Action spectrum of the redox state of the plastoquinone pool defines its function in plant acclimation.

The plastoquinone (PQ) pool mediates electron flow and regulates photoacclimation in plants. Here we report the action spectrum of the redox state of the PQ pool in Arabidopsis thaliana, showing that 470-500, 560 or 650-660 nm light favors Photosystem II (PSII) and reduces the PQ pool, whereas 420-440, 520 or 690 nm light favors Photosystem I (PSI) and oxidizes PQ. These data were used to construct a model predicting the redox state of PQ from the spectrum of any polychromatic light source. Moderate reduction of the PQ pool induced transition to light state 2, whereas state 1 required highly oxidized PQ. In low-intensity PSI light, PQ was more oxidized than in darkness and became gradually reduced with light intensity, while weak PSII light strongly reduced PQ. Natural sunlight was found to favor PSI, which enables plants to use the redox state of the PQ pool as a measure of light intensity.

Pure forms of the singlet oxygen sensors TEMP and TEMPD do not inhibit Photosystem II.

In a recent article (Hakala-Yatkin and Tyystjärvi BBA 1807 (2011) 243-250) it was reported that the singlet oxygen spin traps 2,2,6,6-tetramethylpiperidine (TEMP) and 2,2,6,6-tetramethyl-4-piperidone (TEMPD) inhibit Photosystem II (PSII), the water oxidizing enzyme. O2 evolution, chlorophyll fluorescence and thermoluminescence were measured and were shown to be greatly affected by these chemicals. This work casts doubts over an earlier body of work in which these chemicals were used as spin traps for monitoring ¹O2 production when PSII was inhibited by high light intensities. Here we show that these spin probes hardly affect PSII. We show that the commercial batches of TEMPD and TEMP used by Hakala-Yatkin and Tyystjärvi contained impurities and/or derivatives that inhibited PSII and caused the specific effects on fluorescence. Earlier work that used pure spin traps to measure ¹O2 during photoinhibition, thus remains valid. However, concern must be expressed towards using these spin traps without proper controls.

Magnetic field protects plants against high light by slowing down production of singlet oxygen.

Recombination of the primary radical pair of photosystem II (PSII) of photosynthesis may produce the triplet state of the primary donor of PSII. Triplet formation is potentially harmful because chlorophyll triplets can react with molecular oxygen to produce the reactive singlet oxygen (¹O2). The yield of ¹O2 is expected to be directly proportional to the triplet yield and the triplet yield of charge recombination can be lowered with a magnetic field of 100-300 mT. In this study, we illuminated intact pumpkin leaves with strong light in the presence and absence of a magnetic field and found that the magnetic field protects against photoinhibition of PSII. The result suggests that radical pair recombination is responsible for significant part of ¹O2 production in the chloroplast. The magnetic field effect vanished if leaves were illuminated in the presence of lincomycin, an inhibitor of chloroplast protein synthesis, or if isolated thylakoid membranes were exposed to light. These data, in turn, indicate that ¹O2 produced by the recombination of the primary charge pair is not directly involved in photoinactivation of PSII but instead damages PSII by inhibiting the repair of photoinhibited PSII. We also found that an Arabidopsis thaliana mutant lacking α-tocopherol, a scavenger of ¹O2, is more sensitive to photoinhibition than the wild-type in the absence but not in the presence of lincomycin, confirming that the target of ¹O2 is the repair mechanism.

Inhibition of Photosystem II by the singlet oxygen sensor compounds TEMP and TEMPD.

2,2,6,6-tetramethylpiperidine (TEMP) and 2,2,6,6-tetramethyl-4-piperidinone (TEMPD) have earlier been used to quantify singlet oxygen produced by plant material. Both compounds were found to cause severe side effects on Photosystem II. Addition of TEMP or TEMPD to thylakoids immediately stabilized the reduced state of the Q(A) electron acceptor and destabilized the reduced state of the Q(B) acceptor, causing decrease in the driving force of forward electron transfer. Oxygen evolution, thermoluminescence and fluorescence measurements indicated that the number of functional PSII units decreased during incubation of thylakoids with TEMP or TEMPD. Singlet oxygen determinations in photosynthetic systems with piperidine derivatives should be interpreted with care.

Mahalanobis distance screening of Arabidopsis mutants with chlorophyll fluorescence.

Rapid nondestructive screening of mutants is a common step in many research projects in plant biology. Here we report the development of a method that uses kinetic imaging of chlorophyll fluorescence to detect phenotypes that differ from wild-type plants. The method uses multiple fluorescence features simultaneously in order to catch different types of photosynthesis-related mutants with a single assay. The Mahalanobis distance was used to evaluate the degree of similarity in fluorescence features between the wild-type and test plants, and plants differing strongly from the wild-type were classified as mutants. The method was tested on a collection of photosynthesis-related mutants of Arabidopsis thaliana. The plants were evaluated from images in which the color of each pixel depended on the Mahalanobis distance of the fluorescence features. Two parameters of the color-coding procedure were used to adjust the trade-off between detection of true mutants and erratic classification of wild-type plants as mutants. We found that a large percentage of photosynthesis-related mutants can be detected with this method. Scripts for the free statistics software R are provided to facilitate the practical application of the method.

