Matthias Gilbert

Evidence for the existence of one antenna-associated, lipid-dissolved and two protein-bound pools of diadinoxanthin cycle pigments in diatoms

We studied the localization of diadinoxanthin cycle pigments in the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum. Isolation of pigment protein complexes revealed that the majority of high-light-synthesized diadinoxanthin and diatoxanthin is associated with the fucoxanthin chlorophyll protein (FCP) complexes. The characterization of intact cells, thylakoid membranes, and pigment protein complexes by absorption and low-temperature fluorescence spectroscopy showed that the FCPs contain certain amounts of protein-bound diadinoxanthin cycle pigments, which are not significantly different in high-light and low-light cultures. The largest part of high-light-formed diadinoxanthin cycle pigments, however, is not bound to antenna apoproteins but located in a lipid shield around the FCPs, which is copurified with the complexes. This lipid shield is primarily composed of the thylakoid membrane lipid monogalactosyldiacylglycerol. We also show that the photosystem I (PSI) fraction contains a tightly connected FCP complex that is enriched in protein-bound diadinoxanthin cycle pigments. The peripheral FCP and the FCP associated with PSI are composed of different apoproteins. Tandem mass spectrometry analysis revealed that the peripheral FCP is composed mainly of the light-harvesting complex protein Lhcf and also significant amounts of Lhcr. The PSI fraction, on the other hand, shows an enrichment of Lhcr proteins, which are thus responsible for the diadinoxanthin cycle pigment binding. The existence of lipid-dissolved and protein-bound diadinoxanthin cycle pigments in the peripheral antenna and in PSI is discussed with respect to different specific functions of the xanthophylls.

Bio-optical modelling of oxygen evolution using in vivo fluorescence: comparison of measured and calculated photosynthesis/irradiance (P-I) curves in four representative phytoplankton species

Summary The measurement of variable fluorescence yields relative electron transport rates which can not directly be converted into absolute values of photosynthetic rates. We have developed a measuring device which provides identical optical geometry for simultaneous fluorescence and oxygen measurements in single cell or chloroplast suspensions and also allows the determination of the absorbed photosynthetic radiation (Q phar ). In this set-up we have simultaneously measured photosynthesis-irradiance curves (P-I curves) of algal cell suspensions with both PAM fluorometry and a Clark-type electrode. From the electron flow through photosystem II per absorbed light quanta (Q phar ) we have modeled oxygen based P-I curves. In order to evaluate the validity of the model we have compared modeled with directly measured oxygen based P-I curves. This comparison was extended to four different algal taxa which exhibit significantly different absorption spectra leading to changes in Q phar . The results show that in the initial slope of the P-I curves (α), calculated and measured oxygen production is quite similar on an absolute scale, whereas at the transition part (l k = P max /α) and at maximal photosynthetic rates (P max ) both methods can deviate significantly from each other depending on the species used. Therefore, the conversion of fluorescence based electron transfer rates through photosystem II into oxygen production is a reliable procedure for light intensities below the l k . However, at light intensities close to light saturation, physiological regulation processes can cause problems in the interconversion of fluorescence- and oxygen-based photosynthetic rates.

Dual Role of the Plastid Terminal Oxidase in Tomato

The plastid terminal oxidase (PTOX) is a plastoquinol oxidase whose absence in tomato (Solanum lycopersicum) results in the ghost (gh) phenotype characterized by variegated leaves (with green and bleached sectors) and by carotenoid-deficient ripe fruit. We show that PTOX deficiency leads to photobleaching in cotyledons exposed to high light primarily as a consequence of reduced ability to synthesize carotenoids in the gh mutant, which is consistent with the known role of PTOX as a phytoene desaturase cofactor. In contrast, when entirely green adult leaves from gh were produced and submitted to photobleaching high light conditions, no evidence for a deficiency in carotenoid biosynthesis was obtained. Rather, consistent evidence indicates that the absence of PTOX renders the tomato leaf photosynthetic apparatus more sensitive to light via a disturbance of the plastoquinone redox status. Although gh fruit are normally bleached (most likely as a consequence of a deficiency in carotenoid biosynthesis at an early developmental stage), green adult fruit could be obtained and submitted to photobleaching high light conditions. Again, our data suggest a role of PTOX in the regulation of photosynthetic electron transport in adult green fruit, rather than a role principally devoted to carotenoid biosynthesis. In contrast, ripening fruit are primarily dependent on PTOX and on plastid integrity for carotenoid desaturation. In summary, our data show a dual role for PTOX. Its activity is necessary for efficient carotenoid desaturation in some organs at some developmental stages, but not all, suggesting the existence of a PTOX-independent pathway for plastoquinol reoxidation in association with phytoene desaturase. As a second role, PTOX is implicated in a chlororespiratory mechanism in green tissues.

