Fabrice Franck

Leaf chlorosis in oilseed rape plants (Brassica napus) grown on cadmium-polluted soil : causes and consequences for photosynthesis and growth

Abstract.Brassica napus L. (oilseed rape) was grown from seeds on a reconstituted soil contaminated with cadmium (100 mg Cd kg−1 dry soil), resulting in a marked chlorosis of the leaves which was investigated using a combination of biochemical, biophysical and physiological methods. Spectroscopic and chromatographic analyses of the photosynthetic pigments indicated that chlorosis was not due to a direct interaction of Cd with the chlorophyll biosynthesis pathway. In addition, mineral deficiency and oxidative stress were apparently not involved in the pigment loss. Leaf chlorosis was attributable to a marked decrease in the chloroplast density caused by a reduction in the number of chloroplasts per cell and a change in cell size, suggesting that Cd interfered with chloroplast replication and cell division. Relatively little Cd was found in the chloroplasts and the properties of the photosynthetic apparatus (electron transport, protein composition, chlorophyll antenna size, chloroplast ultrastructure) were not affected appreciably in plants grown on Cd-polluted soil. Depth profiling of photosynthetic pigments by phase-resolved photoacoustic spectroscopy revealed that the Cd-induced decrease in pigment content was very pronounced at the leaf surface (stomatal guard cells) compared to the leaf interior (mesophyll). This observation was consistent with light transmission and fluorescence microscopy analyses, which revealed that stomata density in the epidermis was noticeably reduced in Cd-exposed leaves. Concomitantly, the stomatal conductance estimated from gas-exchange measurements was strongly reduced with Cd. When plants were grown in a high-CO2 atmosphere (4,000 μl CO2 l−1), the inhibitory effect of Cd on growth was not cancelled, suggesting that the reduced availability of CO2 at the chloroplast level associated with the low stomatal conductance was not the main component of Cd toxicity in oilseed rape.

Etioplast differentiation in arabidopsis: both PORA and PORB restore the prolamellar body and photoactive protochlorophyllide-F655 to the cop1 photomorphogenic mutant.

The etioplast plastid type of dark-grown angiosperms is defined by the accumulation of the chlorophyll (Chl) precursor protochlorophyllide (Pchlide) and the presence of the paracrystalline prolamellar body (PLB) membrane. Both features correlate with the presence of NADPH:Pchlide oxidoreductase (POR), a light-dependent enzyme that reduces photoactive Pchlide–F655 to chlorophyllide and plays a key role in chloroplast differentiation during greening. Two differentially expressed and regulated POR enzymes, PORA and PORB, have recently been discovered in angiosperms. To investigate the hypothesis that etioplast differentiation requires PORA, we have constitutively overexpressed PORA and PORB in the Arabidopsis wild type and in the constitutive photomorphogenic cop1-18 (previously det340) mutant, which is deficient in the PLB and Pchlide–F655. In both genetic backgrounds, POR overexpression increased PLB size, the ratio of Pchlide–F655 to nonphotoactive Pchl[ide]–F632, and the amount of Pchlide–F655. Dramatically, restoration of either PORA or PORB to the cop1 mutant led to the formation of etioplasts containing an extensive PLB and large amounts of photoactive Pchlide–F655.

Protochlorophyllide-NADP+ and protochlorophyllide-NADPH complexes and their regeneration after flash illumination in leaves and etioplast membranes of dark-grown wheat

The fast (1 min) regeneration process of the photoactive Pchlide forms after a light flash was studied in etiolated wheat leaves, and this process was simulated in vitro by incubating etioplast inner membranes of wheat with excess NADPH or NADP+. The 77 K fluorescence spectra were recorded after flash illumination, dark incubation and a subsequent flash illumination of the samples. A non-photoactive Pchlide form with an emission maximum at 650 nm was transiently detected in leaves during regeneration of a photoactive Pchlide form with an emission maximum at 654 nm. Gaussian deconvolution of fluorescence spectra of isolated membranes showed that this 650 nm form appeared in conditions of excess NADP+, as suggested in previous studies. Additionally a Pchlide form emitting at 638.5 nm was detected in the same conditions. The analysis of the spectra of leaves at different times after a flash indicated that these two non-photoactive forms are involved as intermediates in the regeneration of photoactive Pchlide. This regeneration is in correlation with the production of the Chlide form emitting at 676 nm. The results demonstrate that, in vivo, part of the NADPH:protochlorophyllide oxidoreductase is reloading with nonphotoactive Pchlide on a fast time-scale and that the 676 nm Chlide form is the released product of the phototransformation in this process.

Room temperature fluorescence spectra of protochlorophyllide and chlorophyllide forms in etiolated bean leaves

Abstract Room-temperature fluorescence emission and excitation spectra of 3-day or 10-day old dark-grown bean (Phaseolus vulgaris L. cv Commodore) leaves were measured. The excitation light was defocused in such way that only a low rate of phototransformation took place and protochlorophyllide (Pchlide) forms could be detected. The spectra were resolved into gaussian components using a new method based on the comparison of the 4th derivative of the experimental spectrum and that of the calculated spectrum, i.e. the sum of the gaussians. In addition to Pchlide emission bands with maxima at 631, 644, 655 and 667 nm which correspond to those described earlier in 77 K spectra, two new and unusually narrow bands were found at 637 and 650 nm. In the Chlide region, emission bands were found at 676, 682, 686 and 695 nm. Changes in the relative amplitudes of the Pchlide and Chlide room temperature emission bands as a function of age, of excitation wavelength and in response to a short light flash were studied. A model is given in which dynamic interconversions of the pigment forms are suggested and the presence of the new forms is explained with the differences in the aggregational states of the pigments and with their interactions with NADPH or NADP+.

