Informe de la CABE-FASEN: Monitoreo de desórdenes por defciencia de iodo (DDI) en la provincia de Santa Fe, Argentina (2007) / CABE-FASEN Report: Screening of Disorders Associated with lodine Defciency in the Province of Santa Fe, Argentina (2007)

Un total de 1.470 alumnos de escolaridad primaria, de ambos sexos, fue estudiado en este monitoreo de bocio endémico en cuatro localidades de la provincia de Santa Fe: Reconquista (n=404) y Villa Ocampo (n=294) ubicadas en el norte de la provincia, y Rufino (n=317) y Venado Tuerto (n=455) ubicadas en el sur provincial. La edad de los escolares osciló entre 6 y 14 años. Se examinó el cuello de todos los escolares; con el fin de aunar criterios con los realizados en otras provincias, se tomó como referencia la palpación de un solo médico (HN). La definición del grado de bocio fue la misma que fuera utilizada en los otros relevamientos. Se determinó la ioduria en muestras casuales de orina emitidas por los niños una vez que fueron palpados (109 de Reconquista, 105 de Villa Ocampo, 113 de Rufino y 139 de Venado Tuerto). Se midió también el contenido de iodo en 337 muestras de sal de consumo hogareño de Reconquista, 227 de Villa Ocampo, 295 de Rufino y 422 de Venado Tuerto (traídas por los niños desde sus viviendas). El examen palpatorio de los niños reveló una prevalencia de bocio del 2,6%, sin diferencias por localidad. Los niveles de ioduria mostraron diferencias entre las localidades del norte y del sur: en Reconquista 238,6±116 Ag/L con mediana de 213 Ag/L, y en Villa Ocampo 241,6±223,5 Ag/L con mediana de 188 Ag/L; mientras que en Rufino 544±349 Ag/L con mediana de 442 Ag/L y en Venado Tuerto 550±375 Ag/L con mediana de 418 Ag/L (media±DS). Los niveles de iodo en sal fueron similares en toda la provincia, promediando los 35,5 mg/kg. La prevalencia de muestras con concentraciones de iodo <15 mg/kg fueron del 0,3% para Reconquista, del 1,3% en Villa Ocampo, del 3 % en Rufino y del 4 % en Venado Tuerto. Concluimos que en la provincia de Santa Fe no existe endemia bociosa. Los autores declaran no poseer conflictos de interés.(AU)

A total of 1,470 primary school students were evaluated to monitor the prevalence of endemic goiter in four cit-ies of Santa Fe province: Reconquista (404) and Villa Ocampo (294) situated in the north, and Rufino (317) and Venado Tuerto (455) situated in the south of the province. Their age ranged from 6 to 14 years. Neck palpation was performed in all students; for the sake of consistency with monitoring performed in other provinces, palpation was performed by only one physician (HN). The goiter grading system was the same used in other surveys. After palpation, urine samples for iodine determination were randomly collected (109 from Reconquista, 105 from Villa Ocampo, 113 from Rufino and 139 from Venado Tuerto). Iodine was also measured in salt samples collected from the childrens homes (337 from Reconquista, 227 from Villa Ocampo, 295 from Rufino and 422 from Venado Tuerto). Prevalence of goiter was 2.6 % without differences among cities. Urine iodine levels were significantly different between northern and southern cities: Reconquista had a mean of 238.6 ± 116 Ag/L (median 213 Ag/L) and Villa Ocampo had a mean of 241.6 ± 223.5 Ag/L (median 188 Ag/L), while in Rufino the mean was 544 ± 349 Ag/L (median 442 Ag/L) and in Venado Tuerto it was 550 ± 375 Ag/L (median 418 Ag/L; mean ± SD). Iodine contents in salt were similar across the province, with a mean of 35.5 mg/kg. Prevalence of salt samples with iodine concentration <15 mg/kg was 0.3 % in Reconquista, 1.3 % in Villa Ocampo, 3 % in Rufino, and 4 % in Venado Tuerto. We conclude that the province of Santa Fe, Argentina, is free from endemic goiter. No financial conflicts of interest exist.(AU)

Informe de la CABE-FASEN: Monitoreo de desórdenes por defciencia de iodo (DDI) en la provincia de Santa Fe, Argentina (2007) / CABE-FASEN Report: Screening of Disorders Associated with lodine Defciency in the Province of Santa Fe, Argentina (2007)

