Elmar Schmälzlin

Regulation of Respiration and Fermentation to Control the Plant Internal Oxygen Concentration

Plant internal oxygen concentrations can drop well below ambient even when the plant grows under optimal conditions. Using pea (Pisum sativum) roots, we show how amenable respiration adapts to hypoxia to save oxygen when the oxygen availability decreases. The data cannot simply be explained by oxygen being limiting as substrate but indicate the existence of a regulatory mechanism, because the oxygen concentration at which the adaptive response is initiated is independent of the actual respiratory rate. Two phases can be discerned during the adaptive reaction: an initial linear decline of respiration is followed by a nonlinear inhibition in which the respiratory rate decreased progressively faster upon decreasing oxygen availability. In contrast to the cytochrome c pathway, the inhibition of the alternative oxidase pathway shows only the linear component of the adaptive response. Feeding pyruvate to the roots led to an increase of the oxygen consumption rate, which ultimately led to anoxia. The importance of balancing the in vivo pyruvate availability in the tissue was further investigated. Using various alcohol dehydrogenase knockout lines of Arabidopsis (Arabidopsis thaliana), it was shown that even under aerobic conditions, alcohol fermentation plays an important role in the control of the level of pyruvate in the tissue. Interestingly, alcohol fermentation appeared to be primarily induced by a drop in the energy status of the tissue rather than by a low oxygen concentration, indicating that sensing the energy status is an important component of optimizing plant metabolism to changes in the oxygen availability.

Decreased Expression of Cytosolic Pyruvate Kinase in Potato Tubers Leads to a Decline in Pyruvate Resulting in an in Vivo Repression of the Alternative Oxidase

The aim of this work was to investigate the effect of decreased cytosolic pyruvate kinase (PKc) on potato (Solanum tuberosum) tuber metabolism. Transgenic potato plants with strongly reduced levels of PKc were generated by RNA interference gene silencing under the control of a tuber-specific promoter. Metabolite profiling showed that decreased PKc activity led to a decrease in the levels of pyruvate and some other organic acids involved in the tricarboxylic acid cycle. Flux analysis showed that this was accompanied by changes in carbon partitioning, with carbon flux being diverted from glycolysis toward starch synthesis. However, this metabolic shift was relatively small and hence did not result in enhanced starch levels in the tubers. Although total respiration rates and the ATP to ADP ratio were largely unchanged, transgenic tubers showed a strong decrease in the levels of alternative oxidase (AOX) protein and a corresponding decrease in the capacity of the alternative pathway of respiration. External feeding of pyruvate to tuber tissue or isolated mitochondria resulted in activation of the AOX pathway, both in the wild type and the PKc transgenic lines, providing direct evidence for the regulation of AOX by changes in pyruvate levels. Overall, these results provide evidence for a crucial role of PKc in the regulation of pyruvate levels as well as the level of the AOX in heterotrophic plant tissue, and furthermore reveal that these parameters are interlinked in vivo.

HRE-Type Genes are Regulated by Growth-Related Changes in Internal Oxygen Concentrations During the Normal Development of Potato (Solanum tuberosum) Tubers

The occurrence of hypoxic conditions in plants not only represents a stress condition but is also associated with the normal development and growth of many organs, leading to adaptive changes in metabolism and growth to prevent internal anoxia. Internal oxygen concentrations decrease inside growing potato tubers, due to their active metabolism and increased resistance to gas diffusion as tubers grow. In the present work, we identified three hypoxia-responsive ERF (StHRE) genes whose expression is regulated by the gradual decrease in oxygen tensions that occur when potato tubers grow larger. Increasing the external oxygen concentration counteracted the modification of StHRE expression during tuber growth, supporting the idea that the actual oxygen levels inside the organs, rather than development itself, are responsible for the regulation of StHRE genes. We identified several sugar metabolism-related genes co-regulated with StHRE genes during tuber development and possibly involved in starch accumulation. All together, our data suggest a possible role for low oxygen in the regulation of sugar metabolism in the potato tuber, similar to what happens in storage tissues during seed development.

Intracellular pH homeostasis and serotonin-induced pH changes in Calliphora salivary glands: the contribution of V-ATPase and carbonic anhydrase.

SUMMARY Blowfly salivary gland cells have a vacuolar-type H+-ATPase (V-ATPase) in their apical membrane that energizes secretion of a KCl-rich saliva upon stimulation with serotonin (5-hydroxytryptamine, 5-HT). We have used BCECF to study microfluometrically whether V-ATPase and carbonic anhydrase (CA) are involved in intracellular pH (pHi) regulation, and we have localized CA activity by histochemistry. We show: (1) mean pHi in salivary gland cells is 7.5±0.3 pH units (N=96), higher than that expected from passive H+ distribution; (2) low 5-HT concentrations (0.3–3 nmol l–1) induce a dose-dependent acidification of up to 0.2 pH units, with 5-HT concentrations >10 nmol l–1, causing monophasic or multiphasic pH changes; (3) the acidifying effect of 5-HT is mimicked by bath application of cAMP, forskolin or IBMX; (4) salivary gland cells exhibit CA activity; (5) CA inhibition with acetazolamide and V-ATPase inhibition with concanamycin A lead to a slow acidification of steady-state pHi; (6) 5-HT stimuli in the presence of acetazolamide induce an alkalinization that can be decreased by simultaneous application of the V-ATPase inhibitor concanamycin A; (7) concanamycin A removes alkali-going components from multiphasic 5-HT-induced pH changes; (8) NHE activity and a Cl–-dependent process are involved in generating 5-HT-induced pH changes; (9) the salivary glands probably contain a Na+-driven amino acid transporter. We conclude that V-ATPase and CA contribute to steady-state pHi regulation and 5-HT-induced outward H+ pumping does not cause an alkalinization of pHi because of cytosolic H+ accumulation attributable to stimulated cellular respiration and AE activity, masking the alkalizing effect of V-ATPase-mediated acid extrusion.

