Benoît Schoefs

Spectroscopic Properties of Protochlorophyllide Analyzed In Situ in the Course of Etiolation and in Illuminated Leaves

Abstract The spectroscopic properties of photoactive (i.e. flash-transformable) and nonphotoactive protochlorophyll(ide)s (Pchl(ide)) were reinvestigated during the development of bean leaves in darkness. Two phases in the process of Pchl(ide) accumulation were apparent from quantitative measurements of pigment content: a lag phase (first week) during which photoactive Pchl(ide) accumulated faster than nonphotoactive Pchl(ide); and a fast phase (second week), showing parallel accumulation of both types of Pchl(ide). ‘Flashed-minus-dark’ absorbance difference spectra recorded in situ at 77 K showed that P650–655 was the predominant form of photoactive protochlorophyllide regardless of developmental stage. Quantitative analysis of energy migration processes between the Pchl(ide) forms showed the existence of energy transfer units containing a 1:8 ratio of nonphotoactive and photoactive Pchl(ide)s during development. Gaussian deconvolution of in situ 77 K fluorescence spectra indicated that the 633 nm band of nonphotoactive Pchl(ide) was made of four bands, at 625, 631, 637 and 643 nm, whose relative amplitudes only slightly changed during development. The emission band of photoactive Pchlide was also analyzed using the same method. Three components were found at 644, 652 and 657 nm. The emission band of P650–655 included the last two components, which become predominant only in fully etiolated plants. Photoactive Pchlide with an emission maximum at 653 nm was detected in the light during development of leaves of photoperiodically grown plants.

Cadmium inhibits epoxidation of diatoxanthin to diadinoxanthin in the xanthophyll cycle of the marine diatom Phaeodactylum tricornutum

Cd has pleiotropic effects on plant physiology and in particular on photosynthesis. It has not been established yet if Cd alters the functioning of the xanthophyll cycle. To answer this question, an exponentially growing culture of the marine diatom Phaeodactylum tricornutum was incubated with Cd (20 mg/l) for 24 h and irradiated with a light activating the xanthophyll cycle, which in diatoms, consists of the reversible deepoxidation of diadinoxanthin to diatoxanthin. The measurements show that the deepoxidation step is not influenced by Cd. In contrast, the Cd concentration used sharply inhibits the epoxidation of diatoxanthin to diadinoxanthin.

Isolation and characterization of photoactive complexes of NADPH:protochlorophyllide oxidoreductase from wheat

Abstract. A photoactive substrate-enzyme complex of the NADPH:protochlorophyllide oxidoreductase (POR; EC 1. 3. 1. 33) was purified from etiolated Triticum aestivum L. by gel chromatography after solubilization of prolamellar bodies by dodecyl-maltoside. Irradiation by a 1-ms flash induced the phototransformation of protocholorophyllide a (Pchlide) with −196 °C absorbance and emission maxima at 640 and 643 nm, respectively. The apparent molecular weight of this complex was 112 ± 24 kDa, which indicates aggregation of enzyme subunits. By lowering the detergent concentration in the elution buffer, a 1080 ± 250-kDa particle was obtained which displayed the spectral properties of the predominant form of photoactive Pchlide in vivo (−196 °C absorbance and fluorescence maxima at 650 and 653 nm). In this complex, POR was the dominant polypeptide. Gel chromatography in the same conditions of an irradiated sample of solubilized prolamellar bodies indicated rapid disaggregation of the complex after Pchlide phototransformation. High performance liquid chromatographic analysis of the POR complexes obtained using two detergent concentrations indicates a possible association of zeaxanthin and violaxanthin with the photoactive complex.

Photoactive protochlorophyllide regeneration in cotyledons and leaves from higher plants.

Abstract Chlorophyll accumulation during greening implies the continuous transformation of photoactive protochlorophyllide (Pchlide) to chlorophyllide. Since this reaction is a light-dependent step, the study of regeneration of photoactive Pchlide under a continuous illumination is difficult. Therefore this process is best studied on etiolated plants during a period of darkness following the initial photoreduction of photoactive Pchlide. In this study, the regeneration process has been studied using spinach cotyledons, as well as barley and bean leaves, illuminated by a single saturating flash. The regeneration was characterized using 77 K fluorescence emission and excitation spectra and high-performance liquid chromatography. The fluorescence data indicated that the same spectral forms of photoactive Pchlide are regenerated by different pathways: (1) photoactive Pchlide regeneration starts immediately after the photoreduction through the formation of a nonphotoactive Pchlide form, emitting fluorescence at approximately 651 nm. This form is similar to the large aggregate of photoactive Pchlide present before the illumination, but it contains oxidized form of nicotinamide adenine dinucleotide phosphate, instead of the reduced form (NADPH), in the ternary complexes; and (2) after the dislocation of the large aggregates of chlorophyllide–light-dependent NADPH:Pchlide a photooxidoreductase–NADPH ternary complexes, the regeneration occurs at the expense of the several nonphotoactive Pchlide spectral forms present before the illumination.

Changes in the photosynthetic pigments in bean leaves during the first photoperiod of greening and the subsequent dark-phase. Comparison between old (10-d-old) leaves and young (2-d-old) leaves

Chlorophyll and carotenoid variations of 2-d-old and 10-d-old bean leaves (Phaseolus vulgaris var Red Kidney) were analyzed by HPLC during the first photoperiod of greening (16 h light + 8 h dark). The HPLC method used is suitable for the separation of cis- and trans-carotenoid isomers, Pchlide a and Chlide a as well as their esters. The main results are (1) before illumination the composition of the carotenoid pool is similar at the two developmental stages; (2) non-illuminated 2-d-old leaves are devoid of Pchlide a ester; (3) chlorophyll and carotenoid accumulation in 2-d-old leaves presented a lag phase twice longer than observed in 10-d-old ones; (4) Chlide a seems directly esterified to Chl a in 2-d-old leaves whereas esterification requires four steps in 10-d-old leaves and, (5) the kinetics of Chl and carotenoid accumulation are different at the two investigated developmental stages.