A new type of thermoluminometer: A highly sensitive tool in applied photosynthesis research and plant stress physiology

Here we describe a newly developed thermoluminescence measuring device that employs flash excitation, peltier heating, and light detection by channel photomultipliers (CPM). The new thermoluminometer is equipped with four sample holders for simultaneous measurements of thermoinduced light emission in the temperature range from -20 degrees C to +180 degrees C. It allows one to measure leaf samples, chloroplasts, thylakoids, algae, or even bioorganic material lacking chlorophyll by means of naturally induced or artificially applied chemilumigenic probes. The temperature range of the thermoluminometer allows one to analyse the thermoinduced radical pair recombination of photosystem II in the lower temperature region as well as chemiluminescence from lipid peroxidation in the higher temperature region. Hence, plant material can be assessed concerning both its photosynthetic and its oxidative stress status. Since the device is equipped with four sample holders and four CPM channels for simultaneous detection of thermoinduced light emission, it facilitates a high throughput. Therefore, the new device is interesting, not only in ecophysiology, but also in the field of plant breeding, as it can be used to study the stress tolerance of various cultivars of cultural crop plants.

Evaluation of carbon tetrachloride-induced stress on rat hepatocytes by 31P NMR and MALDI-TOF mass spectrometry: lysophosphatidylcholine generation from unsaturated phosphatidylcholines.

Carbon tetrachloride (CCl(4)) represents an excellent model to study oxidative injury of cells. It is widely accepted that hepatocellular injury is a consequence of the metabolic conversion of CCl(4) into highly reactive, free radical intermediates. Among the direct toxic effects of CCl(4), stimulation of lipid peroxidation and the binding of the electrophilic radicals to membrane lipids have been suggested to play important roles in the pathogenesis of irreversible cell damage. CCl(4)-induced liver damage was modeled in cultures of rat hepatocytes with the focus on alterations of phosphatidylcholine (PC). The PC acyl chain composition was analyzed by (31)P NMR spectroscopy and MALDI-TOF mass spectrometry. The content of the membrane arachidonoyl PC was decreased by almost 30% after incubation of the cells with CCl(4). This relative decrease was found to correlate with increased concentrations of the corresponding saturated lysophosphatidylcholine (LPC). It is concluded that LPC represents a useful biomarker of CCl(4)-mediated damaging of hepatocytes. It is also speculated that de novo biosynthesis of PC is influenced by CCl(4).

Effects of UV irradiation on barley and tomato leaves: thermoluminescence as a method to screen the impact of UV radiation on crop plants

The effect of different UV intensities and irradiation times on barley and tomato leaves was investigated by analysis of thermoluminescence (TL) and chlorophyll (chl) fluorescence measurements. Epifluorescence microscopy was used to estimate the epidermal UV transmittance of leaves. In barley a strong supression of TL emission from the S2QB- (B-band) and the S2QA- (Q-band) charge recombination was observed increasing with prolonged UV exposure. Primary barley leaves were more sensitive to UV than secondary leaves. In tomato plants a decrease in the B-band only takes place at very high UV intensities and after prolonged exposure times (4 h). The impact of UV in cotyledons was more pronounced than in pinnate leaves of tomato plants. The strong differences in sensitivity to UV in the investigated barley and tomato variety may be due to different concentrations of UV screening pigments in the epidermal layer as demonstrated by epifluorescence measurements. The results show that TL has the same potential to analyse the sensitivity or tolerance of crop plants to UV irradiation as routine fluorescence techniques. Furthermore, TL is directly monitoring the radical pair states of PSII and can distinguish between UV-induced donor and acceptor site-related damage.

Can Chlorophyll-a in-vivo fluorescence be used for quantification of carbon-based primary production in absolute terms?

We tested the Chlorophyll-a in-vivo fluorescence technique as a method to measure absolute values of primary production by the application of a bio-optical model. In this model, absolute values of gross photosynthetic oxygen evolution were calculated on the basis of Chlorophyll-a in-vivo fluorescence, such as photosystem II quantum yield and the amount of absorbed light quanta per time (Q Phar ). Simultaneous measurements of photosynthesis rates, as measured by a Clark-type electrode and estimated via the fluorescence based model, yield similar results. The P-I-curves measured by these two independent methods are nearly identical under light limitation, whereas maximal photosynthetic activities under saturating light are found to be higher using the bio-optical model. Furthermore, we determined the relationship between carbon-based biomass formation and the photosynthesis rate under low and high light. The quantity of electrons consumed for C and N assimilation is the same as the quantity of electrons produced by the photosystems under low light. Under high light, the electron production exceeds the electron consumption for N and C assimilation. We found that an oxygen production of 55-60 μmol O 2 is equivalent to the production of 1 mg dry matter. We conclude that the photosynthetic electron transport rate represents a fairly good estimated measure of newly synthesised organic matter under the applied conditions. Therefore, the use of fluorescence data in an appropriate bio-optical model is a powerful tool for measuring biomass formation in absolute terms but is restricted to replete nutrient and medium light conditions. The validity of this experimental approach for measuring primary production under stress conditions still needs to be elaborated.

