Judith Felten

Vibrational spectroscopic image analysis of biological material using multivariate curve resolution–alternating least squares (MCR-ALS)

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.

Arabidopsis WAT1 is a vacuolar auxin transport facilitator required for auxin homoeostasis

The plant hormone auxin (indole-3-acetic acid, IAA) has a crucial role in plant development. Its spatiotemporal distribution is controlled by a combination of biosynthetic, metabolic and transport mechanisms. Four families of auxin transporters have been identified that mediate transport across the plasma or endoplasmic reticulum membrane. Here we report the discovery and the functional characterization of the first vacuolar auxin transporter. We demonstrate that WALLS ARE THIN1 (WAT1), a plant-specific protein that dictates secondary cell wall thickness of wood fibres, facilitates auxin export from isolated Arabidopsis vacuoles in yeast and in Xenopus oocytes. We unambiguously identify IAA and related metabolites in isolated Arabidopsis vacuoles, suggesting a key role for the vacuole in intracellular auxin homoeostasis. Moreover, local auxin application onto wat1 mutant stems restores fibre cell wall thickness. Our study provides new insight into the complexity of auxin transport in plants and a means to dissect auxin function during fibre differentiation.

Development of the Poplar-Laccaria bicolor Ectomycorrhiza Modifies Root Auxin Metabolism, Signaling, and Response.

Symbiotic ectomycorrhizal interaction leads to the arrest of root growth and is associated with significant changes in auxin metabolism, signaling, and response. Root systems of host trees are known to establish ectomycorrhizae (ECM) interactions with rhizospheric fungi. This mutualistic association leads to dramatic developmental modifications in root architecture, with the formation of numerous short and swollen lateral roots ensheathed by a fungal mantle. Knowing that auxin plays a crucial role in root development, we investigated how auxin metabolism, signaling, and response are affected in poplar (Populus spp.)-Laccaria bicolor ECM roots. The plant-fungus interaction leads to the arrest of lateral root growth with simultaneous attenuation of the synthetic auxin response element DR5. Measurement of auxin-related metabolites in the free-living partners revealed that the mycelium of L. bicolor produces high concentrations of the auxin indole-3-acetic acid (IAA). Metabolic profiling showed an accumulation of IAA and changes in the indol-3-pyruvic acid-dependent IAA biosynthesis and IAA conjugation and degradation pathways during ECM formation. The global analysis of auxin response gene expression and the regulation of AUXIN SIGNALING F-BOX PROTEIN5, AUXIN/IAA, and AUXIN RESPONSE FACTOR expression in ECM roots suggested that symbiosis-dependent auxin signaling is activated during the colonization by L. bicolor. Taking all this evidence into account, we propose a model in which auxin signaling plays a crucial role in the modification of root growth during ECM formation.

Signalling in Ectomycorrhizal Symbiosis

The mechanism by which tree roots and soil fungi interact and form their common, symbiotic organ, the ectomycorrhiza (ECM), involves numerous steps. During this ontogenic process, the developmental programs of both partners are modified in order to enable symbiosis establishment. Both roots and fungus release an array of various metabolites (morphogens and signalling molecules) that establish a molecular cross-talk between symbionts. In contrast to some other plant–microbe interactions, such as rhizobia or arbuscular mycorrhiza symbiosis, the characterization of these signalling molecules and their impact on developmental pathways is poorly known. Recent studies have provided new insights into specific phases and signalling pathways of ECM development on a molecular level and have thereby started to fill the gaps in our understanding of root–fungus communication. Based on this knowledge and recent data from ECM interaction, we will identify possible crosstalk between ECM signalling and root development.

Biology, Chemistry and Structure of Tension Wood

Trees maintain and adjust their stature by developing reaction wood in stems and branches. The physical properties of reaction wood result in a higher strain than in normal wood. Because reaction wood is only formed at one side of the stem, this unilateral strain creates a force and hence a movement of the stem or branches towards a more favorable position. The spectacular modification of cambial growth, cell shape, cell-wall chemistry, and ultrastructure observed in reaction wood has attracted generations of scientists to study its features and molecular regulation. In the early literature, the physiology of reaction wood induction was much studied, especially the relative importance of positional and mechanical sensing for its induction. Even today this is still a matter of debate and confusion, as discussed in the first part of this chapter. In angiosperm trees, reaction wood is denoted tension wood (TW), and in many tree species TW fibers develop an inner cellulose-rich gelatinous layer (G-fibers). Much research has been devoted to understand the chemistry and ultrastructure of the gelatinous layer and its function in creating tension stress in the wood. Less attention has been paid to TW without G-fibers, although it has similar physical properties and function as TW with G-fibers. The chemistry and structural variation of TW, and their importance for TW function, are discussed in the second part of this chapter. Not much is known about the molecular control of TW formation. However, some information has been gained about the role of plant hormones as signaling components in TW induction. The last part of the chapter summarizes this knowledge.

