Ora Canaani

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.

Photoinhibition in Chlamydomonas reinhardtii: Effect on state transition, intersystem energy distribution and Photosystem I cyclic electron flow

The energy distribution, state transitions and photosynthetic electron flow during photoinhibition of Chlamydomonas reinhardtii cells have been studied in vivo using photoacoustics and modulated fluorescence techniques. In cells exposed to 2500 W/m2 light at 21 °C for 90 min, 90% of the oxygen evolution activity was lost while photochemical energy storage as expressed by the parameter photochemical loss (P.L.) at 710–720 nm was not impaired. The energy storage vs. modulation frequency profile indicated an endothermic step with a rate constant of 2.1 ms. The extent of the P.L. was not affected by DCMU but was greatly reduced by DBMIB. The regulatory mechanism of the state 1 to state 2 transition process was inactivated and the apparent light absorption cross section of photosystem II increased during the first 20 min of photoinhibition followed by a significant decrease relative to that of photosystem I. These results are consistent with the inactivation of the LHC II kinase and the presence of an active cyclic electron flow around photosystem I in photoinhibited cells.

Hydroxylamine, hydrazine and methylamine donate electrons to the photooxidizing side of Photosystem II in leaves inhibited in oxygen evolution due to water stress

Abstract When leaf discs are water stressed, they lose the capacity for photosynthetic oxygen evolution and variable (chlorophyll a ) fluorescence. Such a loss of variable fluorescence was previously reported by Govindjee et al. (Plant Sci. Lett. 20 (1981) 191–194). The later activity is not lost if prior to the water-stress treatment the leaf is incubated with typical water analogs known to act as electron donors to Photosystem II, such as hydroxylamine and hydrazine. Methylamine also acts in the same fashion. These results indicate that one of the sites of drought damage is the oxidizing side of Photosystem II, and that electron donors can restore electron transport, at least to the plastoquinone pool, similar to their effect in Tris treatment of isolated chloroplasts.

Photoacoustic detection of oxygen evolution and State 1–State 2 transitions in cyanobacteria

Abstract Photoacoustic detection of modulated oxygen evolution was obtained from algae in vivo. In the cyanobacterium Nostoc muscorum, during State 1 to State 2 transition, the effective absorption cross-section of PS II measured at 580 and 620 nm, decreased by 10–15% concomitant with an equal increase in the cross-section of Photosystem I. Upon incubation of Nostoc cells with a specific inhibitor of phosphatases (NaF) which blocks dephosphorylation of proteins, State 1 was abolished and only State 2 could be observed. These results suggest that in organisms containing phycobiliproteins as efficient antennae and which are missing both light-harvesting chlorophyll a b protein complex and grana thylakoids the level of protein phosphorylation controls the distribution of excitation energy between the two photosystems.

Photosynthetic chromatic transitions and Emerson enhancement effects in intact leaves studied by photoacoustics

The decrease of the quantum yield of photosynthesis in long wavelength light (h > 690 nm; ‘light l’), and its enhancement by addition of short wavelength light, and vice versa (Emerson enhancement [ 1,2]), were among the most decisive experimental results that led to the concept of the two photosystems in photosynthesis. These phenomena are due to an initial, uneven light distribution between the two photosystems, and can be used to estimate such excitation imbalance quantitatively. It was suggested [3-51 that there exists a mechanism to reduce the initial imbalance of light distribution by allowing excitation energy transfer from photosystem (PS) II to PS I. This mechanism, known as ‘spill-over’ [3], operates under short wavelength illumination (‘light 2’, h < 690 nm), when initially there is excess excitation of PS II. The extent to which this ‘spill-over’ mechanism is operating depends on illumination conditions, with slow adaptations occurring to two physiological states; viz. state 1 with no or minimal energy transfer, obtained after adaptation to long wavelength illumination and state 2 with much more effective energy transfer obtained during exposure to short wavelength light. State 2 is characterized by low fluorescence and low Emerson enhancement relative to state 1 [5,6]. The biochemical basis of such transitions between the two states was partly elucidated [7-l 11.

THE ROLE OF CYCLIC ELECTRON FLOW AROUND PHOTOSYSTEM I and EXCITATION ENERGY DISTRIBUTION BETWEEN THE PHOTOSYSTEMS UPON ACCLIMATION TO HIGH IONIC STRESS IN Dunaliella salina

Abstract— The distribution of excitation energy between the two photosystems in the halophylic alga Dunaliella salina has been analyzed under ionic stress. In the transition from state 1 to state 2, it was found that a, the absorption cross‐section of photosystem (PS) I increased from 42 to 49% until an equal distribution between PS I and PS II was obtained in state 2. Acclimation of the algae to different salt concentrations did not change the fractions of light absorbed in PS II and PS I, but slowed down the transition time from state 1 to state 2. A large increase in ΔpH induced fluorescence quenching was observed which was abolished by the uncoupler nigericin. Photoacoustic quantum yield spectra of energy storage indicated a larger energy storage at 700 nm induced upon stress. The additional ΔpH quenching of fluorescence and the additional quantum yield of energy storage at 700 nm, in the stressed algae, are consistent with the operation of a cyclic, energy‐storing pathway in PS I which is uncoupler sensitive.

Photoacoustic study of the green alga Trebouxia in the lichen Ramalina duriaei in vivo.

