Claudia Büchel

Changes in yield ofin-vivo fluorescence of chlorophyll a as a tool for selective herbicide monitoring

Triazines and derivatives of phenylurea, which are often found in outdoor water samples, induce specific changes in the yield of thein-vivo chlorophyll α-fluorescence of PSII. These changes are correlated quantitatively with the concentration of the herbicides and can therefore be used to set-up a low-price monitor system. In order to detect selectively the herbicide-sensitive part of the fluorescence emission a pulse amplitude modulated fluorimeter was used. The bioassay system was optimised with respect to test organism, growing and measuring conditions. The relationship between fluorescence yield and herbicide concentrations were experimentally determined for the triazines atrazine and simazine and the phenylurea herbicide DCMU and mathematically fitted (r=0.99). The I50-values were 0.9 µM for DCMU, 2.2 µM for simazine and 3.3 µM for atrazine. The detection limit of about 0.5 µM clearly shows that the sensitivity of this bioassay system is too low to reach the requirements of the drinking water regulation. However, due to its insensitivity against complex water matrices, there is good hope to combine this fluorometric bioassay with a potent herbicide preconcentration method like a solid-phase extraction procedure.

Organization of the pigment molecules in the chlorophyll a/c light-harvesting complex of Pleurochloris meiringensis (xanthophyceae). Characterization with circular dichroism and absorbance spectroscopy

Abstract By the aid of circular dichroism (CD), absorbance and fluorescence spectroscopy, we studied the molecular organization of the pigment molecules in cells, isolated chloroplasts and the chlorophyll a / c light-harvesting complex (LHC) associated with photosystem II of the chlorphyll c -containing alga, Pleurochloris meiringensis . In cells and chloroplasts, similarly to higher plant chloroplasts, a (+) 693 nm CD band accompanied by a tail outside the absorbance indicated a long-range chiral organization of the chlorophyll molecules. The LHCII of these algae exhibited an intense negative CD band at 679 nm. However, in contrast to the chlorophyll a / b LHCII of higher plants, where the intense, non-conservative (−)684 nm band has been shown to be associated with long-range chiral organization of the macro-aggregates, the intense, non-conservative (−)679 nm band in the chlorophyl a / c LHC originated from the non-aggregated form of the complexes. In sharp contrast to the trimers or monomers of the chlorophyll a / b LHCII, in the chlorophyll a / c LHC no split excitonic CD bands could be detected in the red spectral region, thus CD provided no indication for the occurence of excitonic interactions among the Q Y transition dipoles of the chlorophyll molecules. Gaussian analysis of the absorbance and CD bands showed that the (−)679 nm CD signal is given rise by a small number of long-wavelength absorbing chlorophyll a molecules. Data obtained with LHC treated with low concentrations of acetone or digitonin strongly suggest a specific binding site of chlorophyll a to the protein, which upon binding a chlorophyll a molecule induces a bathochromic shift and asymmetry in the electronic structure of the molecule. These results and literature data strongly suggest that the organization of the pigment molecules in chlorophyll a / c antenna complexes is significantly different from the organization of the chlorophyll a / b complexes of green algae and higher plants.

Isolation and characterization of a photosystem I-associated antenna (LHC I) and a photosystem I—core complex from the chlorophyll c-containing alga Pleurochloris meiringensis (Xanthophyceae)

Abstract A photosystem (PS) I holocomplex was isolated from Pleurochloris meiringensis Vischer (Xanthophyceae) using sucrose density centrifugation. This complex exhibited a fluorescence emission maximum at 715 nm, which is in accordance with the long wavelength emission of whole cells. The complex was further dissociated into a core complex and a light-harvesting protein (LHC I). The core protein contains mainly Chl a and β-carotene, is 8.25 times enriched in P700 and has its main emission maximum at 715 nm. Therefore, the longest wavelength emission of P. meiringensis is due to the PS I core, which is in contrast to higher plants. The LHC I differs from LHC II with regard to its polypeptide pattern as well as its spectral properties. The arrangement of antennae is discussed in relation to the regulation of energy transfer between the photosystems.

A new fluorometric device to measure the in vivo chlorophyll a fluorescence yield in microalgae and its use as a herbicide monitor

A new fluorometric device was developed to measure the fluorescence yield of microalgae in order to detect photosystem II specific herbicides in outdoor water samples. The system was tested with respect to sensitivity and reliability with two species: Chlorella fusca (a green alga) and Phaeodactylum tricornutum (a diatom). The main advantages are the use of cell cultures without preliminary centrifugation, the evaluation of variable fluorescence from steady-state levels, and the rapidity of the measurement. Concentrations around the binding constant of inhibitors (around 10-8 M) can be detected for dichlorophenyldimethylurea, atrazine and simazine. The new system was compared with the commercially available pulse amplitude fluorometer PAM 101; it was shown that the sensitivity was about 10 times greater than with PAM 101, working at optimal chlorophyll concentrations. The advantages and limits of both systems when applied to free-water samples are discussed.