New Challenges in biophotonics: Laser-based fluoroimmuno analysis and in-vivo optical oxygen monitoring

Two examples of our biophotonic research utilizing nanoparticles are presented, namely laser-based fluoroimmuno analysis and in-vivo optical oxygen monitoring. Results of the work include significantly enhanced sensitivity of a homogeneous fluorescence immunoassay and markedly improved spatial resolution of oxygen gradients in root nodules of a legume species.

Distributed Fiber Optical Sensing of Oxygen with Optical Time Domain Reflectometry

In many biological and environmental applications spatially resolved sensing of molecular oxygen is desirable. A powerful tool for distributed measurements is optical time domain reflectometry (OTDR) which is often used in the field of telecommunications. We combine this technique with a novel optical oxygen sensor dye, triangular-[4] phenylene (TP), immobilized in a polymer matrix. The TP luminescence decay time is 86 ns. The short decay time of the sensor dye is suitable to achieve a spatial resolution of some meters. In this paper we present the development and characterization of a reflectometer in the UV range of the electromagnetic spectrum as well as optical oxygen sensing with different fiber arrangements.

Distributed fiber optical sensing of molecular oxygen with OTDR

Spatially resolved sensing of molecular oxygen is important for many biological and environmental applications. For this purpose, time-resolved fluorescence measurements were combined with optical time domain reflectometry (OTDR), a technique which was primarily developed for inspections of optical fiber lines. To achieve spatial resolution of some meters, which are typical for commercial OTDR instruments, the lifetimes of the sensor dyes must be within the range of some nanoseconds, which is much shorter than the decay times of common phosphorescent oxygen probes. Therefore, the measurements were performed with a novel fluorescent triangular-[4]phenylene sensor dye. The fluorescence decay times are around 80 ns in absence of oxygen and around 20 ns in the presence of air. The [4]phenylene sensor dye was applied in toluene solution as well as immobilized in a polymer film. Using a branched model fiber line, oxygen measurements were carried out in a micro- to millimolar concentration range. Oxygen-dependent fluorescence decay times measured with OTDR in toluene were verified by use of time-correlated single photon counting (TCSPC). The Stern-Volmer plots obtained for fluorescence quenching of sensor dyes dissolved in toluene solution and polymer-based sensor spots show good linearity.

The Potsdam MRS spectrograph: heritage of MUSE and the impact of cross-innovation in the process of technology transfer

After having demonstrated that an IFU, attached to a microscope rather than to a telescope, is capable of differentiating complex organic tissue with spatially resolved Raman spectroscopy, we have launched a clinical validation program that utilizes a novel optimized fiber-coupled multi-channel spectrograph whose layout is based on the modular MUSE spectrograph concept. The new design features a telecentric input and has an extended blue performance, but otherwise maintains the properties of high throughput and excellent image quality over an octave of wavelength coverage with modest spectral resolution. We present the opto-mechanical layout and details of its optical performance.

Raman imaging of large-area human tissue

Raman spectroscopy has been successfully applied to medical applications and emerged as a powerful non-invasive technique able to provide information to identify tissue diseases and guiding during image surgery. Of particular interest for medical diagnosis are Raman images in which a complete Raman spectrum is acquired for each pixel of the image enabling the generation of chemical maps. However, Raman images are usually recorded in a time consuming step-by-step scanning process that may take several minutes or even hours. Integral field Spectroscopy (IFS) is a technique used in Astronomy that reduces the recording time by combining imaging and spectroscopic capabilities in a single instrument. Both, spatial and spectral information are provided simultaneously without scanning procedure and with outstanding light collection efficiency [1]. IFS is based on slicing the image using a mirror stack or a bundle of optical fibers and stringing the slices together in front of the slit of a multiplex spectrograph. Raman SERDS (Shifted Excitation Raman Differential Spectroscopy) images of large-area porcine skin patches and the basic principles for a pseudo-real-time video Raman imaging have already been reported using this technique [2].

Minimally invasive fiber probes for optical in-vivo oxygen measurements with integrated optical thermometer

Optical measurements of molecular oxygen are based on phosphorescent dyes with decay times dependent on the ambient oxygen content. Additionally, the phosphorescence decay times are affected by the temperature. In this work, we present miniaturized fiber optical probes, which are able to determine both ambient oxygen content and temperature simultaneously.