Quantitative monitoring of paramagnetic contrast agents and their allocation in plant tissues via DCE-MRI.

BACKGROUND: Studying dynamic processes in living organisms with MRI is one of the most promising research areas. The use of paramagnetic compounds as contrast agents (CA), has proven key to such studies, but so far, the lack of appropriate techniques limits the application of CA-technologies in experimental plant biology. The presented proof-of-principle aims to support method and knowledge transfer from medical research to plant science. RESULTS: In this study, we designed and tested a new approach for plant Dynamic Contrast Enhanced Magnetic Resonance Imaging (pDCE-MRI). The new approach has been applied in situ to a cereal crop (Hordeum vulgare). The pDCE-MRI allows non-invasive investigation of CA allocation within plant tissues. In our experiments, gadolinium-DTPA, the most commonly used contrast agent in medical MRI, was employed. By acquiring dynamic T1-maps, a new approach visualizes an alteration of a tissue-specific MRI parameter T1 (longitudinal relaxation time) in response to the CA. Both, the measurement of local CA concentration and the monitoring of translocation in low velocity ranges (cm/h) was possible using this CA-enhanced method. CONCLUSIONS: A novel pDCE-MRI method is presented for non-invasive investigation of paramagnetic CA allocation in living plants. The temporal resolution of the T1-mapping has been significantly improved to enable the dynamic in vivo analysis of transport processes at low-velocity ranges, which are common in plants. The newly developed procedure allows to identify vascular regions and to estimate their involvement in CA allocation. Therefore, the presented technique opens a perspective for further development of CA-aided MRI experiments in plant biology.

Probing the Metabolic Landscape of Plant Vascular Bundles by Infrared Fingerprint Analysis, Imaging and Mass Spectrometry.

Fingerprint analysis is a common technique in forensic and criminal investigations. Similar techniques exist in the field of infrared spectroscopy to identify biomolecules according to their characteristic spectral fingerprint features. These unique markers are located in a wavenumber range from 1800 to 600 cm-1 in the mid infrared region. Here, a novel bioanalytical concept of correlating these spectral features with corresponding mass spectrometry datasets to unravel metabolic clusters within complex plant tissues was applied. As proof of concept, vascular bundles of oilseed rape (Brassica napus) were investigated, one of the most important and widely cultivated temperate zone oilseed crops. The link between mass spectrometry data and spectral data identified features that co-aligned within both datasets. Regions of origin were then detected by searching for these features in hyperspectral images of plant tissues. This approach, based on co-alignment and co-localization, finally enabled the detection of eight distinct metabolic clusters, reflecting functional and structural arrangements within the vascular bundle. The proposed analytical concept may assist future synergistic research approaches and may lead to biotechnological innovations with regard to crop yield and sustainability.

The tyrosine phosphatase SHP2 increases robustness and information transfer within IL-6-induced JAK/STAT signalling.