Zinc Affects Differently Growth, Photosynthesis, Antioxidant Enzyme Activities and Phytochelatin Synthase Expression of Four Marine Diatoms

Zinc-supplementation (20 μM) effects on growth, photosynthesis, antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase, catalase), and the expression of phytochelatin synthase gene were investigated in four marine diatoms (Amphora acutiuscula, Nitzschia palea, Amphora coffeaeformis and Entomoneis paludosa). Zn-supplementation reduced the maximum cell density. A linear relationship was found between the evolution of gross photosynthesis and total chlorophyll content. The Zn treatment decreased the electron transport rate except in A. coffeaeformis and in E. paludosa at high irradiance. A linear relationship was found between the efficiency of light to evolve oxygen and the size of the light-harvesting antenna. The external carbonic anhydrase activity was stimulated in Zn-supplemented E. paludosa but was not correlated with an increase of photosynthesis. The total activity of the antioxidant enzymes did not display any clear increase except in ascorbate peroxidase activity in N. palea. The phytochelatin synthase gene was identified in the four diatoms, but its expression was only revealed in N. palea, without a clear difference between control and Zn-supplemented cells. Among the four species, A. paludosa was the most sensitive and A. coffeaeformis, the most tolerant. A. acutiuscula seemed to be under metal starvation, whereas, to survive, only N. palea developed several stress responses.

Comparison of the photoreduction of protochlorophyllide to chlorophyllide in leaves and cotyledons from dark-grown bean as a function of age

Abstract77 K fluorescence spectra of bean (Phaseolus vulgaris cv Commodore) leaves and cotyledons show the presence of active and inactive protochlorophyllides. The first detected product of the protochlorophyllide photoreduction is the chlorophyllide emitting fluorescence at 690 nm (C690) which is observed one day (leaves) and three days (cotyledons) after sowing. In cotyledons, C690 undergoes the ‘rapid’ and ‘Shibata’ shifts; in leaves, these spectral changes are age-dependent. In order to characterize the formation of C690, we have recorded 298 K fluorescence kinetics at 690 nm and the corresponding absorbance kinetics at 440 nm. The amplitude of the variations of both kinetics increases with the sample age. The absorbance and fluorescence kinetics can be modelized as a monoexponential law. The rate constant of the absorbance and fluorescence kinetics does not significantly change during the studied period (except for old cotyledons). The results presented in this paper give evidences for a low energy transfer between pigments during the photoreduction of protochlorophyllide at room temperature.

Cadmium, Copper, Sodium and Zinc Effects on Diatoms: from Heaven to Hell — a Review

Abstract Diatom development depends on several environmental factors, including the availability in metals. When micronutrients are present of adequate amount, cells exhibit a strong fitness and develop at their maximum growth rate. In many circumstances, the optimal metal amount in the cell environment is disrupted and cells experience starvation or excess for one or more elements. The metals in excess interfere with biochemical and cellular processes triggering a dysfunctioning that reduces growth and may ultimately lead to cell death. The ability of diatoms to adapt/resist to environmental changes has ecological consequences in term of biodiversity. To survive, diatoms activate defence mechanisms, such as the production of antioxidants or/and metal chelators. In this contribution, the diatom requirements for cadmium, copper, zinc and sodium are briefly reviewed. Then the impacts of an excess or a deprivation in one of these elements on diatom physiology is discussed from the molecular and biochemical point of views. The defence mechanisms enabling diatoms to overcome the metal stress are presented. At the end of this contribution, an assay on the integration of the defence mechanisms is presented.

On the limits of applicability of spectrophotometric and spectrofluorimetric methods for the determination of chlorophyll a/b ratio

The concentration limits for spectrophotometric and spectrofluorimetric determinations of the chlorophyll (Chl) a/b ratio in barley leaves were studied using 80% acetone extracts at room temperature. The optimum sample absorbances (at 663.2 nm – maximum of the QY) band of Chl a) for the Chl a/b determination were determined. For given spectrometers and sample positions, these absorbances ranged between 0.2 and 1.0 and 0.008–0.1 for the absorption and fluorescence methods, respectively. Precision of the measurements and the distorting effects are discussed. The lower limits of both absorption and fluorescence methods depend on sensitivity of the spectrometers for the Chl b detection. The spectrophotometric determination of Chl a/b ratio at higher Chl concentrations can be distorted by the chlorophyll fluorescence signal. The extent of this distortion depends on sample-detector geometry in any given type of the spectrometer. The effect of inner filter of Chl molecules and the detection instrumental function affect the value of the upper limit for the spectrofluorimetric method. Both methods were applied to estimate the Chl a/b ratio in pigment extracts from greening barley leaves, which are characterized by a low Chl concentration and a high Chl a/b ratio at the beginning of greening process.

The formation of chlorophyll from chlorophyllide in leaves containing proplastids is a four-step process

The time course of the different esters of chlorophyllide (Chlide) during the formation of chlorophyll a (Chl) in embryonic bean leaves containing proplastids was investigated by HPLC. After the reduction of photoactive Pchlide (Pchlide) to Chlide, three intermediates, i.e. Chlide geranylgeraniol, Chlide dihydrogeranylgeraniol and Chlide tetrahydrogeranylgeraniol were detected before the formation of Chlide phytol, i.e. authentic Chl. The transformation of Chlide to Chl was found to be much faster in leaves containing proplastids than in etiolated leaves with etioplasts.