 1 diabetes. Further, it enabled detection of a previously undocumented accumulation of collagen fibrils in the leptin deficient ob/ob mouse islets. By generating high quality spectral profiles through the tissue capsule of hydrated human pancreata and by in vivo Raman imaging of pancreatic islets transplanted to the anterior chamber of the eye, we provide critical feasibility studies for the translation of this technique to diagnostic assessments of pancreatic biochemistry in vivo.

Alkalization of dissolving cellulose pulp with highly concentrated caustic at low NaOH stoichiometric excess.

We present a quantitative study, using Raman spectroscopy combined with multivariate data analysis, to determine the degree of activation of softwood sulphite dissolving cellulose pulp by aqueous sodium hydroxide. We have chosen industrially relevant conditions, including low stoichiometric ratio of NaOH/Anhydroglucose Unit (AGU)<2 and highly concentrated caustic (≥45% w/w [NaOH]). A design of experiments is used to investigate the effects of simultaneous variation of a set of key parameters on the degree of activation (i.e. transformation to alkali cellulose, denoted as DoA): (a) the NaOH/AGU stoichiometric ratio, denoted (r); (b) the concentration of NaOH, denoted [NaOH]; (c) temperature, denoted (T); and (d) reaction time, denoted (t). Solid-state 13C CP/MAS NMR spectroscopy was applied to investigate the reproducibility of the experiments and to select the range for (t). According to the model, (r) is found to have a statistically significant effect on DoA (increasing from DoA=6-30% at the lowest (r)=0.8, to DoA=48-87% at the highest (r)=1.8), together with [NaOH]. The influence of [NaOH] depends strongly on (r). The other studied variables are found to be insignificant in the model and has a complicated influence on the activation. In particular, (T) is found to be unimportant in the studied range (30-60°C), but increasing (t) from 5 to 25min shows a positive influence on DoA, depending on both (r) and [NaOH]. A mercerisation mechanism that is controlled by diffusion is proposed to explain these phenomena.

Chemical Imaging of Xylem by Raman Microspectroscopy.

Raman microspectroscopic techniques provide highly detailed chemical information about xylem tissue at submicron spatial resolution. The techniques are generally sensitive and they provide a powerful, yet inexpensive way to probe the chemical composition of individual cells or cell wall layers in situ, non-destructively, in a confocal manner, simultaneously detecting all chemical compounds without the need of external agents (label, dyes, stains). Problems with limited specificity in complex chemical matrices such as cell walls may arise, compounded by fluorescence problems. However, these can often be circumvented. In this chapter, the basics of the technique, including a common instrumental setup, together with the general strengths and limitations of Raman microspectroscopy are discussed. Detailed protocols are provided for single point measurements, as well as for fully customizable raster scan mapping, including sample preparation, setup, and measurement steps. The major steps of the data analysis procedure are discussed as well.