Un total de 1.470 alumnos de escolaridad primaria, de ambos sexos, fue estudiado en este monitoreo de bocio endémico en cuatro localidades de la provincia de Santa Fe: Reconquista (n=404) y Villa Ocampo (n=294) ubicadas en el norte de la provincia, y Rufino (n=317) y Venado Tuerto (n=455) ubicadas en el sur provincial. La edad de los escolares osciló entre 6 y 14 años. Se examinó el cuello de todos los escolares; con el fin de aunar criterios con los realizados en otras provincias, se tomó como referencia la palpación de un solo médico (HN). La definición del grado de bocio fue la misma que fuera utilizada en los otros relevamientos. Se determinó la ioduria en muestras casuales de orina emitidas por los niños una vez que fueron palpados (109 de Reconquista, 105 de Villa Ocampo, 113 de Rufino y 139 de Venado Tuerto). Se midió también el contenido de iodo en 337 muestras de sal de consumo hogareño de Reconquista, 227 de Villa Ocampo, 295 de Rufino y 422 de Venado Tuerto (traídas por los niños desde sus viviendas). El examen palpatorio de los niños reveló una prevalencia de bocio del 2,6%, sin diferencias por localidad. Los niveles de ioduria mostraron diferencias entre las localidades del norte y del sur: en Reconquista 238,6±116 µg/L con mediana de 213 µg/L, y en Villa Ocampo 241,6±223,5 µg/L con mediana de 188 µg/L; mientras que en Rufino 544±349 µg/L con mediana de 442 µg/L y en Venado Tuerto 550±375 µg/L con mediana de 418 µg/L (media±DS). Los niveles de iodo en sal fueron similares en toda la provincia, promediando los 35,5 mg/kg. La prevalencia de muestras con concentraciones de iodo <15 mg/kg fueron del 0,3% para Reconquista, del 1,3% en Villa Ocampo, del 3 % en Rufino y del 4 % en Venado Tuerto. Concluimos que en la provincia de Santa Fe no existe endemia bociosa. Los autores declaran no poseer conflictos de interés.

A total of 1,470 primary school students were evaluated to monitor the prevalence of endemic goiter in four cit-ies of Santa Fe province: Reconquista (404) and Villa Ocampo (294) situated in the north, and Rufino (317) and Venado Tuerto (455) situated in the south of the province. Their age ranged from 6 to 14 years. Neck palpation was performed in all students; for the sake of consistency with monitoring performed in other provinces, palpation was performed by only one physician (HN). The goiter grading system was the same used in other surveys. After palpation, urine samples for iodine determination were randomly collected (109 from Reconquista, 105 from Villa Ocampo, 113 from Rufino and 139 from Venado Tuerto). Iodine was also measured in salt samples collected from the children's homes (337 from Reconquista, 227 from Villa Ocampo, 295 from Rufino and 422 from Venado Tuerto). Prevalence of goiter was 2.6 % without differences among cities. Urine iodine levels were significantly different between northern and southern cities: Reconquista had a mean of 238.6 ± 116 µg/L (median 213 µg/L) and Villa Ocampo had a mean of 241.6 ± 223.5 µg/L (median 188 µg/L), while in Rufino the mean was 544 ± 349 µg/L (median 442 µg/L) and in Venado Tuerto it was 550 ± 375 µg/L (median 418 µg/L; mean ± SD). Iodine contents in salt were similar across the province, with a mean of 35.5 mg/kg. Prevalence of salt samples with iodine concentration <15 mg/kg was 0.3 % in Reconquista, 1.3 % in Villa Ocampo, 3 % in Rufino, and 4 % in Venado Tuerto. We conclude that the province of Santa Fe, Argentina, is free from endemic goiter. No financial conflicts of interest exist.

Regulation of psbA and psaE expression by light quality in Synechocystis species PCC 6803. A redox control mechanism.

We investigated the influence of light of different wavelengths on the expression of the psbA gene, which encodes the D1 protein of the photosystem II and the psaE gene, which encodes the subunit Psa-E of the photosystem I, in Synechocystis sp PCC 6803. In an attempt to differentiate between a light-sensory and a redox-sensory signaling processes, the effect of orange, blue, and far-red light was studied in the wild-type and in a phycobilisome-less mutant. Transferring wild-type cells from one type of illumination to another induced changes in the redox state of the electron transport chain and in psbA and psaE expression. Blue and far-red lights (which are preferentially absorbed by the photosystem I) induced an accumulation of psbA transcripts and a decrease of the psaE mRNA level. In contrast, orange light (which is preferentially absorbed by the photosystem II) induced a large accumulation of psaE transcripts and a decrease of psbA mRNA level. Transferring mutant cells from blue to orange light (or vice versa) had no effect either on the redox state of the electron transport chain or on the levels of psbA and psaE mRNAs. Thus, light quality seems to regulate expression of these genes via a redox sensory mechanism in Synechocystis sp PCC 6803 cells. Our data suggest that the redox state of one of the electron carriers between the plastoquinone pool and the photosystem I has opposite influences on psbA and psaE expression. Its reduction induces accumulation of psaE transcripts, and its oxidation induces accumulation of psbA mRNAs.

