Annals of RSCB Vol. XVIII, Issue 1/2013

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1 INDUCTION OF PHOTOSYNTHETICAL STATE TRANSITIONS IN THE CYANOBACTERIUM Microcystis aeruginosa AICB72 M. Mituleţu 1, V. Bercea 1, C. Coman 1, B. Drugă 1, C. Sicora 1,2 1 INSTITUTE OF BIOLOGICAL RESEARCH CLUJ-NAPOCA 2 BIOLOGICAL RESEARCH CENTER, JIBOU Summary The cyanobacterium Mycrocystis aeruginosa AICB 72 was grown at room temperature, on GZ medium, in air-lifted cultures, with a light intensity of 26 µe.m - 2.s -1, for 14 days. In State 1, under far-red and actinic light (62nm), there was an increase in F and Fm. Under blue light (469nm), in the presence of 3 µm DCMU the fluorescence of PS II was inhibited. Far-red light increased the maximal PS II quantum yield (Fv/Fm) and the effective PS II quantum yield (Y II ) and the blue and actinic light inhibited them. The quantum yield of non-regulated energy dissipation (Y NO ), increased under blue and actinic light and decreased under far-red light. The qp and ql coefficients, kept on their maximal value, except for the blue light experiment. For the PS I, under far red light we could observe an increase in the oxidation state of the P 7 centre. A decrease in the oxidation state of the P 7 centre under blue and actinic light was observed. Also the photochemical quantum yield of PS I (Y I ) decreased under farred light and increased under blue and actinic light. The non-photochemical quantum yield of PS I (Y ND ), increased under far-red light and decreased under blue and actinic light. In State 2, the F, Fm and Fv parameters increased under actinic light (62nm) and decreased under blue light (46 nm). The maximal PS II quantum yield (Fv/Fm) and the effective PS II quantum yield (Y II ) decreased under blue and actinic light but the quantum yield of non-regulated energy dissipation (Y NO ), increased. The qp and ql coefficients, kept on their maximal value, except for the blue light experiment. For the PS I in state 2, low levels under actinic and blue light were measured and the oxidized state of P 7 center decreased. The photochemical quantum yield (Y I ) increased under actinic light and decreased under blue light. Also the non-photochemical quantum yield of PS I (Y ND ), was diminished under actinic light and increased under blue light. Under red light illumnation the fraction of reduced P 7 centers increased and the fraction of oxidized P 7 centers limited by their donor side decreased. State 1-to state 2 and state 2- to state 1 transitions lead to an increase of F and Fm. Also the maximal and the effective quantum yield were decreased and the quantum yield of non-regulated energy dissipation (Y NO ) was increased. The inhibition of the PS I activity in both type of transition states, shows a drop in the oxidized state of the P 7 reaction center. The photochemical quantum yield of PS I (Y I ) increased and it is a measure of the fraction of overall P 7 centers that are reduced and not limited by acceptor side The nonphotochemical quantum yield of PS I (Y ND ), was diminished in both types of transition. Keywords: chlorophyll fluorescence, coefficient of photochemical quenching (qp, ql), effective PS II quantum yield (Y II ), maximal fluorescence, minimal fluorescence, maximal PS II quantum yield (Fv/Fm), maximal P 7 change (Pm), non-photochemical quantum yield of PS I (Y ND ), photochemical quantum yield of PS I (Y I ), quantum yield of non-regulated energy dissipation (Y NO ), photoinhibition, state 1 and state 2, state 2 to state 1 transition. cosmin.sicora@gmail.com 56

2 Introduction While light is essential for growth of photosynthetic organisms, excess light energy leads to the production of reactive oxygen species and to eventual inactivation of photosynthesis. To avoid such damage, photosynthetic organisms must adapt to high-light (HL) conditions by altering their photosynthetic apparatus (Muramatsu et al., 29). State 1 State 2 transitions ( state transitions ) are a rapid mechanism for reconfiguring the photosynthetic lightharvesting apparatus in response to changing conditions (Mullineaux et al., 24). Illumination conditions which lead to excess excitation of photosystem II (PSII) compared with photosystem I (PSI) induce a transition to State 2, in which more absorbed excitation energy is diverted to PSI. When PSI is over-excited relative to PSII this induces a transition to State 1, in which more energy is