Denis Charlebois

Differential Effects of Nitrogen Limitation on Photosynthetic Efficiency of Photosystems I and II in Microalgae.

The effects of nitrogen starvation on photosynthetic efficiency were examined in three unicellular algae by measuring changes in the quantum yield of fluorescence with a pump-and-probe method and thermal efficiency (i.e. the percentage of trapped energy stored photochemically) with a pulsed photoacoustic method together with the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea to distinguish photosystems I and II (PSI and PSII). Measured at 620 nm, maximum thermal efficiency for both photosystems was 32% for the diatom Thalassiosira weissflogii (PSII:PSI ratio of 2:1), 39% for the green alga Dunaliella tertiolecta (PSII:PSI ratio of 1:1), and 29% for the cyanobacterium Synechococcus sp. PCC 7002 (PSII:PSI ratio of 1:2). Nitrogen starvation decreased total thermal efficiency by 56% for T. weissflogii and by 26% for D. tertiolecta but caused no change in Synechococcus. Decreases in the number of active PSII reaction centers (inferred from changes in variable fluorescence) were larger: 86% (T. weissflogii), 65% (D. tertiolecta), and 65% (Synechococcus). The selective inactivation of PSII under nitrogen starvation was confirmed by independent measurements of active PSII using oxygen flash yields and active PSI using P700 reduction. Relatively high thermal efficiencies were measured in all three species in the presence of the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, suggesting the potential for significant cyclic electron flow around PSI. Fluorescence or photoacoustic data agreed well; in T. weissflogii, the functional cross-sectional area of PSII at 620 nm was estimated to be the same using both methods (approximately 1.8 x 102 A2). The effects of nitrogen starvation occur mainly in PSII and are well represented by variable fluorescence measurements.

Energy storage and optical cross-section of PS I in the cyanobacterium Synechococcus PCC 7002 and a psaE- mutant

In vivo electron flow in the unicellular cyanobacterium Synechococcus sp. PCC 7002 was studied by pulsed, time- resolved photoacoustics (PTRPA). Using 1-μs, 2 μJ or 1013 hνcm-2 pulses at 695 nm, we observed large (42 ± 2%) Photosystem I (PS I) cyclic energy storage (ES) in the period of 2 to 12 ms after excitation with wild type (WT) intact cells. This cyclic ES was insensitive to flash interval from 0.3 to 10 s and to the presence of 1 μm DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). At this low flash energy and in the absence of continuous background light (in the dark), antimycin A, carbonylcyanide-m- chlorophenylhydrazone (CCCP), 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB), DCMU, 2- n-heptyl-4- hydroxyquinoline-N-oxide (HQNO), myxothiazol and N- ethylmaleimide (NEM) caused little or no inhibition of PS I cyclic electron flow. When continuous background far-red light (λ > 715 nm) was added during the measurement, strong inhibition by DBMIB and NEM and less by HQNO was observed, the amplitude of which was related to both concentration and the intensity of the background light. Analysis of the data with DBMIB yields its binding constant, 1 μm, and the turnover time of the system (> 20 ms). A turnover time of the uninhibited system of 2–3 ms was obtained by a pump-probe method. A dramatic lifting of the partial inhibition in the presence of far-red light was caused by antimycin A and a smaller effect by myxothiazol. The rescuing effect was assigned to a ‘short circuiting’ of the electron flow about the cytochrome (cyt) b6/f system. Progressively increasing the laser pulse energy allowed us to calculate the PS I optical cross-section (54 ± 2 Å). Analysis by the sensitive method of convolutions revealed a possible energy loss on the few ms time scale by antimycin A in the dark. The analysis also revealed a similar effect or artifact in uninhibited samples using the same sample illuminated with saturating continuous light, the standard procedure in photoacoustics (PA). A psaE- mutant showed more inhibition in the dark by DBMIB and with far-red light by HQNO, but less inhibition in the far-red light by myxothiazol than the WT. Under normal growth conditions, maximum ES for the psaE- mutant (38 ± 2%) was similar to that of the WT (42 ± 2%). However, under mild heat stress, maximum ES for the psaE- mutant dropped to 26% while the WT maximum ES stayed unchanged at 41%, within batch-to-batch variation. These results would indicate that the PsaE protein is not essential for PS I cyclic electron flow under our experimental conditions but plays a stabilizing role in the PS I complex under a mild thermal stress.