Redox control of ntcA gene expression in Synechocystis sp. PCC 6803. Nitrogen availability and electron transport regulate the levels of the NtcA protein.

In this work we have studied the influence of the cellular redox status in the expression of the Synechocystis sp. PCC 6803 ntcA gene. Two different ntcA transcripts with different 5' ends were detected, depending on the different dark/light or nitrogen availability conditions. Accumulation of a 0.8-kb ntcA message was light and nitrogen dependent, whereas a longer 1.2-kb ntcA transcript was neither light nor nitrogen regulated. NtcA protein levels increased concomitantly with the accumulation of the 0.8-kb ntcA transcript. The light-dependent accumulation of the ntcA gene and the NtcA protein was sensitive to electron transport inhibitors. In addition, Glc-grown Synechocystis sp. cells showed a similar ntcA expression pattern in darkness to that observed under illumination. These data suggested that electron transport, and not light per se may regulate ntcA gene expression. Primer extension analysis, together with gel mobility-shift assays, demonstrated that in vitro, the Synechocystis sp. NtcA protein specifically bound to the putative promoter region from the light/nitrogen-dependent ntcA transcript but not to that from the constitutive 1.2-kb ntcA mRNA. Band-shift experiments carried out in the presence of thiol oxidizing/modifiying agents and different reducing/oxidizing conditions suggested that NtcA binding to its own promoter was under a thiol-dependent redox mechanism. Our results suggest that the cellular redox status plays a central role in the autoregulatory mechanism of the NtcA protein.

UV-B radiation induced exchange of the D1 reaction centre subunits produced from the psbA2 and psbA3 genes in the Cyanobacterium synechocystis sp. PCC 6803.

UV-B irradiation of Synechocystis 6803 cells inhibits photosystem II activity, which can be restored via de novo synthesis of the D1 (and D2) reaction center subunits. Recently we have shown that of the two psbA genes that encode identical D1 proteins in Synechocystis 6803, UV-B preferentially enhances the transcription of psbA3 compared to that of psbA2 [Máté, Z., Sass, L., Szekeres, M., Vass, I. and Nagy, F. (1998) J. Biol. Chem. 273, 17439-17444]. Here we studied the effect of UV-B on the synthesis of the D1 protein from the psbA2 and psbA3 genes in the P7 mutant of Synechocystis 6803. In this mutant, psbA2 carries the Ala251-->Val point mutation, which confers resistance to the photosystem II electron transport inhibitor metribuzin, but psbA3 is the same as in the wild-type. By applying variable chlorophyll fluorescence measurements to distinguish between metribuzin-sensitive and metribuzin-resistant photosystem II centers we quantified the amount of the D1 protein produced from each of the psbA3 and psbA2 genes. When the cells were exposed to UV-B light, the fraction of D1 protein produced from the psbA3 gene was increased from 15-20 to 32-40% of the total D1. This effect was reversible by transferring the cells to visible light. The rate of D1 production from psbA3 increased with increasing UV-B intensities, and was a transient phenomenon at low UV-B levels (0.1 microE x m-2 x s-1). It is concluded that the enhancement of psbA3 gene transcription by UV-B light leads to enhanced D1 protein synthesis from this gene. Our findings demonstrate that the main role of psbA3 transcription activated by UV-B is to increase the size of the psbA mRNA pool available for translation when a rapid repair of the D1 protein is needed under UV-B stress.

Photosystem II fluorescence quenching in the cyanobacterium Synechocystis PCC 6803: involvement of two different mechanisms.

