Shmuel Malkin

Fluorescence induction studies in isolated chloroplasts I. Number of components involved in the reaction and quantum yields

Abstract 1. 1. A quantitative analysis of the fluorescence induction in isolated chloroplasts is presented. The rise of the fluorescence yield with time is ascribed to the photoreduction of a primary oxidant “Q” of photosystem II. The analysis yields a method of determining the amount of light actually utilized in the process. By comparing results obtained in the absence or presence of Hill oxidants, the quantum yield of results obtained in the absence or presence of Hill oxidants, the quantum yield of the primary photoreduction and the concentrations of all internal oxidants were estimated. The average value of the yield was 0.5 equiv/Einstein (for green light, 510–640 mμ). The total oxidant concentration was 1 equiv per 35 moles chlorophyll. 2. 2. The shape of the rise curve as well as its intensity and temperature dependence suggest that, besides Q, a second electron carrier, P, is involved, reacting in a consecutive dark step: Q and P are present in a ratio 1:1, the concentration of each being 1:70 chlorophyll. 3. 3. The restoration of the fluorecsence induction, i.e. the reoxidation of Q and P in the dark, which is accelerated by far-red light (700–740 mμ), was also subjected to a quantitative analysis. The quantum yield of the far-red reaction proved to be close to 1. The maximum (saturation) rate of the far-red effect was about I/50 of the Hill-reaction saturation rate. The results are discussed in terms of two photosystems and electron carriers arranged between them.

Enhanced oxidative-stress defense in transgenic potato expressing tomato Cu,Zn superoxide dismutases.

SummaryThe two cDNAs coding for the cytosolic (cyt) and the chloroplast-located (chl) Cu,Zn superoxide dismutases (SODs) of tomato (Perl-Treves et al. 1988) were cloned into respective binary vectors and mobilized into Agrobacterium strains. Potato tuber discs were infected with either of the two agrobacterial strains and cultured on selective medium containing kanaymcin. The integration of either of the cyt or the chl SOD transgenes was verified by Southern-blot hybridization. The enzymatic activity of the additional tomato chl Cu,Zn SOD could be distinguished from endogenous SOD activity since the latter isozyme migrated faster on SOD-activity gels. Several transgenic potato lines harboring either the cyt or the chl SOD genes of tomato showed elevated tolerance to the superoxide-generating herbicide paraquat (methyl viologen). After exposure of shoots to paraquat, tolerance was recorded either by scoring symptoms visually or by measurements of photosynthesis using the photoacoustic method. Root cultures from transgenic lines that harbored the additional cyt Cu,Zn SOD gene of tomato were tolerant to methyl viologen up to 10-5 M; a lower tolerance was recorded in roots of transgenic lines that expressed the additional chl Cu,Zn SOD of tomato.

Photoacoustic measurements of photosynthetic activities in whole leaves. Photochemistry and gas exchange

Abstract Photosynthetic activities of sections of intact tobacco leaves ( Nicotina tabacum L. var. Xanthi) were monitored by the photoacoustic effect. A reference signal was obtained using brief simultaneous illumination of the leaf with continuous light of saturating intensity. 1. The effect of the direct continuous light depends on the modulation frequency: A decrease in the photoacoustic signal, ‘negative effect’, is observed at low frequency (under approx. 200 Hz) and an increase of the photoacoustic signal, ‘positive effect’, at high frequency (above approx. 200 Hz). Both effects are reversible. 2. When leaves are water-infiltrated only a ‘positive effect’: of the direct continuous light is observed, at any frequency. Leaves infiltrated with aqueous DCMU solution (50 μM) do not show any direct continuous light effect. 3. The ‘negative effect’ develops, as the leaf is illuminated, in parallel to the (low frequency) photoacoustic signal itself which increases gradually after an initial lag period. This transient phenomenon takes a few minutes under our conditions. The differential increase of the signal during the induction period matches the size of the negative effect obtained by applying the direct continuous light at various times during the induction or in the steady state. 4. In a similar way, the ‘positive effect’ increases during the induction period, due to a transient decrease of the photoacoustic signal measured at high frequency. This change in the ‘positive effect’ is less noticeable, as a sizeable effect is already observed in a dark-adapted leaf, upon illumination, and the extent of the photoacoustic transient is relatively small. 5. No photoacoustic transients are observed when direct continuous light is used continuously. 6. It is concluded that the photoacoustic signal is due to two contributions: At low frequency there is a considerable contribution from modulated oxygen evolution, which tends to zero as the direct continuous light is applied. At high frequency the main contribution is from the conversion of photon energy to heat, and direct continuous light effects reflect mainly photochemical energy storage. Both low and high frequency photoacoustic transients reflect the induction period of photosynthesis. 7. Preliminary calculations on the relative damping of modulated oxygen evolution and thermal photoacoustic contributions with the modulation frequency, are consistent with the above model, taking into account diffusion and the rate-limiting step in O 2 evolution. 8. Photoacoustic data were used to construct a relative quantum yield spectrum for both oxygen evolution and photochemical energy storage.