BACKGROUND: Cell-to-cell heterogeneity is an inherent feature of multicellular organisms and is central in all physiological and pathophysiological processes including cellular signal transduction. The cytokine IL-6 is an essential mediator of pro- and anti-inflammatory processes. Dysregulated IL-6-induced intracellular JAK/STAT signalling is associated with severe inflammatory and proliferative diseases. Under physiological conditions JAK/STAT signalling is rigorously controlled and timely orchestrated by regulatory mechanisms such as expression of the feedback-inhibitor SOCS3 and activation of the protein-tyrosine phosphatase SHP2 (PTPN11). Interestingly, the function of negative regulators seems not to be restricted to controlling the strength and timely orchestration of IL-6-induced STAT3 activation. Exemplarily, SOCS3 increases robustness of late IL-6-induced STAT3 activation against heterogenous STAT3 expression and reduces the amount of information transferred through JAK/STAT signalling. METHODS: Here we use multiplexed single-cell analyses and information theoretic approaches to clarify whether also SHP2 contributes to robustness of STAT3 activation and whether SHP2 affects the amount of information transferred through IL-6-induced JAK/STAT signalling. RESULTS: SHP2 increases robustness of both basal, cytokine-independent STAT3 activation and early IL-6-induced STAT3 activation against differential STAT3 expression. However, SHP2 does not affect robustness of late IL-6-induced STAT3 activation. In contrast to SOCS3, SHP2 increases the amount of information transferred through IL-6-induced JAK/STAT signalling, probably by reducing cytokine-independent STAT3 activation and thereby increasing sensitivity of the cells. These effects are independent of SHP2-dependent MAPK activation. CONCLUSION: In summary, the results of this study extend our knowledge of the functions of SHP2 in IL-6-induced JAK/STAT signalling. SHP2 is not only a repressor of basal and cytokine-induced STAT3 activity, but also ensures robustness and transmission of information. Plain English summary Cells within a multicellular organism communicate with each other to exchange information about the environment. Communication between cells is facilitated by soluble molecules that transmit information from one cell to the other. Cytokines such as interleukin-6 are important soluble mediators that are secreted when an organism is faced with infections or inflammation. Secreted cytokines bind to receptors within the membrane of their target cells. This binding induces activation of an intracellular cascade of reactions called signal transduction, which leads to cellular responses. An important example of intracellular signal transduction is JAK/STAT signalling. In healthy organisms signalling is controlled and timed by regulatory mechanisms, whose activation results in a controlled shutdown of signalling pathways. Interestingly, not all cells within an organism are identical. They differ in the amount of proteins involved in signal transduction, such as STAT3. These differences shape cellular communication and responses to intracellular signalling. Here, we show that an important negative regulatory protein called SHP2 (or PTPN11) is not only responsible for shutting down signalling, but also for steering signalling in heterogeneous cell populations. SHP2 increases robustness of STAT3 activation against variable STAT3 amounts in individual cells. Additionally, it increases the amount of information transferred through JAK/STAT signalling by increasing the dynamic range of pathway activation in heterogeneous cell populations. This is an amazing new function of negative regulatory proteins that contributes to communication in heterogeneous multicellular organisms in health and disease. Video Abstract.

A mechanistic view on lodging resistance in rye and wheat: a multiscale comparative study.

The development of crop varieties that are resistant to lodging is a top priority for breeding programmes. Herein, we characterize the rye mutant ´Stabilstroh' ('stable straw') possessing an exceptional combination of high lodging resistance, tall posture and high biomass production. Nuclear magnetic resonance imaging displayed the 3-dimensional assembly of vascular bundles in stem. A higher number of vascular bundles and a higher degree of their incline were the features of lodging-resistant versus lodging-prone lines. Histology and electron microscopy revealed that stems are fortified by a higher proportion of sclerenchyma and thickened cell walls, as well as some epidermal invaginations. Biochemical analysis using Fourier-transform infrared spectroscopy and inductively coupled plasma-optical emission spectrometry further identified elevated levels of lignin, xylan, zinc and silicon as features associated with high lodging resistance. Combined effects of above features caused superior culm stability. A simplistic mathematical model showed how mechanical forces distribute within the stem under stress. Main traits of the lodging-resistant parental line were heritable and could be traced back to the genetic structure of the mutant. Evaluation of lodging-resistant wheat 'Babax' ('Baviacora') versus contrasting, lodging-prone, genotype ´Pastor´ agreed with above findings on rye. Our findings on mechanical stability and extraordinary culm properties may be important for breeders for the improvement of lodging resistance of tall posture cereal crops.

Cellular Plasticity in Response to Suppression of Storage Proteins in the Brassica napus Embryo.