transferred to PSII. State transitions can be triggered by changes in the redox state of electron carriers between PSII and PSI. The biochemical mechanism of state transitions in cyanobacteria is not known, but it is likely to be significantly different from that in green plants (Mullineaux et al., 24). In cyanobacteria grown under standard conditions, the phycobilisomes are the major accessory light-harvesting complexes. Phycobilisomes can transfer energy directly to PSI as well as to PSII. State transitions change the relative energy transfer from phycobilisomes to PSI and PSII, and also the distribution of chlorophyll-absorbed energy (van Thor et al., 1998). Phycobilisomes are mobile complexes, diffusing rapidly on the surface of the thylakoid membrane. By contrast, PSII is completely immobile under normal conditions (Mullineaux et al., 1997; Sarcina et al., 21). This indicates that the association between phycobilisomes and reaction centers is transient and unstable. From time-resolved fluorescence data it can be estimated that up to 5 6% of phycobilisomes are decoupled from PSII during transition to State 2 (Mullineaux et al., 199), followed by a functional coupling to PSI as shown by energy storage studies (Mullineaux et al., 1991). The biochemical mechanism involved is not known, but one gene specifically required for the process has been identified. The present study shows the results obtained regarding the photochemical activity of the main components of the photosynthetical apparatus (PSI and PSII) when state transitions are induced in the cyanobacterium Microcystis aeruginosa AICB 72. Material and methods The cyanobacterium Microcystis aeruginosa AICB 72 was grown at room temperature, on GZ medium, in air-lifted cultures, at a medium light intensity of 26 µe.m -2.s -1 for 14 days. The cyanobacterial cells reaching the exponential phase of growth were then exposed to different light intensities to induce state transitions. The chlorophyll fluorescence measurements were performed with Walz Dual-PAM- fluorometer using light exposed probes. Cyanobacterial cultures grown under standard conditions were used as control probes. Results and discussions Usually, state transitions are induced by exposure to light 1 (7 nm) or light 2 (68 nm) in order to preferentially excite PSI or PSII, respectively. Because the difference between the wavelengths is small, we would not expect a full transition (Takahashi et al., 26). The methods to induce state 1 and state 2 for the photosynthetic apparatus are very diverse (Table 1). In our experiments, in order to induce state 1, we used three methods: R1: - dark incubation for 24 hours in air-lifted cultures, 5 minutes exposure to far-red light and then a saturation pulse was applied. 57

3 Table 1 Methods for inducing state transitions State 1 State 2 Reference red light incubation (71 nm) 5 min dark incubation 5 min Allen et al., 1989 in the presence of DCMU dark adaptation exposure to light 1 (71 nm) exposure to blue light (425 nm) dark adaptation 5 min and exposure to a 45 nm light illumination with light 2 and background light 1 (715 nm) for 25 min exposure to light 2 (68 nm) dark adaptation 5 min and exposure to an orange light 61 nm illumination with light 2 (65 nm) for 25 min Bissati et al., 2 Veeranjaneyulu et al.,1991 light 1 (715 nm) incubation 3 min light 2 (65 nm) incubation 3 min Veeranjaneyulu et al.,1994 dark incubation light incubation Vallon et al., 1991 light exposure (4-lux) for 2 min in the presence of 1 µm DCMU cells illuminated with 5 µmol.m -2.s M DCMU to oxidize the plastoquinone pool dark incubation under aerobic conditions for 2 hours dark adaptation for 2 min in the presence of 2µM antimycin A and 4 mm salicylhydroxamic acid (which block ATP production) reduction of the plastoquinone pool by dark incubation in anaerobiosis obtained with the addition of glucose and gulucose-oxidase dark incubation under anaerobic conditions (nitrogen bubbling) for 2 min Hamel et al., 2 Turkina et al., 26 induced by far-red light (>7nm) induced by red light (>64 nm) Su and Shen, 23 dark incubation for 16 hours and exposure to far-red light for 1 hour dark incubation for 16 hours and exposure to red light for 1 hour Takahashi et al., 26 dark adaptation and 3 min exposure to far-red light cells exposed for 15 min to light 1 (72 nm) cells treated with 1-5 M DCMU and exposed to white light 6 µe.m -2.s -1 for 15 min