PHOTOACOUSTIC SPECTROSCOPY AND RADIANT ENERGY CONVERSION: THEORY OF THE EFFECT WITH SPECIAL EMPHASIS ON PHOTOSYNTHESIS

Abstract—The use of photoacoustic spectroscopy in the study of photoactive samples (photochemical, luminescent, photovoltaic) is considered. After a short explanation of the parameters important in this technique and ways of measuring them, photochemical processes are considered in detail. Relations are derived for the determination of the energy stored in photochemical products and the quantum yield for the photochemical processes.

Photoacoustic detection of photosynthetic oxygen evolution from leaves. Quantitative analysis by phase and amplitude measurements

Abstract The photoacoustic signal from an intact leaf was analyzed as a vectorial summation of photothermal and photosynthetic oxygen-evolution contributions. A method is outlined to estimate each contribution separately. The amplitude of the oxygen-evolution component relative to that of the photothermal singnal decreases as the modulation frequency increases due to two processes which specifically damp the oxygen-evolution modulation: (1) diffusion of oxygen from the chloroplasts to the cell boundary, and (2) electron-transfer reactions occurring between the photochemical act and oxygen evolution. The effects of the two processes are well separated and are observed over different ranges of modulation frequency. Analysis of the data leads to a consistent estimation of the oxygen diffusion coefficient and also to a preliminary idea on the limiting time constant on the donor side of Photosystem II. The dependence of the photoacoustic oxygen-evolution signal on the intensity of added nonmodulated background light is used to construct the light saturation curve of (gross) Photsynthesis, with an estimation of the ratio maximal rate / maximal quantum yield. The photoacoustic method is distinguished by its sensitivity and rapidity (a single measurement takes approx. 1 s), far better than any other method to measure gross photosynthesis. The only disadvantage is in the fact that the quantum yield of oxygen evolution is determined in a relative basis only. Attempts to calibrate the photoacoustic measurements in an absolute sense are underway.

Distribution of light excitation in an intact leaf between the two photosystems of photosynthesis. Changes in absorption cross-sections following state 1-state 2 transitions

Abstract Using the photoacoustic technique, state 1-state 2 transitions were studied in an intact leaf by direct monitoring of modulated oxygen evolution, excited by modulated light. States 1 and 2 were characterized by the extent of immediate enhancement of the modulated oxygen evolution — ‘Emerson enhancement’ — and the concomitant fluorescence quenching, resulting from the addition of continuous far-red light (greater than 700 nm), absorbed primarily in Photosystem I (light 1). The extent of Emerson enhancement as well as the saturation curve of this effect by far-red light are very sensitive and quantitative indicators for the ratio of light excitation distributed between Photosystems I and II. The enhancement ratios at 650 nm light, a typical light 2, were in a range 1.4–1.8 in state 1, while values as low as 1.06 were observed in state 2. During the transition from state 2 to state 1, monitored in presence of modulated light 2 and background continuous light 1, the modulated oxygen yield increased considerably, indicating a major increase in excitation flux into Photosystem II. Conversely, with modulated light 2 alone in state 1, the modulated oxygen evolution yield was smaller than in state 2, indicating a decrease of the excitation flux in Photosystem I. In a typical example, of the transition to state 1, the fraction of light absorbed by Photosystem II, β, increased from 0.46 to 0.64, while that absorbed by Photosystem II, α, decreased from 0.43 to 0.36. State 1-state 2 transitions, thus, reflect reciprocal changes in the cross-sections of the two photosystems for light absorption. There is no evidence for the operation of a ‘spill-over’ mechanism. The enhancement effect displayed maxima at 480 and 650 nm, related to chlorophyll-b absorption, as well as another band at 500–550 nm. In a chlorophyll-b-less barley mutant, state 1-state 2 transitions, as monitored by modulated oxygen evolution, were absent, and the resulting enhancement corresponded to state 2. These observations are consistent with the model that the light-harvesting chlorophyll - a b complex plays a role in regulating the distribution of light to the photosystems. It is probable that this complex migrates reversibly in the thylakoid membrane in such a way that it is mainly associated with Photosystem II in state 1, but is more evenly distributed in the two photosystems in state 2.

Sulfide inhibition of photosystem II in cyanobacteria (blue-green algae) and tobacco chloroplasts.

