Alaka Srivastava

POLYPHASIC CHLOROPHYLL a FLUORESCENCE TRANSIENT IN PLANTS AND CYANOBACTERIA

Abstract— The variable chlorophyll (Chl) a fluorescence yield is known to be related to the photochemical activity of photosystem II (PSII) of oxygen‐evolving organisms. The kinetics of the fluorescence rise from the minimum yield, F0, to the maximum yield, Fm, is a monitor of the accumulation of net reduced primary bound plastoquinone (QA) with time in all the PSII centers. Using a shutter‐less system (Plant Efficiency Analyzer, Hansatech, UK), which allows data accumulation over several orders of magnitude of time (40 μs to 120 s), we have measured on a logarithmic time scale, for the first time, the complete polyphasic fluorescence rise for a variety of oxygenic plants and cyanobacteria at different light intensities. With increasing light intensity, the fluorescence rise is changed from a typical O‐I‐P characteristic to curves with two intermediate levels J and I, both of which show saturation at high light intensity but different intensity dependence. Under physiological conditions, Chl a fluorescence transients of all the organisms examined follow the sequence of O‐J‐I‐P. The characteristics of the kinetics with respect to light intensity and temperature suggest that the O‐J phase is the photochemical phase, leading to the reduction of QA to QA‐. The intermediate level I is suggested to be related to a heterogeneity in the filling up of the plastoquinone pool. The P is reached when all the plastoquinone (PQ) molecules are reduced to PQH2. The addition of 3‐(3–4‐dichlorophenyl)‐1,1‐dimethylurea leads to a transformation of the O‐J‐I‐P rise into an O‐J rise. The kinetics of O‐J‐I‐P observed here was found to be similar to that of O‐I1‐I2‐P, reported by Neubauer and Schreiber (Z. Naturforsch.42c, 1246–1254, 1987). The biochemical significance of the fluorescence steps O‐J‐I‐P with respect to the filling up of the plastoquinone pool by PSII reactions is discussed.

Regulation of antenna structure and electron transport in Photosystem II of Pisum sativum under elevated temperature probed by the fast polyphasic chlorophyll a fluorescence transient: OKJIP

Abstract Chlorophyll a (Chl a) florescence induction kinetics from the minimum yield F0 to the maximum yield Fm provide information on the filling up of the plastoquinone pool with reducing equivalents. In this paper, we have examined the effect of high temperature (above 40°C) on Chl a fluorescence rise kinetics starting from 40 μs (to 1 s) in pea leaves (Pisum sativum). The variable Chl a fluorescence is strongly quenched after heat treatment. With increasing temperature or the duration of heat treatment a typical O-J-I-P transient (Strasser et al. (1995) Photochem. Photobiol., 61, 32–42) is transformed into an O-K-J-I-P transient, with an additional rapid step called K detected in the 200–300 μs range. After prolonged heat treatment, the K-step becomes a dominant peak in the Chl a fluorescence transient followed by a large dip. We have investigated the origin and the possible interpretation of these changes by using NH2OH which acts as an electron donor to PS II, and DCMU which is known to block the PS II electron transport chain by displacing QB. From the present data we propose that the appearance of this K-step is due to two effects: (1) inhibition of the water splitting system that leads to a much slowed turn over of the reduction of QA; (2) changes in the architecture of the antenna of PS II which affect the energy migration properties within the photosynthetic unit. The K-step can thus be used as an indicator of the heterogeneity of photosynthetic units and as an indicator for the physiological state of the photosynthetic sample.

Multiple effects of chromate on the photosynthetic apparatus of Spirodela polyrhiza as probed by OJIP chlorophyll a fluorescence measurements

Chromate (Cr) decreases the growth of Spirodela polyrhiza. The fronds lost their pigments. The O2 evolution was also decreased. The Cr effect was found to be dose dependent. The toxic effects of Cr have further been studied on the photosynthetic activity of Spirodela polyrhiza by means of the chlorophyll a (Chl a) fluorescence transient O-J-I-P. The Chl a fluorescence transients were recorded in vivo with high time resolution and analyzed according to the JIP-test which can quantify the photosystem II behavior. Cr treated plants show a decrease in yield for primary photochemistry, phi Po. The performance index of PSII, PIABS, which is the combination of the indexes of three independent parameters, (1) the total number of active reaction centers per absorption (RC/ABS), (2) yield of primary photochemistry (phi Po) and (3) efficiency with which a trapped exciton can move an electron into the electron transport chain (psi 0), decreased due to Cr treatment. Chromate sensitivity varies within plant populations. In summary Cr affects several targets of PSII. More specifically, the main targets of Cr, according to the JIP-test, can be listed as a decrease in the number of active reaction centers and damage to the oxygen-evolving complex.