The tradeoff between protein and oil storage in oilseed crops has been tested here in oilseed rape (Brassica napus) by analyzing the effect of suppressing key genes encoding protein storage products (napin and cruciferin). The phenotypic outcomes were assessed using NMR and mass spectrometry imaging, microscopy, transcriptomics, proteomics, metabolomics, lipidomics, immunological assays, and flux balance analysis. Surprisingly, the profile of storage products was only moderately changed in RNA interference transgenics. However, embryonic cells had undergone remarkable architectural rearrangements. The suppression of storage proteins led to the elaboration of membrane stacks enriched with oleosin (sixfold higher protein abundance) and novel endoplasmic reticulum morphology. Protein rebalancing and amino acid metabolism were focal points of the metabolic adjustments to maintain embryonic carbon/nitrogen homeostasis. Flux balance analysis indicated a rather minor additional demand for cofactors (ATP and NADPH). Thus, cellular plasticity in seeds protects against perturbations to its storage capabilities and, hence, contributes materially to homeostasis. This study provides mechanistic insights into the intriguing link between lipid and protein storage, which have implications for biotechnological strategies directed at improving oilseed crops.

Micro Imaging Displays the Sucrose Landscape within and along Its Allocation Pathways.

Sucrose (Suc) is the major transport sugar in plants and plays a primary role as an energy source and signal in adaptive and stress responses. An ability to quantify Suc over time and space would serve to advance our understanding of these important processes. Current technologies used for Suc mapping are unable to quantitatively visualize its distribution within tissues. Here, we present an infrared-based microspectroscopic method that allows for the quantitative visualization of Suc at a microscopic level of resolution (∼12 µm). This method can successfully model the sugar concentration in individual vascular bundles and within a complex organ such as the stem, leaf, or seed. The sensitivity of the assay ranges from 20 to 1,000 mm We applied this method to the cereal crop barley (Hordeum vulgare) and the model plant Arabidopsis (Arabidopsis thaliana) to highlight the potential of the procedure for resolving the spatial distribution of metabolites. We also discuss the relevance of the method for studies on carbon allocation and storage in the context of crop improvement.

A functional imaging study of germinating oilseed rape seed.

Germination, the process whereby a dry, quiescent seed springs to life, has been a focus of plant biologist for many years, yet the early events following water uptake, during which metabolism of the embryo is restarted, remain enigmatic. Here, the nature of the cues required for this restarting in oilseed rape (Brassica napus) seed has been investigated. A holistic in vivo approach was designed to display the link between the entry and allocation of water, metabolic events and structural changes occurring during germination. For this, we combined functional magnetic resonance imaging with Fourier transform infrared microscopy, fluorescence-based respiration mapping, computer-aided seed modeling and biochemical tools. We uncovered an endospermal lipid gap, which channels water to the radicle tip, from whence it is distributed via embryonic vasculature toward cotyledon tissues. The resumption of respiration is initiated first in the endosperm, only later spreading to the embryo. Sugar metabolism and lipid utilization are linked to the spatiotemporal sequence of tissue rehydration. Together, this imaging study provides insights into the spatial aspects of key events in oilseed rape seeds leading to germination. It demonstrates how seed architecture predetermines the pattern of water intake, which sets the stage for the orchestrated restart of life.

The homeodomain transcription factor TaHDZipI-2 from wheat regulates frost tolerance, flowering time and spike development in transgenic barley.

Homeodomain leucine zipper class I (HD-Zip I) transcription factors (TFs) play key roles in the regulation of plant growth and development under stresses. Functions of the TaHDZipI-2 gene isolated from the endosperm of developing wheat grain were revealed. Molecular characterization of TaHDZipI-2 protein included studies of its dimerisation, protein-DNA interactions and gene activation properties using pull-down assays, in-yeast methods and transient expression assays in wheat cells. The analysis of TaHDZipI-2 gene functions was performed using transgenic barley plants. It included comparison of developmental phenotypes, yield components, grain quality, frost tolerance and the levels of expression of potential target genes in transgenic and control plants. Transgenic TaHDZipI-2 lines showed characteristic phenotypic features that included reduced growth rates, reduced biomass, early flowering, light-coloured leaves and narrowly elongated spikes. Transgenic lines produced 25-40% more seeds per spike than control plants, but with 50-60% smaller grain size. In vivo lipid imaging exposed changes in the distribution of lipids between the embryo and endosperm in transgenic seeds. Transgenic lines were significantly more tolerant to frost than control plants. Our data suggest the role of TaHDZipI-2 in controlling several key processes underlying frost tolerance, transition to flowering and spike development.