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.

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.

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.

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.

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.

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.

Populus GT43 family members group into distinct sets required for primary and secondary wall xylan biosynthesis and include useful promoters for wood modification.

The plant GT43 protein family includes xylosyltransferases that are known to be required for xylan backbone biosynthesis, but have incompletely understood specificities. RT-qPCR and histochemical (GUS) analyses of expression patterns of GT43 members in hybrid aspen, reported here, revealed that three clades of the family have markedly differing specificity towards secondary wall-forming cells (wood and extraxylary fibres). Intriguingly, GT43A and B genes (corresponding to the Arabidopsis IRX9 clade) showed higher specificity for secondary-walled cells than GT43C and D genes (IRX14 clade), although both IRX9 and IRX14 are required for xylosyltransferase activity. The remaining genes, GT43E, F and G (IRX9-L clade), showed broad expression patterns. Transient transactivation analyses of GT43A and B reporters demonstrated that they are activated by PtxtMYB021 and PNAC085 (master secondary wall switches), mediated in PtxtMYB021 activation by an AC element. The high observed secondary cell wall specificity of GT43B expression prompted tests of the efficiency of its promoter (pGT43B), relative to the CaMV 35S (35S) promoter, for overexpressing a xylan acetyl esterase (CE5) or downregulating REDUCED WALL ACETYLATION (RWA) family genes and thus engineering wood acetylation. CE5 expression was weaker when driven by pGT43B, but it reduced wood acetyl content substantially more efficiently than the 35S promoter. RNAi silencing of the RWA family, which was ineffective using 35S, was achieved when using GT43B promoter. These results show the utility of the GT43B promoter for genetically engineering properties of wood and fibres.

MYB103 is required for FERULATE-5-HYDROXYLASE expression and syringyl lignin biosynthesis in Arabidopsis stems.

The transcription factor MYB103 was previously identified as a member of the transcriptional network regulating secondary wall biosynthesis in xylem tissues of Arabidopsis, and was proposed to act on cellulose biosynthesis. It is a direct transcriptional target of the transcription factor SECONDARY WALL ASSOCIATED NAC DOMAIN PROTEIN 1 (SND1), and 35S-driven dominant repression or over-expression of MYB103 modifies secondary wall thickness. We identified two myb103 T-DNA insertion mutants and chemically characterized their lignocellulose by pyrolysis/GC/MS, 2D NMR, FT-IR microspectroscopy and wet chemistry. The mutants developed normally but exhibited a 70-75% decrease in syringyl (S) lignin. The level of guaiacyl (G) lignin was co-ordinately increased, so that total Klason lignin was not affected. The transcript abundance of FERULATE-5-HYDROXYLASE (F5H), the key gene in biosynthesis of S lignin, was strongly decreased in the myb103 mutants, and the metabolomes of the myb103 mutant and an F5H null mutant were very similar. Other than modification of the lignin S to G ratio, there were only very minor changes in the composition of secondary cell-wall polymers in the inflorescence stem. In conclusion, we demonstrate that F5H expression and hence biosynthesis of S lignin are dependent on MYB103.

Deficient sucrose synthase activity in developing wood does not specifically affect cellulose biosynthesis, but causes an overall decrease in cell wall polymers.

The biosynthesis of wood in aspen (Populus) depends on the metabolism of sucrose, which is the main transported form of carbon from source tissues. The largest fraction of the wood biomass is cellulose, which is synthesized from UDP-glucose. Sucrose synthase (SUS) has been proposed previously to interact directly with cellulose synthase complexes and specifically supply UDP-glucose for cellulose biosynthesis. To investigate the role of SUS in wood biosynthesis, we characterized transgenic lines of hybrid aspen with strongly reduced SUS activity in developing wood. No dramatic growth phenotypes in glasshouse-grown trees were observed, but chemical fingerprinting with pyrolysis-GC/MS, together with micromechanical analysis, showed notable changes in chemistry and ultrastructure of the wood in the transgenic lines. Wet chemical analysis showed that the dry weight percentage composition of wood polymers was not changed significantly. However, a decrease in wood density was observed and, consequently, the content of lignin, hemicellulose and cellulose was decreased per wood volume. The decrease in density was explained by a looser structure of fibre cell walls as shown by increased wall shrinkage on drying. The results show that SUS is not essential for cellulose biosynthesis, but plays a role in defining the total carbon incorporation to wood cell walls.

Aspen SUCROSE TRANSPORTER3 allocates carbon into wood fibers.