The structural changes associated to non-photochemical quenching in cyanobacteria is still a matter of discussion. The role of phycobilisome and/or photosystem mobility in this mechanism is a point of interest to be elucidated. Changes in photosystem II fluorescence induced by different quality of illumination (state transitions) or by strong light were characterized at different temperatures in wild-type and mutant cells, that lacked polyunsaturated fatty acids, of the cyanobacterium Synechocystis PCC 6803. The amplitude and the rate of state transitions decreased by lowering temperature in both strains. Our results support the hypothesis that a movement of membrane complexes and/or changes in the oligomerization state of these complexes are involved in the mechanism of state transitions. The quenching induced by strong blue light which was not associated to D1 damage and photoinhibition, did not depend on temperature or on the membrane state. Thus, the mechanism involved in the formation of this type of quenching seems to be unrelated to the movement of membrane complexes. Our results strongly support the idea that the mechanism involved in the fluorescence quenching induced by light 2 is different from that involved in strong blue light induced quenching.

Redox control of psbA gene expression in the cyanobacterium Synechocystis PCC 6803. Involvement of the cytochrome b(6)/f complex.

We investigated the role of the redox state of the photosynthetic and respiratory electron transport chains on the regulation of psbA expression in Synechocystis PCC 6803. Different means to modify the redox state of the electron carriers were used: (a) dark to oxidize the whole electron transport chain; (b) a shift from dark to light to induce its reduction; (c) the chemical interruption of the electron flow at different points to change the redox state of specific electron carriers; and (d) the presence of glucose to maintain a high reducing power in darkness. We show that changes in the redox state of the intersystem electron transport chain induce modifications of psbA transcript production and psbA mRNA stability. Reduction of the intersystem electron carriers activates psbA transcription and destabilizes the mRNA, while their oxidation induces a decrease in transcription and a stabilization of the transcript. Furthermore, our data suggest that the redox state of one of the electron carriers between the plastoquinone pool and photosystem I influences not only the expression of the psbA gene, but also that of other two photosynthetic genes, psaE and cpcBA. As a working hypothesis, we propose that the occupancy of the Q(0) site in the cytochrome b(6)/f complex may be involved in this regulation.

Recovery of photosystem II activity in photoinhibited synechocystis cells: light-dependent translation activity is required besides light-independent synthesis of the D1 protein.

Irreversible photoinactivation of photosystem II (PSII) results in the degradation of the reaction center II D1 protein. In Synechocystis PCC 6714 cells, recovery of PSII activity requires illumination. The rates of photoinactivation and recovery of PSII activity in the light are similar in cells grown in minimal (MM) or glucose-containing medium (GM). Reassembly of PSII with newly synthesized proteins requires degradation of the D1 protein of the photoinactivated PSII. This process may occur in darkness in both types of cells. The degraded D1 protein is, however, only partially replaced by newly synthesized protein in MM cells in darkness while a high level of D1 protein synthesis occurs in darkness in the GM cells. The newly synthesized D1 protein in darkness appears to be assembled with other PSII proteins. However, PSII activity is not recovered in such cells. Illumination of the cells in absence but not in the presence of protein synthesis inhibitors allows recovery of PSII activity.

UV-B radiation-induced donor- and acceptor-side modifications of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803.

We studied the effect of UV-B radiation (280-320 nm) on the donor- and acceptor-side components of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803 by measuring the relaxation of flash-induced variable chlorophyll fluorescence. UV-B irradiation increases the t(1/2) of the decay components assigned to reoxidation of Q(A)(-) by Q(B) from 220 to 330 micros in centers which have the Q(B) site occupied, and from 3 to 6 ms in centers with the Q(B) site empty. In contrast, the t(1/2) of the slow component arising from recombination of the Q(A)Q(B)(-) state with the S(2) state of the water-oxidizing complex decreases from 13 to 1-2 s. In the presence of DCMU, fluorescence relaxation in nonirradiated cells is dominated by a 0.5-0.6 s component, which reflects Q(A)(-) recombination with the S(2) state. After UV-B irradiation, this is partially replaced by much faster components (t(1/2) approximately 800-900 micros and 8-10 ms) arising from recombination of Q(A)(-) with stabilized intermediate photosystem II donors, P680(+) and Tyr-Z(+). Measurement of fluorescence relaxation in the presence of different concentrations of DCMU revealed a 4-6-fold increase in the half-inhibitory concentration for electron transfer from Q(A) to Q(B). UV-B irradiation in the presence of DCMU reduces Q(A) in the majority (60%) of centers, but does not enhance the extent of UV-B damage beyond the level seen in the absence of DCMU, when Q(A) is mostly oxidized. Illumination with white light during UV-B treatment retards the inactivation of PSII. However, this ameliorating effect is not observed if de novo protein synthesis is blocked by lincomycin. We conclude that in intact cyanobacterium cells UV-B light impairs electron transfer from the Mn cluster of water oxidation to Tyr-Z(+) and P680(+) in the same way that has been observed in isolated systems. The donor-side damage of PSII is accompanied by a modification of the Q(B) site, which affects the binding of plastoquinone and electron transport inhibitors, but is not related to the presence of Q(A)(-). White light, at the intensity applied for culturing the cells, provides protection against UV-B-induced damage by enhancing protein synthesis-dependent repair of PSII.