Greening of Peas: Parallel Measurements of 77 K Emission Spectra, OJIP Chlorophyll a Fluorescence Transient, Period Four Oscillation of the Initial Fluorescence Level, Delayed Light Emission, and P700

Irradiation of etiolated leaves leads to their greening. Although this problem has a long history, the question of whether the intermittent irradiation (IMI) grown plants have fully functional reaction centres as well as the oxygen clock, before exposure to continuous irradiation (CI), had not been resolved. To answer this question, as well as to analyze the development of the two photosystems, the following parallel measurements were made: (1) Emission spectra at 77 K; (2) OJIP chlorophyll (Chl) a fluorescence transient; (3) period 4 oscillation in the flash number dependence of initial fluorescence F0 (at 50 µs) and FJ (at 2 ms); and (4) P700. In the 1-ms-flash (FL) grown pea, that has a different biogenesis of the photosynthetic apparatus, delayed light emission (DLE) and Chl a fluorescence transient were measured in parallel. Quantitative analysis of Chl a fluorescence values provided the following conclusions: (1) IMI, not FL, plants have almost fully developed reaction centres and the oxygen clock. (2) Further greening of IMI plants under CI involves two phases: (a) during 3-4 h of CI, the number of PS2 units and connectivity between them increase, and then (b) light-harvesting antenna increases. (3) In FL, 10 min CI activates fully the oxygen clock.

Action of the allelochemical, fischerellin A, on photosystem II

The cyanobacterium, Fischerella muscicola, produces a secondary metabolite named fischerellin A (FS) that strongly inhibits the growth of cyanobacteria and other photosynthetic organisms. The compound exhibits a unique structure and is composed of two cyclic amines and a C15 substituent that contains a double bond in the (Z) configuration and two triple bonds [L. Hagmann, F. Jüttner, Tetrahedron Lett., 37 (1996) 6539-6542]. The site of FS action is located in photosystem II (PSII). The chlorophyll fluorescence induction transient and O2 evolution methods have been used to determine the site of action of FS in PSII. FS affects the fluorescence transients, as well as O2 evolution by the cyanobacterium, Anabaena P9. The green alga, Chlamydomonas reinhardtii, and higher plants were also affected by FS in a concentration- and time-dependent fashion. FS acts at several sites which appear with increasing half-time of interaction in the following sequence: (1) effect on the rate constant of QA- reoxidation; (2) primary photochemistry trapping; (3) inactivation of PSII reaction center; and (4) segregation of individual units from grouped units. FS does not affect the photosynthetic activity of purple bacteria, Rhodospirillum rubrum.

Stress and Stress Management of Land Plants During a Regular Day

In vivo photochemical activity of photosystem II (PSII) was measured by Chlorophyll a fluorescence intensity of pea leaves exposed to different types of stresses of daily time course. Pea plants grown at 22 °C show a dramatical decrease in the activity of PSII when exposed to higher temperatures. High light exacerbates the damage of PSII when imposed together with high temperature. But the low light acts as an efficient protector of the photochemical activity against its inactivation by heat. The thermal tolerance of PSII was also triggered by exposing the leaves to moderately elevated temperature and low light (30 °C and 30 Wm -2 ) before exposing them to higher temperatures. The increase in thermo-tolerance was more than 5 to 10 °C. Pre-heat treatment causes an upward shift in the photoinhibitory light. The stability of PSII against heat was increased strongly in water stressed leaf discs. The experimental results show the existence of an adaptive mechanism of land plants to protect themselves against heat and strong light, the usual changes during the diurnal cycle of the day. A comparable behavior has not been found in unicellular organisms. Our results demonstrate the existence of an antagonism between different stresses (heat, light, water deficit) which are associated to the time course of the day.

Polyphasic rise of chlorophyll a fluorescence in herbicide-resistant D1 mutants of Chlamydomonas reinardtii

Chlorophyll (Chl) a fluorescence transient, a sensitive and non-invasive probe of the kinetics and heterogeneity of the filling up of the electron acceptor pool of Photosystem II (PS II), was used to characterize D1-mutants of Chlamydomonas reinhardtii. Using a shutter-less system (Plant Efficiency Analyzer, Hansatech, UK), which provides the first measured data point at 10 μs and allows data accumulation over several orders of magnitude of time, we have characterized, for the first time, complete Chl a fluorescence transients of wild type (WT), cell wall less (CW-15) C. reinhardtii and several herbicide-resistant mutants of the D1 proteins: D1-V219IThe mutants are labeled as follows: the single letter code for the wild type amino acid, followed by the residue number, then the code for the mutated amino acid. A251V, F255Y, S264A G256D and L275F. In all cases, the Chl a fluorescence induction transients follow a pattern of O-J-I-P where J and I appear as two steps between the minimum Fo (O) and the maximum Fmax (Fm, P) levels. The differences among the mutants are in the kinetics of the filling up of the electron acceptor pool of PS II (this paper) in addition to those in the re-oxidation kinetics of QA− to QA, published elsewhere (Govindjee et al. (1992) Biochim Biophys. Acta: 1101: 353–358; Strasser et al. (1992) Archs. Sci. Genève 42: 207–224) and not in the ratio of the maximal fluorescence Fm to the initial fluorescence Fo. The value of this experimental ratio is Fm/Fo = 4.4±0.21 independent of the mutation. At 600 W m−2 of 650 nm excitation, distinct hierarchy in the fraction of variable Chl a fluorescence at the J level is observed: S264A > A251V ∼ G256A > L275F ∼ V219I > F255Y ∼ CW-15 ∼ WT. At 300 and 60 W m−2 excitation, a somewhat similar hierarchy among the mutants was observed for the intermediate levels J and I. Addition of bicarbonate-reversible inhibitor formate did not change the O to J phases, slowed the I to P rise, and in many cases, slowed the decay of fluorescence beyond the P level. These observations are interpreted in terms of formate effect being on the acceptor rather than on the donor side (S-states) of PS II. The formate effect was different in different mutants, with L275F being the most insensitive mutant followed by others (V219I, F255Y, WT, A251V and S264A). Further, in the presence of high concentrations of DCMU, identical transients were observed for all the mutants and the WT.The quantum yield of photochemistry of PS II, calculated from 1-(Fo/Fm), is in the range of 0.73 to 0.82 for the WT as well as for the mutants examined. Thus, in contrast to differences in the kinetics of the electron acceptor side of PS II, there were no significant differences in the maximum quantum yield of PS II, among the mutants tested. We suggest that earlier photochemistry yield values were much lower (0.4−0.6) than those reported here due to either higher measured values of Fo by instruments using camera shutters, or due to the use of cells grown in less than-optimal conditions.