Contributions of visible and ultraviolet parts of sunlight to photoinhibition.

Photoinhibition is light-induced inactivation of PSII, and action spectrum measurements have shown that UV light causes photoinhibition much more efficiently than visible light. In the present study, we quantified the contribution of the UV part of sunlight in photoinhibition of PSII in leaves. Greenhouse-grown pumpkin leaves were pretreated with lincomycin to block the repair of photoinhibited PSII, and exposed to sunlight behind a UV-permeable or UV-blocking filter. Oxygen evolution and Chl fluorescence measurements showed that photoinhibition proceeds 35% more slowly under the UV-blocking than under the UV-permeable filter. Experiments with a filter that blocks UV-B but transmits UV-A and visible light revealed that UV-A light is almost fully responsible for the UV effect. The difference between leaves illuminated through a UV-blocking and UV-transparent filter disappeared when leaves of field-grown pumpkin plants were used. Thylakoids isolated from field-grown and greenhouse-grown plants were equally sensitive to UV light, and measurements of UV-induced fluorescence from leaves indicated that the protection of the field-grown plants was caused by substances that block the passage of UV light to the chloroplasts. Thus, the UV part of sunlight, especially the UV-A part, is potentially highly important in photoinhibition of PSII but the UV-screening compounds of plant leaves may offer almost complete protection against UV-induced photoinhibition.

Acclimation of photosynthesis to nitrogen deficiency in Phaseolus vulgaris.

Nitrogen deficiency diminishes consumption of photosynthates in anabolic metabolism. We studied adjustments of the photosynthetic machinery in nitrogen-deficient bean plants and found four phenomena. First, the number of chloroplasts per cell decreased. Chloroplasts of nitrogen starved leaves contained less pigments than those of control leaves, but the in vitro activities of light reactions did not change when measured on chlorophyll basis. Second, nitrogen deficiency induced cyclic electron transfer. The amounts of Rubisco and ferredoxin-NADP(+) reductase decreased in nitrogen starved plants. Low activities of these enzymes are expected to lead to increase in reduction of oxygen by photosystem I. However, diaminobenzidine staining did not reveal hydrogen peroxide production in nitrogen starved plants. Measurements of far-red-light-induced redox changes of the primary donor of photosystem I suggested that instead of producing oxygen radicals, nitrogen starved plants develop a high activity of cyclic electron transport that competes with oxygen for electrons. Nitrogen starvation led to decrease in photochemical quenching and increase in non-photochemical quenching, indicating that cyclic electron transport reduces the plastoquinone pool and acidifies the lumen. A third effect is redistribution of excitation energy between the photosystems in favor of photosystem I. Thus, thylakoids of nitrogen starved plants appeared to be locked in state 2, which further protects photosystem II by decreasing its absorption cross-section. As a fourth response, the proportion of non-Q(B)-reducing photosystem II reaction centers increased and the redox potential of the Q(B)/Q(B)(-) pair decreased by 25 mV in a fraction of photosystem II centers of nitrogen starved plants.

Short flashes and continuous light have similar photoinhibitory efficiency in intact leaves.

Lincomycin-treated pumpkin leaves were illuminated with either continuous light or saturating single-turnover xenon flashes to study the dependence of photoinactivation of photosystem II (PSII) on the mode of delivery of light. The flash energy and the time interval between the flashes were varied between the experiments, and photoinactivation was measured with oxygen evolution and the ratio of variable to maximum fluorescence (F(v)/F(m)). The photoinhibitory efficiency of saturating xenon flashes was found to be directly proportional to flash energy and independent of the time interval between the flashes. These findings indicate that a low-light-specific mechanism, based on charge recombination between PSII electron acceptors and the oxygen-evolving complex, is not the main cause of photoinactivation caused by short flashes in vivo. Furthermore, the relationship between the rate constant of photoinactivation and photon flux density was similar for flashes and continuous light when F(v)/F(m) was used to quantify photoinactivation, suggesting that continuous-light photoinactivation has a mechanism in which the quantum yield does not depend on the mode of delivery of light. A similar quantum yield of photoinhibition for flashes and continuous light is compatible with the manganese-based photoinhibition mechanism and with mechanisms in which singlet oxygen, produced via a direct photosensitization reaction, is the agent of damage. However, the classical acceptor-side and donor-side mechanisms do not predict a similar quantum yield for flashes and continuous light.