cells incubated with 1 µm DCMU for 2 min cells were placed in state 1 either by an incubation for 2 min under continuous illumination (27 lx) in the presence of DCMU (DCMU+light) or in darkness under strong aeration dark adaptation with the addition of 5 µm DCMU and exposure to far-red light (76 nm) for 1 min cells exposed to a blue light in the presence of 1 µm DCMU dark incubation of aerated cultures and exposure to far-red light incubation of the cells in the dark under strong agitation incubation of the cells in the dark under continuous agitation dark adaptation and exposure to blue light cells exposed for 5 min to a 476 nm light cells treated with 1-5 M DCMU and the samples were flushed with nitrogen in the absence of light cells incubated with 5 µm FCCP for 2 min anaerobic incubation in darkness under nitrogen atmosphere for 3 min dark adaptation with the addition of 5 µm DCMU and exposure to blue light (76 nm) for 1 min cells are dark incubated longer than 1 min dark incubation in anaerobiosis obtained with the addition of glucose and gulucose-oxidase and exposure to a 59 nm light induced through anaerobiosis or through FCCP addition to the aerobic cell cuture obtained through dark incubation in anaerobic conditions obtained by argon bubbling Shapiguzov et al., 21 Iwaia et al., 21 Lefebvre-Legendre et al., 27 Iwai et al., 28 Bulté et al., 199 Hodges and Barber, 1983 Fujimori et al., 25 Finazzi et al., 21 Finazzi et al., 22 Finazzi et al., 21 58

4 R2: - dark incubation for 24 hours in air-lifted cultures, then illumination 753 µe.m -2.s -1 ) for 5 minutes in the presence of 3µM DCMU. R3: - dark incubation for 24 hours in air-lifted cultures and 5 minutes exposure to actinic light (62 nm; 1952 µe.m -2.s -1 ) in the presence of DCMU. State 1 represents the structural condition in which most of the excitation energy is used in the photochemistry of PSII. The photochemical activity of PSII in state 1 for the cyanobacterium Microcystis aeruginosa AICB72 is shown in Fig.1. The minimal F and maximal Fm fluorescence and also the variable fluorescence Fv have increased significantly under far-red (R1) and actinic light (62nm)(R3) (Fig. 1 A). The slightly lower values of Fm related to F for the actinic light experiment (R3), led to a diminishing of the variable fluorescence (Fv). In the presence of 3µM DCMU, which inhibits the photosynthetic electron transfer from Q A to Q B and under blue light (46nm)_(R2), the chlorophyll fluorescence parameters decreased significantly compared to the control, which showed an inhibition of the PSII activity. The maximal quantum yield (Fv/Fm) and the effective quantum yield (Y II )_of PS II had the same value as the control under far-red light (R1)(Fig.1 B). The exposure to blue light (R2) and actinic light (R3) led to a significant drop of the quantum yields. Also, the quantum yield of non-regulated energy dissipation (Y NO ) increased significantly under blue light (R2) and actinic light (R3), with blue light (46 nm; and had a slight decrease under far-red light (R1). The quantum yield of non-regulated energy dissipation is an expression of the energy dissipation process in the photosystems antenna. The coefficients of photochemical quenching, qp and ql, kept on their maximal value with the exception of R2 (Fig.1 C). The high values registered indicate the maximal opening state and the high number of reaction centres involved in an intense photochemical process with low fluorescence emissions. For the R2 experiment using blue light, the amount of the open reaction centres was slightly diminished. The state 1 induction requires the oxidation of the intersystem chain through the photochemistry of the PS I (Forti & Caldiroli, 25). The activity of PSI in the cyanobacterium Microcystis aeruginosa AICB72 in state 1 is shown in Fig. 2. Pm represents the maximal change of the P 7 signal upon quantitative transformation of P 7 from the fully reduced to the fully oxidized state and it is analogue to the Fm fluorescence. Pm registered slightly higher values than the control under far-red light (R1), which points to the oxidation of the P 7 reaction center. For the blue light (R2) and actinic light (R3) exposure, the Pm signal dropped significantly, reflecting a decrease in the oxidation state of the P 7 center. 59