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.

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.

Localization of NADPH-protochlorophyllide reductase in plastids of barley at different greening stages

The localization of protochorophyllide (Pchlide) and of NADPH-protochlorophyllide oxidoreductase (POR, EC 1.6.99.1) within (etio)chloroplasts has been investigated at selected stages of greening of barley seedlings. Pchlide pigment and POR protein contents were evaluated in different plastid membrane fractions by fluorescence spectroscopy and immunoblot analysis using a monospecific polyclonal antibody raised against the purified enzyme. Fluorescence analysis showed the presence of Pchlide in both the envelope and thylakoid membranes. During greening, the Pchlide content, expressed on a total protein basis, decreased in thylakoid membranes, whereas it increased in the envelope membranes. POR proteins were detected mainly in thylakoid membranes at early greening stages. In contrast, the weak amount of POR proteins was associated more specifically with envelope membranes of mature chloroplasts. Whatever the greening stage, thylakoid-bound Pchlide and POR proteins were more abundant in the thylakoid regions which remained unsolubilized after mild Triton treatment used as standard procedure to prepare PS II particles. This suggests the preferential association of Pchlide and POR to the appressed regions of thylakoids.

Studies on the O-J-I-P transient of chlorophyll fluorescence in relation to photosystem II assembly and heterogeneity in plastids of greening barley

Abstract The polyphasic variable fluorescence in saturating light (O-J-I-P transient, Strasser et al. (1995) Photochem. Photobiol. 61: 21–42) has been investigated in etiochloroplasts during the greening of etiolated leaves of Hordeum vulgare . The initial photochemical phase (O-J) due to reduction of the primary quinone acceptor Q A was found to represent a constant proportion (65–70%) of total variable fluorescence independent of greening time. The partial fluorescence quenching in the Q A -reduced state seems, therefore, to represent a basic property of PSII electron transport. The biphasic character of the slower J-I-P transient due to reduction of the plastoquinone pool developped progressively during the first hours of greening. In the same period of time the proportion and rate constant of rapid PSIIα sub-population increased, as calculated from the induction curve in the presence of DCMU. Etiochloroplasts or chloroplasts resuspended in low salt medium showed a low I level, which was restored upon readdition of 5 MM MgCl 2 and NaCl. Salts also increased the apparent proportion of PSIIα. These results suggest that the J-I and I-P phases of the induction curve are related to different rates of plastoquinone photoreduction by two distinct PSII populations. The effects of DMQ and of DCBQ on the O-J-I-P transient were also studied in (etio-)chloroplasts. In addition to the already reported quenching of the initial ( F O ) and variable fluorescence by DCBQ, a slow fluorescence increase phase was found to appear upon the addition of DCBQ but not of DMQ. The latter observations confirm that DCBQ differs from DMQ by its higher efficiency as PSII electron acceptor.

TEMPERATURE DEPENDENCE OF CHLOROPHYLL(IDE) SPECTRAL SHIFTS and PHOTOACTIVE PROTOCHLOROPHYLLIDE REGENERATION AFTER FLASH IN ETIOLATED BARLEY LEAVES

Abstract— Absorbance spectroscopy at 77 K was used to investigate the effect of temperature on in vivo chlorophyllide shifts and photoactive protochlorophyllide regeneration after a saturating flash, which transformed all protochlorophyllide to chorophyllide. Photoactive protochlorophyllide present in darkness was stable up to 40°C. The rate of Shibata shift and protochlorophyllide regeneration after flash were strongly temperature dependent in the range 0–25°C. At 0°C, the shift was still observed but no regeneration occurred. Only slight effects were observed in the range 25–40°C. At all temperatures, the process of protochlorophyllide regeneration was significantly slower than the Shibata shift. The final chlorophyll shift from 672 to 674 nm was observed up to 40°C. The implication of these results concerning the pigment‐protein interactions during the Shibata shift are discussed.

Formation of long-wavelength chlorophyllide (Chlide695) is required for the assembly of Photosystem II in etiolated barley leaves

Chlorophyll(ide) spectroscopic properties and Photosystem II assembly, monitored by 77 K variable fluorescence, were studied in etiolated barley leaves as a function of the extent of protochlorophyllide photoreduction by a single millisecond light flash of different intensities. Variable fluorescence, measured 2 hours after the flash, was only detected when the extent of phototransformation was higher than a threshold value of 0.4. Its development paralleled the formation of a chlorophyll emission component at 685 nm, which itself derived from long-wavelength chlorophyllide with an emission maximum at 695 nm. At low flash intensities, short-wavelength chlorophyllide forms preferentially accumulated and no Photosystem II fluorescence was detected after 2 hours. Chlorophyllide esterification was independent of the extent of phototransformation. These results suggested that the formation of long-wavelength chlorophyllide was essential for further assembly of Photosystem II. This interpretation was strengthened by the observed inhibition of both long-wavelength chlorophyllide formation and of variable fluorescence development in leaves treated with δ-aminolevulinic acid or in untreated leaves subjected to repeated flashes of low intensity.