The present study shows that in the presence of 600 nm light, sulfide acts as a specific inhibitor of photosynthetic electron transport between water and Photosystem II in the cyanobacteria Aphanothece halophytica and Synechococcus 6311 as well as in tobacco chloroplasts. In the presence of 600 nm light sulfied affects the fast fluorescence transients as does a low concentration (10 mM) of hydroxylamine; the fluorescence yield decreases in the presence of either chemical and can be restored by the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. In chloroplasts, however, NH2OH, an electron donor at high concentrations (40 mM), relieves the sulfide effect. In the dark, sulfide affects the cyanobacterial fluorescence transients through decrease of oxygen tension. The fluorescence yield increases in a similar pattern to that observed under nitrogen flushing. Upon omission of sulfide in A. halophytica, the characteristic aerobic fluorescence transients return, consistent with the ease of alternation between oxygenic and sulfide-dependent anoxygenic photosynthesis in many cyanobacteria.

Quantitative analysis of State 1–State 2 transitions in intact leaves using modulated fluorimetry — evidence for changes in the absorption cross-section of the two photosystems during state transitions

Abstract Using a combination of modulated and non-modulated light with synchronized detection it has been possible to monitor State 1–State 2 transitions in intact leaves as changes in the yield of modulated chlorophyll fluorescence. In the presence of excess far-red non-modulated light (713 nm) absorbed mainly by Photosystem I (PS I), the modulated fluorescence intensity was taken to represent F o — the emission yield which occurs when the reaction centres of Photosystem II (PS II) are all open. On the other hand, superimposing saturating non-modulated wide-band, blue-green light resulted in a transitory maximum yield of modulated chlorophyll fluorescence, F m , due to the total closure of the PS II reaction centres. In the absence of these additional lights the fluorescence level assumed a steady-state value, F s , between F o and F m . All these parameters changed as the leaf slowly adapted to light of a given spectral composition. It was found that both F o and F m increased reversibly (by about 15–20%) during the transition from State 2 to State 1 such that the ratio of F m to F o remained constant, indicative of changes in absorption cross-section of PS II and PS I rather than alterations in ‘spillover’ which would cause preferential changes in F m . It was also possible to estimate the fractions of light, β and α, channeled to PS II and PS I, respectively, from the values of F o , F m and F s . In one approach, β was estimated in State 1, using the assumption that α + β = 1, and its variation during the subsequent state transition was assumed to follow proportional changes in F o (or F m ). It was found that in State 2 there is a small loss (about 4%) of the total utilization of light in both photosystems. However, if such loss is neglected, assuming α + β is always unity, the calculated β was found to vary in the same direction and almost with the same magnitude as F o (or F m ), indicating independently that a change in absorption cross-section in PS II (and PS I) had occurred. Consistent with these data were the light-saturation curves for the non-modulated far-red light-quenching effect in bringing the fluorescence from F s to F o in States 1 and 2. The ratio of the initial slopes of these curves indicates quantitatively both redistribution of light between PS I and PS II during the State 1–State 2 transitions and a partial loss of excitation energy in State 2.

The effect of copper on chlorophyll organization during greening of barley leaves

The effect of copper on chlorophyll organization and function during greening of barley was examined, using chlorophyll fluorescence and photoacoustic techniques. Copper was found to inhibit pigment accumulation and to retard chlorophyll integration into the photosystems, as evident from low temperature (77 K) fluorescence spectra. Resolution of the minimal fluorescence (F0) into active and inactive parts, indicated a higher inactive fraction with copper treatment. This was attributed to chlorophyll molecules which failed to integrate normally, a conclusion supported by the longer fluorescence lifetime observed in copper treated plants. A lower ratio of chlorophyll a to b and fluorescence induction transients, showing accelerated Photosystem II closure, both indicate that copper treatment resulted in a larger light-harvesting antenna. Another effect of copper treatment was the suppression of oxygen evolution, indicating a decrease in photosynthetic capacity. We suggest that the non-integrated chlorophyll fraction sensitizes photodamage in the membrane, contributing to disruption of electron flow and pigment accumulation.

Fluorescence induction studies in isolated chloroplasts I. Kinetic analysis of the fluorescence intensity dependence on time

Abstract 1. 1. Fluorescence induction curves (intensity versus time) obtained by irradiation of isolated chloroplasts were observed and subjected to a detailed kinetic analysis. This analysis was based on a photosystem II model consisting of independent units, each of which contains one pigment aggregate connected to one electron-transport chain. During irradiation, two electron carriers, which exist initially in the oxidized state, are reduced in a light reaction followed by a dark reaction, both being first order: , where Q is the primary oxidant of photosystem II. Q and P are present in a 1:1 ratio1. The experimental curves agree with the calculated ones over a wide range of light intensity and temperature. 2. 2. Using the analysis outlined above, it was possible to estimate the first-order rate constant of the reaction between Q− and P as k = 30–40 sec −1 at 22° and 2–2.5 sec −1 at 0° . Comparison with the saturation rate of the Hill reaction shows that the above reaction between Q− and P may be rate limiting in the Hill reaction.