Photosynthetic parameters of the lichen Ramalina duriael were investigated in vivo, by the photoacoustic method using intensity—modulated exciting light (frequency range 5–300 Hz). The photoacoustic signal in intact lichens was similar in its general characteristics to that obtained from intact leaves of higher plants (Poulet et al., Biochim. Biophys. Acta 724, 433–446, 1983). It included two components interpreted to be due to modulated heat and modulated oxygen evolution. The quantum yield of the oxygen evolution component was maximal in the red spectral region and exhibited the ‘red drop’ decrease at wavelengths larger than 680 nm, similar to observations in higher plants. In contrast to those however, there was a pronounced decrease in this yield in the region below about 600 nm, indicating that pigments absorbing at shorter wavelengths are inefficient energy transfer agents. Similar results were observed for the quantum yield spectrum of photochemical energy storage. Analysis of oxygen diffusion in the symbiont alga, from the modulation frequency dependence of the ratio of oxygen evolution to photothermal signal component is consistent with an average diffusion path of about 4 μm, compared to a smaller, 1 μm, average diffusion path obtained in green leaves.

Physiological adaptation to a newly observed low light intensity state in intact leaves, resulting in extreme imbalance in excitation energy distribution between the two photosystems

Abstract In intact leaves, a new physiological state is obtained reversibly at low light intensity (typically 1 W / m2), in which oxygen evolution yield, monitored by the photoacoustic method, approaches zero. In this ‘low-light’ state, irradiation with far-red (λ > 700 nm) background light immediately restores the normal oxygen yield, resulting in an unusually high Emerson enhancement ratio. Quantitative analysis of the enhancement ratio and the saturation curve of enhancement by far-red light shows that in the new state, short wavelength excitation does not reach PS I reaction centers, resulting in an extreme imbalance between the two photosystems. We suggest that adaptation to the low-light state occurs through loss of excitonic interaction between antennae of PS I and their reaction-centers. It appears also that the ‘far-red’ absorbing pigments do not participate in the disconnection and remain closely attached to the reaction centers of PS I. Their number is estimated to be less than 30 per reaction center. The disconnection of the antennae from the reaction center appears to be reversed by readaptation to ‘normal’ light levels, as well as by a brief preillumination with broad band (400–600 nm) light, acting as a trigger. In the last case, the transition to high oxygen yield state is transient. The quantum requirement of this recovery process is very small (approx. 10 hv / reaction center). The adaptation times after switching from higher to lower intensities and vice versa are in the range of minutes. The fluorescence yield remains virtually constant during adaptation to the low-light state in contrast to expectations, suggesting the possibility of cyclic electron flow around PS II in this state. In a chlorophyll-b-less barley mutant, which lacks the light-harvesting chlorophyll - a b protein (LHC) (and possibly the newly discovered light-harvesting chlorophyll - a b protein associated with PS I (LHC-I)), the ‘low-light’ state was absent. These results are consistent with the hypothesis that these antennae complexes participate directly in the adaptation to low light intensities.

The use of photothermal radiometry in assessing leaf photosynthesis. I : General properties and correlation of energy storage to P700 redox state

Energy storage measurements by modulated photothermal radiometry (PTR) were carried out on intact leaves to assess the value of the PTR method for photosynthesis research. In particular, correlations to the redox state of P700 under various conditions were examined. PTR monitors modulated light conversion to heat by sensing the resulting modulated infra-red radiation emitted from the leaf. It is, therefore, a complementary method to photoacoustics for estimating energy storage and its time variation, particularly under controlled leaf atmosphere.With modulated light-1 (λ>690 nm) the energy storage approached zero and P700 was maximally oxidized. When background light of shorter wavelength (λ<690 nm-light-2) was added, energy storage momentarily increased (a manifestation of Emerson enhancement) while P700 was reduced. The values of both parameters varied as a function of the background light intensity, keeping a mutual linear relationship. Following the initial change, there was a slow reversal transient of P700 oxidation with a parallel decrease in energy storage. Temporal correlation to P700 redox state after dark adaptation was observed also for the energy storage measured in modulated light 2 when combined with background actinic light of medium intensity (about 50 W m2). Under these circumstances P700 was almost totally oxidized initially and then gradually reduced while energy storage was initially low and then increased parallel to P700 reduction.A comparison between the maximum energy storage in modulated light 1, enhanced by background light 2, to the energy storage with short wavelength light (where light tends to be more evenly distributed) indicates a comparable contribution to energy storage from each active photosystem. The above experiments indicate that energy storage contribution from PS I is directly related to the extent of openness of its reaction-centers.While some aspects of the data call for more experimentation, these experiments already establish PTR as a valuable method to monitor photosynthetic energy storage activity in vivo, particularly when used simultaneously with other non-invasive methods.

Rapid screening for heat tolerance in Phaseolus species using the photoacoustic technique

Abstract The potential utility of photoacoustic measurements in vivo for rapid estimation of heat tolerance was investigated in seven Phaseolus genotypes with known and contrasting heat resistance. When small leaf discs of 1 cm diameter were heated at 40.5°C, both relative quantum yield for oxygen evolution (as estimated by the ratio of the amplitude of the oxygen evolution signal (Aox) to the amplitude of the photothermal signal (Apt)) and photochemical energy storage were reduced drastically within a few minutes in the heat sensitive genotypes. In contrast, in the genotypes processing heat tolerance characteristics, these photoacoustic parameters remained constant for at least 20 min. Photochemical energy storage appeared to be more resistant towards heat stress conditions than oxygen evolution, suggesting differential heat sensitivity of PS2 compared to PS1. Within the heat resistant species, we were able to further select for high temperature tolerance when the rate of decrease of Aox/Apt was measured in leaf discs incubated at a higher temperature (42°C). The results suggest that the photoacoutic technique could be used as a rapid and easy screening test for heat tolerance in crop plants.