Intra- and interspecific differences of 10 barley and 10 tomato cultivars in response to short-time UV-B radiation: A study analysing thermoluminescence, fluorescence, gas-exchange and biochemical parameters

The impact of UV-B radiation on 10 genotypically different barley and tomato cultivars was tested in a predictive study to screen for potentially UV-tolerant accessions and to analyze underlying mechanisms for UV-B sensitivity. Plant response was analyzed by measuring thermoluminescence, fluorescence, gas exchange and antioxidant status. Generally, barley cultivars proved to be much more sensitive against UV-B radiation than tomato cultivars. Statistical cluster analysis could resolve two barley groups with distinct differences in reaction patterns. The UV-B sensitive group showed a stronger loss in PSII photochemistry and a lower gas-exchange performance and regulation after UV-B radiation compared to the more tolerant group. The results indicate that photosynthetic light and dark reactions have to play optimally in concert to render plants more tolerant against UV-B radiation. Hence, measuring thermoluminescence/fluorescence and gas exchange in parallel will have much higher potential in identifying tolerant cultivars and will help to understand the underlying mechanisms.

Thermoluminescence as a tool for monitoring ozone-stressed plants

The effect of ozone (6 h, various concentrations from 0 to 350 ppb) on barley (Hordeum vulgare L., cv. Bomi) and tomato (Lycopersicon esculentum L., cv. Yellow Cherry) leaves was investigated in parallel by thermoluminescence (TL) and fluorescence (FL) methods. Several significant changes were found in TL glow curves measured after excitation by one single turnover flash at +2 degree C in the temperature range from 2 to 170 degree C immediately after ozone exposure. Contrary to TL, ozone induced only negligible changes in FL parameters F0, FM and Fv/FM. Measurements done 24 h after ozone exposure showed partial recovery of ozone-induced changes. The extent of recovery was not the same in different parts of TL curves. Fluorescence parameters were not significantly changed. The results demonstrate that TL parameters are more sensitive to ozone than conventially used FL parameters F0, FM and Fv/FM. Moreover, TL measurements seem to give information not only about the PSII electron transport, but also about the extent of oxidative damage and membrane lipid peroxidation. It is concluded, that TL can be a highly informative tool for monitoring the impact of ozone on plants.

The mechanism of the ozone-induced changes in thermoluminescence glow curves of barley leaves

The changes in thermoluminescence (TL) signals induced by short-term ozone exposure of leaves are characterized by a down-shift of the peak-temperature of the TLB-band and an increase of a TL band at 55°C. We investigated the relationship of these changes to photosystem 2 (PS2) photochemistry. The changes were not only detectable in the presence of ozone, but also after irradiation of dark-adapted leaves and after aging of irradiated detached leaf segments. The opposite effect on TL, an up-shift of the peak-temperature of the B-band and the decrease of the intensity of the band at 55°C were found after infiltration of leaves with nigericin, antimycin A, and diphenyleneiodonium chloride (DPI). Propyl gallate down-shifted the peak-temperature of the B-band. 2,5-dimethyl-1,4-benzoquinone up-shifted the peak-temperature of the B-band and decreased the intensity of the 55°C band. The intensity of the 55°C band did not change significantly in the presence of oxygen in comparison to that in nitrogen atmosphere. It decreased with time of dark adaptation (50% intensity was observed after 3 h of dark adaptation at room temperature), however, it was reactivated to its initial value (at 5 min of dark adaptation) after 1 single-turnover flash. The 55°C band was not significantly changed in the presence of DCMU. Thus the ozone-induced band at 55°C is assigned to charge recombination in PS2. Changes in the electron transport chain at the acceptor side of PS2, probably related to the cyclic electron transport around photosystem 1 and/or chlororespiration, could play an important role in the increase of the 55°C band and the down-shift of the B-band. The changes at the acceptor side indicated by TL can be an ex pression of a physiological regulatory mechanism functional under stress conditions.