Annette Naumann

Downregulation of cinnamoyl-coenzyme A reductase in poplar: multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure.

Cinnamoyl-CoA reductase (CCR) catalyzes the penultimate step in monolignol biosynthesis. We show that downregulation of CCR in transgenic poplar (Populus tremula × Populus alba) was associated with up to 50% reduced lignin content and an orange-brown, often patchy, coloration of the outer xylem. Thioacidolysis, nuclear magnetic resonance (NMR), immunocytochemistry of lignin epitopes, and oligolignol profiling indicated that lignin was relatively more reduced in syringyl than in guaiacyl units. The cohesion of the walls was affected, particularly at sites that are generally richer in syringyl units in wild-type poplar. Ferulic acid was incorporated into the lignin via ether bonds, as evidenced independently by thioacidolysis and by NMR. A synthetic lignin incorporating ferulic acid had a red-brown coloration, suggesting that the xylem coloration was due to the presence of ferulic acid during lignification. Elevated ferulic acid levels were also observed in the form of esters. Transcript and metabolite profiling were used as comprehensive phenotyping tools to investigate how CCR downregulation impacted metabolism and the biosynthesis of other cell wall polymers. Both methods suggested reduced biosynthesis and increased breakdown or remodeling of noncellulosic cell wall polymers, which was further supported by Fourier transform infrared spectroscopy and wet chemistry analysis. The reduced levels of lignin and hemicellulose were associated with an increased proportion of cellulose. Furthermore, the transcript and metabolite profiling data pointed toward a stress response induced by the altered cell wall structure. Finally, chemical pulping of wood derived from 5-year-old, field-grown transgenic lines revealed improved pulping characteristics, but growth was affected in all transgenic lines tested.

A novel procedure for strain classification of fungal mycelium by cluster and artificial neural network analysis of Fourier transform infrared (FTIR) spectra

Fourier transform infrared spectroscopy (FTIR) was used to discriminate important wood-destroying fungi. Mycelia of 26 fungal strains belonging to 24 different species were grown on agar plates and subjected to FTIR attenuated total reflection (ATR) measurements. To classify the FTIR spectra, cluster analysis--an unsupervised multivariate data analysis method--was compared with artificial neural network (ANN) analysis--a supervised approach. By internal validation, both methods classified 99% of the spectra correctly. External validation with independent test set spectra resulted in 95% correctly classified spectra, demonstrating the high potential of this method for fungal strain identification.

FTIR spectroscopy in combination with principal component analysis or cluster analysis as a tool to distinguish beech (Fagus sylvatica L.) trees grown at different sites

Abstract FTIR spectroscopy was used to distinguish between beech (Fagus sylvatica L.) trees grown at five different sites; one in middle Germany close to Göttingen (forest district Reinhausen), three located in the southwest (two in Rhineland-Palatinate: forest districts Saarburg and Hochwald, and one in Luxembourg), and one in North-Rhine Westfalia. Detailed investigation of the spectra in the fingerprint region (1800–600 cm-1) revealed 16 distinct peaks and shoulders, most of which were assignable to wavenumbers previously shown to represent wood compounds. Differences in peak heights and peak ratios indicated differences in wood composition of beech trees from different sites. To determine if the wood of individual trees could be distinguished, principal component analysis (PCA) and cluster analysis were performed using FTIR spectra as input data. With both PCA and cluster analysis, trees from four of the five different sites were separated. It was not possible to distinguish between trees from Saarburg and Hochwald, where similar edaphic and climatic conditions exist, while wood spectra from trees from all other areas clearly segregated. Wood collected at different positions in the stem (bottom, crown, center and outer year rings) of trees grown at the same site was not distinguishable. Therefore, FTIR spectral analysis in combination with multivariate statistical methods can be used to distinguish wood of trees from different growth habitats. Extension of this method to other species may be of great interest for wood certification, as it may be possible to distinguish wood, of a given species, originating from different regions.

Ectomycorrhizal identification in environmental samples of tree roots by Fourier-transform infrared (FTIR) spectroscopy

Roots of forest trees are associated with various ectomycorrhizal (ECM) fungal species that are involved in nutrient exchange between host plant and the soil compartment. The identification of ECM fungi in small environmental samples is difficult. The present study tested the feasibility of attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy followed by hierarchical cluster analysis (HCA) to discriminate in situ collected ECM fungal species. Root tips colonized by distinct ECM fungal species, i.e., Amanita rubescens, Cenococcum geophilum, Lactarius subdulcis, Russula ochroleuca, and Xerocomus pruinatus were collected in mono-specific beech (Fagus sylvatica) and mixed deciduous forests in different geographic areas to investigate the environmental variability of the ECM FTIR signatures. A clear HCA discrimination was obtained for ECM fungal species independent of individual provenance. Environmental variability neither limited the discrimination between fungal species nor provided sufficient resolution to discern species sub-clusters for different sites. However, the de-convoluted FTIR spectra contained site-related spectral information for fungi with wide nutrient ranges, but not for Lactarius subdulcis, a fungus residing only in the litter layer. Specific markers for distinct ECM were identified in spectral regions associated with carbohydrates (i.e., mannans), lipids, and secondary protein structures. The present results support that FTIR spectroscopy coupled with multivariate analysis is a reliable and fast method to identify ECM fungal species in minute environmental samples. Moreover, our data suggest that the FTIR spectral signatures contain information on physiological and functional traits of ECM fungi.

Fourier Transform Infrared (FTIR) Microscopy and Imaging of Fungi

This chapter will begin with the basic principles of Fourier transform infrared (FTIR) spectroscopy, microscopy and imaging followed by highlighted examples of applications.

Genetic Variation of the Host Plant Species Matters for Interactions with Above- and Belowground Herbivores

Plants are challenged by both above- and belowground herbivores which may indirectly interact with each other via herbivore-induced changes in plant traits; however, little is known about how genetic variation of the host plant shapes such interactions. We used two genotypes (M4 and E9) of Solanum dulcamara (Solanaceae) with or without previous experience of aboveground herbivory by Spodoptera exigua (Noctuidae) to quantify its effects on subsequent root herbivory by Agriotes spp. (Elateridae). In the genotype M4, due to the aboveground herbivory, shoot and root biomass was significantly decreased, roots had a lower C/N ratio and contained significantly higher levels of proteins, while the genotype E9 was not affected. However, aboveground herbivory had no effects on weight gain or mortality of the belowground herbivores. Root herbivory by Agriotes increased the nitrogen concentration in the roots of M4 plants leading to a higher weight gain of conspecific larvae. Also, in feeding bioassays, Agriotes larvae tended to prefer roots of M4 over E9, irrespective of the aboveground herbivore treatment. Fourier-Transform Infrared Spectroscopy (FT-IR) documented differences in metabolic profiles of the two plant genotypes and of the roots of M4 plants after aboveground herbivory. Together, these results demonstrate that previous aboveground herbivory can have genotype-specific effects on quantitative and qualitative root traits. This may have consequences for belowground interactions, although generalist root herbivores might not be affected when the root biomass offered is still sufficient for growth and survival.