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Chemical compositional information can be extracted from Raman and infrared microscopic images by MCR-ALS. The algorithm finds the spectral profiles of compounds contributing to each image pixel and their relative concentrations. Raman and Fourier transform IR (FTIR) microspectroscopic images of biological material (tissue sections) contain detailed information about their chemical composition. The challenge lies in identifying changes in chemical composition, as well as locating and assigning these changes to different conditions (pathology, anatomy, environmental or genetic factors). Multivariate data analysis techniques are ideal for decrypting such information from the data. This protocol provides a user-friendly pipeline and graphical user interface (GUI) for data pre-processing and unmixing of pixel spectra into their contributing pure components by multivariate curve resolution–alternating least squares (MCR-ALS) analysis. The analysis considers the full spectral profile in order to identify the chemical compounds and to visualize their distribution across the sample to categorize chemically distinct areas. Results are rapidly achieved (usually <30–60 min per image), and they are easy to interpret and evaluate both in terms of chemistry and biology, making the method generally more powerful than principal component analysis (PCA) or heat maps of single-band intensities. In addition, chemical and biological evaluation of the results by means of reference matching and segmentation maps (based on k -means clustering) is possible.

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.

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.

Programmed Cell Death (PCD) in eukaryotes is a regulated process occurring during development, cell differentiation and aging. Apoptosis is a particularly well studied morphotype of PCD, only observed in animal cells (metazoan). Its most definitive hallmark is the formation and release of membrane-enclosed extracellular vesicles called Apoptotic Bodies (ABs). Although apoptotic-like features have been described in plants, yeast, protozoa and phytoplankton, the production of ABs has been thought to be limited to multicellular animals. Here we report the production and release of extracellular ABs in a non-metazoan unicellular eukaryote, the cryptophyte alga Guillardia theta . Morphologies of G. theta cells during aging and pharmacologically-induced cell death confirm the presence of ABs and apoptosis in phytoplankton. G. theta ABs have similar composition to metazoan ABs, carrying DNA, proteins, lipids, carbohydrates, fragments of organelles and cytosol portions. Our results demonstrate that G. theta , a microalga that arose from secondary endosymbiosis, experiences apoptotic cell death in physiological conditions, similar to animal cells. Since secondary endosymbiosis occurred prior to the origin of multicellularity, our discovery questions the evolutionary origin of PCD. Here the authors show that a single-celled, photosynthetic alga produces apoptotic bodies during programmed cell death. This mechanism, which was thought to be unique to animals, seems to be more ancient and widespread than previously believed.

The chemical composition of wood is one of the key features that determine wood quality. The focus of this study was on identifying differences between juvenile and mature woods in Scots pine ( Pinus sylvestris L.) and developing models for predicting the chemical composition of these two wood types. Chemical traits, determined by traditional wet chemistry techniques, included the proportion of lignin, polysaccharides and extractives. Partial least squares regression of Fourier transform infrared (FTIR) spectra was used for model building. The model performance was primarily evaluated by root mean squared error of predictions (RMSEP). High predictive power was attained for the content of lignin (RMSEP of 0.476 and 0.495 for juvenile and mature woods, respectively) and extractives (0.302 and 0.471), good predictive power for cellulose (0.715 and 0.696) and hemicelluloses in juvenile wood (0.719) and low predictive power for hemicelluloses in mature wood (0.823). A distinct band was observed at 1693 cm −1 , and its intensity was strongly associated with the content of extractives ( r  = 0.968 and 0.861 in juvenile and mature woods, respectively). FTIR has proved suitable for the rapid, non-destructive, cost-efficient assessment of the chemical composition of juvenile and mature woods in Scots pine. The band at 1693 cm −1 is to be further investigated to unravel its link with individual extractive components.

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 9 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 ethylenemediated regulation of wood development.

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.

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 (Populustremula × tremuloides) and studied secondary cell wall anatomy, chemistry and ultrastructure. We furthermore analyzed the transcriptome (RNA Seq) after ACC application to wildtype 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.

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.