Photosynthetic energy storage of Photosystems I and II in the spectral range of photosynthetically active radiation in intact sugar maple leaves

The relative activity of Photosystems (PS) I and II in the spectral range between 400 and 720 nm was studied by measuring photosynthetic energy storage (ES) of an intact sugar maple leaf using photoacoustic spectroscopy. ES, determined with a modulated (80 Hz) monochromatic light beam in the presence of saturating intensity of background non-modulated white light, indicated the total energy stored by both photosystems (EST). Using background far-red light, ES of PS I (ESPS I) was quantified. ESPS II was derived from EST-ESPS I. EST dependence on intensity and wavelength of modulated light was studied at 470, 560, 640 and 680 nm. EST was maximum in red light and minimum in blue light. It decreased with an increase in modulated light intensity. The ratio ESPS II/ESPS I, measured at 640 nm, remained nearly constant with an increase in modulated light intensity. The relative quantum yield of EST spectrum showed two peaks around 610 and 660 nm, and declined sharply after 680 nm, revealing a clear red drop. ESPS I spectrum presented peaks around 610 and 670 nm, and a minimum between 440 and 470 nm. ESPS I was observed beyond 700 nm up to 720 nm, indicating the energy stored by cyclic electron transport. ESPS II spectrum showed broad peaks, around 460, 490, 600 and 660 nm, and a shoulder between 530 and 560 nm. ESPS II was always higher than ESPS I between 400 and 690 nm and reached zero around 700 nm.

Photoacoustic signal generation in leaves: are O2-consuming processes involved?

The photoacoustic signal from a green leaf is the vectorial sum of photothermal (heat emitted by non-radiative deexcitation of molecules) and photobaric (photosynthetic O 2 exchanges) contributions. With sugar maple leaf, we have observed a decline of the O 2 signal in the course of time, once the leaf attained a steady-state photosynthesis. This decline of the O 2 signal was not due to a decrease in photochemical activity as was evident by a stable fluorescence signal and constant energy storage. The rate and the extent of decline were larger at low modulation frequency and low modulated light intensity. The decline continued even in the presence of background far-red light (λ>715 nm). The above observations lead us to conclude that the photoacoustic O 2 signal from leaves is influenced by O 2 -consuming processes. Data are discussed in relation to a possible occurrence of an equilibrium between oxygen-evolving and oxygen-consuming processes to account for the O 2 signal in the plant cell. Moreover, the oxygen-uptake component may have to be included in the vectorial model of photoacoustic signal generation in leaves.

Effect of sulfur dioxide and sulfite on photochemical energy storage of isolated chloroplasts--a photoacoustic study.

Photoacoustic spectroscopy was used to study the effect of sulfite and SO(2) on isolated corn mesophyll chloroplasts by monitoring the photochemical energy storage. Sulfite incubation of isolated chloroplasts, either in light or in darkness, caused a decrease in photochemical energy storage. The more pronounced decrease in light indicates a light-dependent sulfite inhibitory site(s) in chloroplasts. Also diphenylcarbazide caused a partial recovery of energy storage in sulfite treated chloroplasts indicating a possible site of damage at the water oxidizing system. Although the chloroplast membranes were found to be insensitive to high concentrations of SO(2) for relatively short exposure periods (10 min) in light, exposure of chloroplasts to 28.5 ng cm(-3) SO(2) for 10 min caused a decrease in energy storage. An attempt was made to explain the mechanism of action of sulfite and SO(2) in chloroplasts.