Ethylene signaling via Ethylene Response Factors (ERFs) modifies wood development in hybrid aspen

Background The phytohormone ethylene (ET) has the potential to regulate secondary growth of plants and wood formation in trees. Application of exogenous ethylene or its in planta precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), to wood forming tissues of hybrid aspen (Populus tremula x Populus tremuloides) enhances xylem growth [1]. In the same study it was demonstrated that stimulation of enhanced xylem formation (tension wood, TW) at the upper side of leaning stems is mediated by endogenous ET. The production of endogenous ET in TW forming tissues is further supported by the increase of ACC oxidase gene transcript and enzyme activity on the TW side [2]. The ET perception and signal transmission cascade in Arabidopsis has been linked to the transcriptional activation of Ethylene Response Factors (ERFs) [3,4]. As transcription factors, ERFs regulate the expression of various specific downstream target genes by binding to cis-elements in their promoters [5]. We hypothesize that ERFs participate in xylem development through ethylene signaling and that they are involved in ET responses during TW formation.

Sharing resources for mutual benefit: crosstalk between disciplines deepens the understanding of mycorrhizal symbioses across scales

Mycorrhizal scientists from 53 countries gathered in the city of Prague from 30 July until 4 August 2017 for the 9th International Conference on Mycorrhiza (ICOM9). They came to discuss an ancient symbiosis based on the exchange of resources between plant and fungal partners, with many impacts on plant health (van der Heijden et al., 2015). Much like this mutualistic interaction, delegates from disparate disciplines united with a strong focus on integration and sharing of resources for mutual benefit. By exchanging knowledge among researchers from the fields of molecular biology, physiology and ecology, the participants of ICOM9 made a leap forward in our understanding of symbiotic structure and function at multiple scales.

Ethylene-Related Gene Expression Networks in Wood Formation

Thickening of tree stems is the result of secondary growth, accomplished by the meristematic activity of the vascular cambium. Secondary growth of the stem entails developmental cascades resulting in the formation of secondary phloem outwards and secondary xylem (i.e., wood) inwards of the stem. Signaling and transcriptional reprogramming by the phytohormone ethylene modifies cambial growth and cell differentiation, but the molecular link between ethylene and secondary growth remains unknown. We addressed this shortcoming by analyzing expression profiles and co-expression networks of ethylene pathway genes using the AspWood transcriptome database which covers all stages of secondary growth in aspen (Populus tremula) stems. ACC synthase expression suggests that the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is synthesized during xylem expansion and xylem cell maturation. Ethylene-mediated transcriptional reprogramming occurs during all stages of secondary growth, as deduced from AspWood expression profiles of ethylene-responsive genes. A network centrality analysis of the AspWood dataset identified EIN3D and 11 ERFs as hubs. No overlap was found between the co-expressed genes of the EIN3 and ERF hubs, suggesting target diversification and hence independent roles for these transcription factor families during normal wood formation. The EIN3D hub was part of a large co-expression gene module, which contained 16 transcription factors, among them several new candidates that have not been earlier connected to wood formation and a VND-INTERACTING 2 (VNI2) homolog. We experimentally demonstrated Populus EIN3D function in ethylene signaling in Arabidopsis thaliana. The ERF hubs ERF118 and ERF119 were connected on the basis of their expression pattern and gene co-expression module composition to xylem cell expansion and secondary cell wall formation, respectively. We hereby establish data resources for ethylene-responsive genes and potential targets for EIN3D and ERF transcription factors in Populus stem tissues, which can help to understand the range of ethylene targeted biological processes during secondary growth.

WAT1 (WALLS ARE THIN1) defines a novel auxin transporter in plants and integrates auxin signaling in secondary wall formation in Arabidopsis fibers

Background Our knowledge of signaling mechanisms involved in secondary cell wall (SCW) formation is quite limited. To discover novel markers of SCW, a genomics approach using Zinnia elegans xylogenic cultures was undertaken that identified hundreds of gene candidates expressed at the onset of secondary wall formation [1]. Arabidopsis homologs and the corresponding T-DNA mutants for each Zinnia gene were identified and the panel of Arabidopsis cell wall mutants was subjected to developmental and wall-related phenotyping.