5 F lu o re s c e n c e % Fo Fm Fv A qp ql R 3 C R 3 Fv/Fm Y(II) Y(NO) F lu o re s c e n c e % B Fig. 1 Evolution of fluorescence parameters (A), quantum yields (B) and coefficients of photochemical quenching (C) of PSII in Microcystis aeruginosa AICB 72 during the photosynthetic state 1 (R 1, R 2 and R 3 -see text). R 3 The photochemical quantum yield_(y I ) decreased under far-red light exposure and had a significant increase under blue (R2) and actinic light (R3). F lu o re s c e n c e % Pm Y(I) Y(ND) R 3 Fig. 2. Photochemical activity of PS I in Microcystis aeruginosa AICB 72 during the photosynthetic state 1 (R 1,R 2 and R 3 -see text) The photochemical quantum yield of PS I, Y(I), is defined by the fraction of overall P 7 centres that in a given state are reduced and not limited by acceptor side.the fraction of P 7 centers in a certain state can vary between (the P 7 centers are totally oxidized) and 1 (the P 7 centers 6 are totally reduced, usually - in the dark). Under far-red light, the reduction state of the P 7 centers decreased, the oxidation state being dominant. The non-photochemical quantum yield of PS I, (Y ND ), increased under illumination with far-red light (R1), but decreased under blue (R2) and actinic (R3) light (Fig. 2). The non-photochemical quantum yield of PS I, Y(ND), represents the fraction of the overall P 7 that is oxidized in a given state. Centers with oxidized P 7 converts the excitation energy quantitatively into heat. The quantum yield (Y ND ) is a measure of donor side limitation, which is enhanced by a trans-thylakoid proton gradient and damages at the level of PS II. After Finazzi et al. (21a), state transition is controlled by the intracellular ATP content: dark adapted cells are in state 2 when the intracellular ATP content is low. In state 2, the PS II activity was more inhibited by light than in state 1. In state 2, however, the D1 subunit was not degraded, whereas a substantial degradation was observed in state 1. In state 2, only

6 cyclic photosynthetic electron transport is active. The activity of PS I and of cytochrome b 6 f was not affected by illumination under the same conditions. State 2 would represent a structural condition where most of the excitation energy is utilized by PS I photochemistry so that cyclic electron transport around PS I is likely to prevail over linear electron flow that involves both PS I and PS II. Cytochrome b 6 f was inhibited by DCMU in state 1, whereas no effect was observed in state 2. This shows that in state 2 the reducing equivalents involved in the reduction of the cytochrome b 6 f do not occur at the level of PS II but rather at the level of PS I (Finazzi et al., 21b) Fo Fm Fv A qp ql C Fv/Fm Y(II) Y(NO) Under these conditions PSII is not connected to the intersystem electron carriers but is still photochemically active (Finazzi et al., 21b). In our experiments, in order to induce the photosynthetic state 2, we used two methods: R1: - dark adaptation for 24 hours under anaerobic conditions induced by argon bubbling, then 5 min exposure to red light (62 nm) with the intensity of 1952 µe.m -2.s -1. R2: - dark adaptation for 24 hours under anaerobic conditions induced by argon bubbling, then 5 min exposure to blue light (46 nm) with the intensity of 753 µe.m -2.s -1. The photochemical activity of the PS II in state 2 conditions is shown in Fig. 3. The minimal F and maximal Fm B Fig. 3. Evolution of fluorescence parameters (A), quantum yields (B) and coefficients of photochemical quenching (C) of PSII in Microcystis aeruginosa AICB 72 during the photosynthetic state 2 (R 1 and R 2 -see text). fluorescence increased significantly under red light (62 nm)(r1) and decreased under blue light (46 nm)(r2), compared to the control (Fig.3 A). The maximal quantum yield (Fv/Fm) and the effective quantum yield (Y II ) of PSII decreased in both experiments R1 and R2, but the drop was more significant for the blue light exposure (R2) (Fig.3 B). The low values showed that the absorbed energy was used as chemically fixed energy through the photochemical charge separation at the reaction centers. The quantum yield of non-regulated energy dissipation (Y NO ) increased for both R1 and R2 experiments (Fig.3 B). The coefficients of photochemical quenching, qp and ql, maintained their maximal value for R1 but not for R2 (Fig.3 C). The high values registered in the red light exposure experiment indicate the 61