Changes in the photosynthetic activities during several stages of vegetative growth of Spirodela polyrhiza : Effect of chromate

Summary The photosynthetic behavior of fresh green plants, plants carrying turions and the detached turions of Spirodela polyrhiza , was studied by using chlorophyll a (Chl a ) fluorescence. The fresh green fronds showed a normal Chl a fluorescence transient, OJIP. However, the mother fronds carrying turions did not show an OJIP transient. Instead, a clear OKJIP with an additional K step, which usually appears due to limitation on the water splitting system, was detected. The K step again disappeared in matured turions. The photosynthetic performance index (PI) of turions was more than 46% lower than the fresh green plants, which has been correlated with a decrease in the probability that an absorbed photon will move an electron into the electron transport chain. Cr 2 O 7 2- inhibited the plant growth, though it induces the turion formation. Cr 2 O 7 2- also induces the K step in green fronds. The overall photosynthetic efficiency of PS II and PS I of green fronds was found to be decreased after Cr 2 O 7 2- treatment.

DIFFERENTIAL EFFECTS OF DIMETHYLBENZOQUINONE AND DICHLOROBENZOQUINONE ON CHLOROPHYLL FLUORESCENCE TRANSIENT IN SPINACH THYLAKOIDS

In isolated thylakoids, the differential effects of 2,5-dimethyl-p-benzoquinone (DMQ) and 2,6-dichloro-p-benzoquinone (DCBQ) on chlorophyll a (Chl a) fluorescence transients and 02 evolution data have been used to differentiate between active and inactive photosystem II (PSII) centers. This conclusion was challenged by Lavergne and Leci (Photosynth. Res., 35 (1993) 323-343). Thus we have systematically re-investigated this phenomenon using different concentrations of DMQ and DCBQ in thylakoids exposed to various light intensities. We show that the differential effects of DMQ and DCBQ on Chl a fluorescence transients in spinach thylakoids include a larger decrease in the v mable Chl a fluorescence by DCBQ than by DMQ and a decrease in the/7o level with increasing [DCBQ] but not [DMQ] in the 5-150 /aM range. These differential effects confirm the results of Lavergne and Leci, and thus sustain the conclusion that the effects of DMQ and E,CBQ on Chl a fluorescence cannot be used to distinguish between active and inactive PSII centers. The data show that the decrease in Chl a fluorescence follows Stern-Volmer behavior. Furthermore, our data confirm that the rate of 02 evolution is higher in the DCBQ-supported E ill reaction than in the DMQ- supported Hill reaction. These results can be explained by a more efficient ability of DCBQ to oxidize the pkastoquinol pool or a more efficient ability of DCBQ to displace QB (secondary bound plastoquinone, a two-electron acceptor of PSII), rather than activating the inactive PSII centers. However, our results do not challenge the existence of inactive PSII. 1~ eywords: Chlorophyll a fluorescence; Active/inactive photosystem ll; Dichlorobenzoquinone; Dimethylbenzoquinone

Interaction of EDTA and iron on the accumulation of Cd2+ in Duckweeds (Lemnaceae)

Summary The influence of free and total concentrations of Cd 2+ in the presence and absence of EDTA on the accumulation in Spirodela polyrhiza (L.) Schleiden, strain SP 20 , and Lemna aequinoctialis Welwitsch, strain LP 6 , was investigated. Accumulation by both plant species correlated with the concentration of free Cd 2+ rather than with total concentration. However, when the ion was removed by the plants from the solution, the EDTA-Cd 2+ complex provided additional free Cd 2+ and accumulation was higher than expected from the calculated free Cd 2+ pool. Because of the high complex stability constant of Fe III -EDTA, Fe 3+ competed with Cd 2+ for EDTA in the solution and, thus, cadmium accumulation was increased by the presence of Fe 3+ . Only after reduction of Fe 3+ to Fe 2+ (low complex stability) or iron uptake by the plants, could the additional EDTA complex Cd 2+ in solution and inhibit its accumulation.