Assessment of Strawberry Maturity by Photoacoustic Spectroscopy

Reliable estimates of the stages of maturity of fruits are important for evaluating the efficiency of post-harvest treatments applied to delay senescence. Many objective criteria for judging maturity of strawberries have been used, for example, flesh firmness, titrable acidity, and determination of total anthocyanins.

Sulfite inhibition of photochemical activity of intact pea leaves

Sulfite treatment of pea leaf disks in light caused a significant decrease in the relative quantum yield of photosynthetic oxygen evolution and energy storage (ES) as measured by photoacoustic (PA) spectroscopy. The inhibition was concentration dependent and was less in darkness than in light, indicating light-dependent inhibitory site(s) on the photosynthetic electron transport chain. Further, in darksulfite-treated leaves, the energy storage was more affected than the relative quantum yield of oxygen evolution, suggesting that photophosphorylation and/or cyclic electron transport around PS I are sites of sulfite action in darkness. The Rfd values, the ratio of fluorescence decrease (fd) to the steady-state fluorescence (fs), decreased significantly in leaves treated with sulfite in light but were not affected in dark-treated ones, confirming the photoacoustic observations. Similarly, the ratio of variable fluorescence (Fv) to maximum fluorescence (Fm), a measure of PS II photochemical efficiency, was affected by sulfite treatment in light and not changed by treatment in darkness. An attempt was made to explain the mechanism of sulfite action on photosynthetic electron transport in light and in darkness.

Effects of acid watering of the soil on the photosynthetic activity, growth, and foliar pigments of sugar maple saplings

Abstract Photoacoustic spectroscopy has been used to monitor the photosynthetic activity of the leaves of sugar maple saplings treated by watering the soil with simulated acid rain at different pH levels (2.S, 3.0, 3.5, 4.0, 4.5, 5.0 and 5.6). We also measured the relative growth rate of the plants and the pigment content of leaves. The results indicated an ambivalent effect of acidity: there was a linear decline (r = 0.92) in the photosynthetic O2 evolution as the pH of the simulated rain was lowered from 5.6 to 3.0 and a jump when it reached 25, with a stimulation effect on the relative growth rate at lower pH levels. The photochemical energy storage and the relative growth rate increased from pH 5.6 to pH 4.0–4.5, then decreased at pH 3.0 and increased again at pH 2.5. The pattern of variation of the photochemical energy storage was fairly well correlated with the pigment content of the leaves (r = 0.83). It was concluded that acid rain preferentially affects photosystem II (PSII), inhibiting O2 evolut...

In Vivo State Transitions as Followed by Photoacoustically Measured Energy Storage

State 1-state 2 transitions in intact sugar maple leaves were monitored by following changes in energy storage (ES) of PSI (ESPSI) and PSII (ESPSII). ES of both photosystems (EST) was recorded under saturating background white light, while ESPSI was determined under saturating far-red light. The difference between EST and ESPSI represented ESPSII. Adaptation of the leaf to far-red light (light 1, λ > 715 nm) and red light (light 2, 650 nm) led to state 1 where light 2 was distributed mainly to PSII. Exposure of the leaf to state 2 conditions (light 2 alone) reversed the phenomenon. These state transitions needed about 20 min to occur. From our experiments on light to dark transitions, and vice-versa, we suggest that the dark state was state 2. We also studied the effect of NaF, a known inhibitor of state 2 to state 1 transition. Our results support the concept of phosphorylationand migration of light harvesting complexes, as responsible for the light energy distribution between both photosystems in vivo.

Impact of Air Pollutants and Acid Rain on Corn and Sugar Maple Seedlings Assessed by Photoacoustic Spectroscopy

The problem of air pollution and forest decline has become one of the most controversial subjects today. This is mainly due not only to economical and political implications but also to conflicting results obtained by many research groups using different methods which are more or less sensitive and reliable [1,2]. It therefore appears that more sensitive techniques used under controlled experimental conditions are needed. We have utilized the photo-acoustic spectroscopy (PAS) to study the effects of O3, SO2 and simulated acid rains on the photosynthetic activity of corn and sugar maple leaves.