Evidence for the operation of a cyanide-sensitive oxidase in chlororespiration in the thylakoids of the chlorophyll c-containing alga Pleurochloris meiringensis (Xanthophyceae)

For characterisation of chlororespiration in the chlorophyll c-containing alga Pleurochloris meiringensis, we measured the flash-induced electrochromic absorbance changes between 470 and 545 nm and the redox changes of cytochrome f and cytochrome c553. Cytochrome c553 was shown to be present in high amounts (1 mol cytochrome c553 per 300 mol chlorophyll) in this alga and to function as the obligate electron donor for photosystem I instead of plastocyanin. Whereas salicylhydroxamic acid had no effect on the flash-induced absorbance transients, cyanide enhanced the slow-rising (t1/2≈10 ms) kinetic component of the electrochromic absorbance change. Cyanide also accelerated the re-reduction of the cytochrome f+/c553+electron pool following the photooxidation by repetitive single-turnover flashes. These data suggest that an oxidase competes with the cytochromes for electrons. The KCN concentration needed to induce these effects was 0.25 mM at half-saturation, whereas mitochondrial respiration was completely blocked at 0.1 mM. Therefore, the oxidase cannot be identical to the cytochrome aa3-oxidase of mitochondria and is most likely located in the chloroplast of P. meiringensis.

Electrochromic absorbance changes in the chlorophyll-c-containing alga Pleurochloris meiringensis (Xanthophyceae).

Flash-induced absorbance changes were measured in the Chl-c-containing alga Pleurochloris meiringensis (Xanthophyceae) between 430 and 570 nm. In addition to the bands originating from redox changes of cytochromes, three major positive and tow negative transient bands were observed both 0.7 and 20 ms after the exciting flash. These transient bands peaking at 520, 480 and 451 nm and 497 and 465 nm, respectively, could be assigned to an almost homogeneous shift of the absorbance bands with maxima at 506, 473 and 444 nm, respectively. The shape of the absorbance transients elicited from PS I or PS II was identical, and the two photosystems contributed nearly equally to the absorbance changes. Furthermore, the decay transients were sensitive to the preillumination of the cells. These data strongly suggest that the absorbance transients originate from an electrochromic response of carotenoid molecules. The pigment species responsible for the 506 nm absorption band, probably heteroxanthin or diatoxanthin, transferred excitation energy to both photosystems as shown by the aid of 77 K fluorescence excitation spectra.

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae).

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q0, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. Km-values for both pyridine nucleotides were in the μmolar range (Km[NADH]=9.8 μM, Km[NADPH]=3.2 μM calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP+ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP+ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.

The molecular architecture of the thylakoid membrane from various classes of eukaryotic algae

There is convincing consensus that the photosynthetic apparatus is of prokaryotic origin. The wide variety of algal plastids is mostly assumed to be the result of different endocytological events. Chloroplasts surrounded by two membranes as in rhodophytes and chlorophytes were considered as the association of a prokaryotic symbiont and a eukaryotic host, whereas algae having a chloroplast surrounded with more than two membranes can be delineated from an endocytological event of two eukaryotes (see S. Gibbs in this volume). Since chlorophyll b was neither combined with chlorophyll c nor with phycobiliproteins it was proposed that all present day chloroplasts can be integrated in three lines. Based on recent progress in sequencing the 28 S RNA from various algal classes (Perasso et al., 1989; Adoutte, this volume) it was suggest that these lines emerge as three distinct groups, although within the branches deep demarcations can be observed.

The Interaction of State Transitions and Chlororespiration in the Xanthophycean Alga Pleurochloris Meiringensis

Wavelength dependent State I-State II-transitions have been shown to exist in chlorophytes and red algae. Little is known about the regulation of energy distribution between the photosystems of chlorophyll c-containing plants. Previously it was shown that in the xanthophycean alga Pleurochloris meiringensis two states of energy distribution could be established [l]: In state “D” light is preferentially transferred to PS II, whereas in state “L” PS I is favoured. These state regulations strictly depend on the intensity and not on the wavelength of prei1lumination. In this paper we give new evidence that chlororespiration is involved in the mechanism of state “L”-state “D”-transitions.