7 maximal opening and the high amount of the RCII centers. In the case of the blue light illumination, the number of open reaction centers was significantly diminished. The activity of PS I in the state 2 conditions is shown in fig. 4. The Pm signal registered low values for both types of light (red and blue), pointing out a decrease in the oxidation state of the P 7 center. Also the photochemical quantum yield (Y I ) increased under red light exposure (R1) and had a significant decrease under blue light (R2). The fraction of the reduced P 7 centers also increased in red light. The non-photochemical quantum yield of PS I, (Y ND ), was low under illumination with red light (R1), but had an increase under blue light (R2) (Fig. 4). In our case, under red light (R1), the fraction of P 7 centers that are oxidized and limited by their donor side decreased while under blue light (R2), the donor side becomes non-limitative leading to the increase in the proportion of oxidized P 7 centers. The phenomenon of state transitions in the photosynthetic apparatus, discovered by Bonaventura and Myers (1969), involves the reversible transfer of a fraction of the PS II outer antenna to PS I, and is understood as a mechanism for balancing the absorption cross-section size of both photosystems under natural illumination conditions and therefore their photochemical activities (Forti & Caldiroli, 25) Pm Y(I) Y(ND) Fig. 4. Photochemical activity of PS I in Microcystis aeruginosa AICB 72 during the photosynthetic state 2 (R 1 and R 2 -see text). 62 Photosynthetic organisms alter their photosynthetic apparatus in order to adapt to high-light (HL) conditions. (Hihara & Sonoike,21). In cyanobacteria, the decrease of PSI content is more prominent than that of PS II, leading to the decrease of photosystem stoichiometry (PSI/ PSII ratio) under HL conditions (Hihara & Sonoike, 21). State transitions involve a reversible redistribution of the light-harvesting antenna between PS I/PS II and optimize light energy utilization in photosynthesis whereas the cyclic electron flow modulates the photosynthetic yield (Finazzi et al., 22). The methods employed to induce transitions from one state to the other are very diverse. Shapiguzov et al. (21) exposed the samples to far-red light or transfered them into dark conditions for state 2 to state 1 transition. Bulté et al. (199) induced state 1 to state 2 transition by illumination in the presence of DCMU followed by anaerobiosis and state 2 to state 1 transition through illumination of algal cells in state 2 with 27 lx /2 min in the presence of DCMU. Hodges & Barber(1983), exposed the algal cells to light absorbed preferrentially by PS II and induced state 1 to state 2 transition. The exposure of PS I to excess light or dark incubation induced state 2 to state 1 transition (Hodges & Barber, 1983). In our experiments, the methods employed in order to induce the transition from one state to the other were: R1: state 1 to state 2 transition: cells were incubated in dark anaerobic conditions in the presence of 3 µm DCMU and then exposed to a red actinic light (62 nm, 1952 µe.m -2.s -1 ) for 5 min. R2: state 2 to state 1 transition: cells in photosynthetic state 2 were submitted to a saturation pulse in the presence of DCMU. The photochemical activity of PS II in state transitions is shown in Fig. 5. The minimal fluorescence, increased significantly in both state transitions which

8 indicates the openness of the reaction centers. The maximal fluorescence is higher than the control, but its actual values are below the ones of F. Fm is an expression of the state of PS II when all the Q A (the primary quinone electron acceptor in PS II) molecules are reduced. Because of the high increase of F, the variable fluorescence, Fv has low values (Fig.5 A) Fo Fm Fv A qp ql C Fv/Fm Y(II) Y(NO) B Fig. 5. Evolution of fluorescence parameters (A), quantum yields (B) and coefficients of photochemical quenching (C) of PS II in Microcystis aeruginosa AICB 72 during state 1 to state 2 (R 1 ) and state 2 to state 1 (R 2 ) transitions A lower value of the Fv/Fm parameter (.832) is an expression of an increase of F due to the initially blocked reaction centers. An increase of F under stress conditions, when Fm remained constant, is probably caused by a decrease in the overall rate constant for the utilisation of excitons for photochemistry. This is in agreement with the results that a slight increase in F is caused by a partially reversible decrease in the quantum yield of PS II photochemistry, whereas