Wood formation in trees requires carbon import from the photosynthetic tissues. In several tree species, including Populus species, the majority of this carbon is derived from sucrose (Suc) transported in the phloem. The mechanism of radial Suc transport from phloem to developing wood is not well understood. We investigated the role of active Suc transport during secondary cell wall formation in hybrid aspen (Populus tremula × Populus tremuloides). We show that RNA interference-mediated reduction of PttSUT3 (for Suc/H(+) symporter) during secondary cell wall formation in developing wood caused thinner wood fiber walls accompanied by a reduction in cellulose and an increase in lignin. Suc content in the phloem and developing wood was not significantly changed. However, after (13)CO2 assimilation, the SUT3RNAi lines contained more (13)C than the wild type in the Suc-containing extract of developing wood. Hence, Suc was transported into developing wood, but the Suc-derived carbon was not efficiently incorporated to wood fiber walls. A yellow fluorescent protein:PttSUT3 fusion localized to plasma membrane, suggesting that reduced Suc import into developing wood fibers was the cause of the observed cell wall phenotype. The results show the importance of active Suc transport for wood formation in a symplasmically phloem-loading tree species and identify PttSUT3 as a principal transporter for carbon delivery into secondary cell wall-forming wood fibers.

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.

Fructokinase is required for carbon partitioning to cellulose in aspen wood.

Sucrose is the main transported form of carbon in several plant species, including Populus species. Sucrose metabolism in developing wood has therefore a central role in carbon partitioning to stem biomass. Half of the sucrose-derived carbon is in the form of fructose, but metabolism of fructose has received little attention as a factor in carbon partitioning to walls of wood cells. We show that RNAi-mediated reduction of FRK2 activity in developing wood of hybrid aspen (Populus tremula × tremuloides) led to the accumulation of soluble neutral sugars and a decrease in hexose phosphates and UDP-glucose, indicating that carbon flux to cell-wall polysaccharide precursors is decreased. Reduced FRK2 activity also led to thinner fiber cell walls with a reduction in the proportion of cellulose. No pleiotropic effects on stem height or diameter were observed. The results establish a central role for FRK2 activity in carbon flux to wood cellulose.

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.

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.

Cell-specific chemotyping and multivariate imaging by combined FT-IR microspectroscopy and orthogonal projections to latent structures (OPLS) analysis reveals the chemical landscape of secondary xylem.

Fourier-transform infrared (FT-IR) spectroscopy combined with microscopy enables chemical information to be acquired from native plant cell walls with high spatial resolution. Combined with a 64 × 64 focal plane array (FPA) detector, 4096 spectra can be simultaneously obtained from a 0.3 × 0.3 mm image; each spectrum represents a compositional and structural 'fingerprint' of all cell wall components. For optimal use and analysis of such a large amount of information, multivariate approaches are preferred. Here, FT-IR microspectroscopy with FPA detection is combined with orthogonal projections to latent structures discriminant analysis (OPLS-DA). This allows for: (i) the extraction of spectra from single cell types, (ii) identification and characterization of different chemotypes using the full spectral information, and (iii) further visualization of the pattern of identified chemotypes by multivariate imaging. As proof of concept, the chemotypes of Populus tremula xylem cell types are described. The approach revealed unknown features about chemical plasticity and patterns of lignin composition in wood fibers that would have remained hidden in the dataset with traditional data analysis. The applicability of the method to Arabidopsis xylem and its usefulness in mutant chemotyping is also demonstrated. The methodological approach is not limited to xylem tissues but can be applied to any plant organ/tissue also using other techniques such as Raman and UV microspectroscopy.

Constitutive expression of a fungal glucuronoyl esterase in Arabidopsis reveals altered cell wall composition and structure.

A family 15 carbohydrate esterase (CE15) from the white-rot basidiomycete, Phanerochaete carnosa (PcGCE), was transformed into Arabidopsis thaliana Col-0 and was expressed from the constitutive cauliflower mosaic virus 35S promoter. Like other CE15 enzymes, PcGCE hydrolyzed methyl-4-O-methyl-d-glucopyranuronate and could target ester linkages that contribute to lignin-carbohydrate complexes that form in plant cell walls. Three independently transformed Arabidopsis lines were evaluated in terms of nine morphometric parameters, total sugar and lignin composition, cell wall anatomy, enzymatic saccharification and xylan extractability. The transgenic lines consistently displayed a leaf-yellowing phenotype, as well as reduced glucose and xylose content by as much as 30% and 35%, respectively. Histological analysis revealed 50% reduction in cell wall thickness in the interfascicular fibres of transgenic plants, and FT-IR microspectroscopy of interfascicular fibre walls indicated reduction in lignin cross-linking in plants overexpressing PcGCE. Notably, these characteristics could be correlated with improved xylose recovery in transgenic plants, up to 15%. The current analysis represents the first example whereby a fungal glucuronoyl esterase is expressed in Arabidopsis and shows that the promotion of glucuronoyl esterase activity in plants can alter the extent of intermolecular cross-linking within plant cell walls.