a higher increase in F probably originates from an irreversible disconnection of the small light harvesting complex of PS II (Lazár, 1999). The maximal quantum yield (Fv/Fm) and the effective quantum yield (Y II ) of PS II, dropped significantly in both transition types compared to the control. On the other hand, the quantum yield of non-regulated energy dissipation (Y NO ), had a significant increase in both state transitions. A high Y(NO) value indicates that both photochemical energy conversion and protective regulatory mechanisms are inefficient. (Fig. 5 B). The coefficients of photochemical quenching, qp and ql registered maximal values in state 1 to state 2 transition (Fig. 5 C). These high values indicate the maximal opening state and the high proportion of the reaction centres RC II. In state 2 to state 1 transition however the open state of the reaction centres was significantly diminished. The photochemical activity of PS I in state transitions is shown in Fig. 6. The 63

9 maximal P 7 change (Pm) has lower values compared to the control in both state transitions, which indicates a decrease in the oxidation state of the P 7 reaction center. Also the photochemical quantum yield (Y I ) increased significantly in both transition states. The reduced state of PSI centers not limited by acceptor side is dominant during state transitions. The non-photochemical quantum yield of PS I, (Y ND ), decreased in both transition states suggesting an increase in the donor side limitation and a decrease of the oxidized P 7 centers. State transitions appear to act as a mechanism to balance excitation of the two photosystems under changing light regimes. State transitions can be triggered by changes in the redox state of electron carriers between PS II and PS I (Mullineaux & Emlyn-Jones, 24) Pm Y(I) Y(ND) Fig. 6. Photochemical activity of PS I in Microcystis aeruginosa AICB 72 during state 1 to state 2 (R 1 ) and state 2 to state 1 (R 2 ) transitions. To maintain maximal rates of photosynthesis at limiting light intensities, plants have evolved a mechanism which enables them to optimize the balance of incoming light energy between PS I and PS II (Hodges & Barber, 1983). Conclusions For the photosynthetic state 1 under far-red and blue light (46 nm), the fluorescence of the chlorophyll increased. Far-red light stimulates the quantum yield of PS II and the opening of the reaction centers decreasing the non-regulated 64 dissipation of the excitation energy. On the other hand, blue and actinic light in the presence of DCMU lead to a diminuation of the quantum yields and open centers of PS II, increasing the non-regulated dissipation of energy. Far-red light also favors the photochemistry of PS II and reduces fluorescence emission. For the PS I photosystem, far-red light leads to an increase in the oxidized state because of the increase in the Pm signal, the diminuation of the photochemical quantum yield and because of the increase in the non-photochemical quantum yield. Blue and actinic light in the presence of DCMU lead to an increase of the reduced state of PS I because of the decrease of the Pm signal and the nonphotochemical quantum yield and because of the increase in the photochemical quantum yield which measures the reduction state. In the photosynthetic state 2, under actinic light (62 nm) there was an increment of the fluorescence parameters, open reaction centers and non-regulated energy dissipation and a diminuation of the quantum yields of the PS II. Blue light exposure decreased fluorescence and opening of the reaction centers. PS I activity in state 2 conditions registered a drop in the oxidized state of the P 7 center because of the decrease of the Pm signal. Actinic light favors the photochemical quantum yield (Y I ) and also the reduced state of the photosystem. Under blue light, the donor and acceptor sides of the P 7 centers become limitative. The photochemical activity of the PS II, in state 1 to state 2 and state 2 to state 1 transitions, was characterized by the increase of the fluorescence parameters, the diminuation of the quantum yields and the increase of the non-regulated energy dissipation. Also in state 2 to state 1 transition the opening of the reaction centers decreases. State transitions had a synergic effect on the PS I photochemistry. There was a decrease in the oxidation of the P 7

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