DeltapH-dependent photosystem II fluorescence quenching induced by saturating, multiturnover pulses in red algae

We have previously shown that in the red alga Rhodella violacea, exposure to continuous low intensities of light 2 (green light) or near-saturating intensities of white light induces a DeltapH-dependent PSII fluorescence quenching. In this article we further characterize this fluorescence quenching by using white, saturating, multiturnover pulses. Even though the pulses are necessary to induce the DeltapH and the quenching, the development of the latter occurred in darkness and required several tens of seconds. In darkness or in the light in the presence of 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, the dissipation of the quenching was very slow (more than 15 min) due to a low consumption of the DeltapH, which corresponds to an inactive ATP synthase. In contrast, under far-red illumination or in the presence of 3-(3,4-dichlorophenyl)-1,1'-dimethylurea (only in light), the fluorescence quenching relaxed in a few seconds. The presence of N, N'-dicyclohexyl carbodiimide hindered this relaxation. We propose that the quenching relaxation is related to the consumption of DeltapH by ATP synthase, which remains active under conditions favoring pseudolinear and cyclic electron transfer.

Expression of the psbA gene during photoinhibition and recovery in Synechocystis PCC 6714: inhibition and damage of transcriptional and translational machinery prevent the restoration of photosystem II activity.

The D1 reaction center protein of the photosystem II complex is very sensitive to light. It is continuously being damaged, degraded and resynthesized. Under high light, photosystem II inactivation is observed. This is because the rate of D1 damage is faster than that of its replacement. This process can be reversed if exposure to high light is not too long. In this work we study the changes that occur in the transcriptional and translational machinery that could lead to irreversible photoinhibition in Synechocystis PCC 6714. In the first minutes of photoinhibition, high light induced an accumulation of psbA mRNA due to an increase in psbA transcription initiation. Although the transcription rate of other photosynthetic genes (e.g. psaE and cpcB-cpcA) declined, the high turnover of the psbA transcript was maintained for a long time. When the light stress was too long, the stability of psbA mRNA increased and the psbA transcription rate appeared to decrease. A high level of psbA mRNA was maintained even though translation no longer occurred and the cells were unable to recover. Experiments to measure newly synthesized D1 incorporation into the thylakoid membranes during recovery in the presence of rifampicin showed that the initiation of transcription was not required for translation of psbA mRNA when photoinhibition was still reversible. Since psbA translation did not depend on the level of psbA transcript or on the initiation of psbA transcription, we propose that damage to the translational machinery also occurred during light stress, leading to the inhibition of D1 synthesis and to irreversible photoinhibition.

Induced new mutation of D1 serine-268 in soybean photosynthetic cell cultures produced atrazine resistance, increased stability of S2QB- and S3QB- states, and increased sensitivity to light stress.

We have isolated several herbicide-resistant cell lines from photosynthetic cell suspensions of soybean (Glycine max) that possessed different levels of herbicide resistance, photosystem II activity, and chlorophyll a/b ratio. We have further studied the STR7 mutant, which showed the highest level of resistance to atrazine as well as a cross-resistance to 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (50- and 3-fold, respectively, compared with the wild type). Sequencing of the psbA gene (coding for the D1 polypeptide of photosystem II) from this mutant revealed a single change, serine-268 to proline, in the D1 protein. To our knowledge, this substitution has not previously been described in any photosynthetic organism. In addition to affecting atrazine resistance, this single amino acid change caused a decrease in the electron transfer rate between the secondary acceptors QA and QB and a stabilization of the S2QB- and S3QB- states. The mutant also showed a larger antenna size, an increase in non-QB-reducing centers, and a higher sensitivity to light stress. The unusual stability of the S2QB- and S3QB- states indicates that STR7 belongs to a new class of QB-site mutants.

State transitions or delta pH-dependent quenching of photosystem II fluorescence in red algae.

Fluorescence changes attributed to state transitions have been shown to exist in phycobilisome-containing organisms. Contradictory conclusions have been derived from studies about the mechanism of state transitions carried out either in cyanobacteria or in red algae. In this paper, fluorescence changes induced by light 1 and light 2 are reinvestigated in a unicellular red alga, Rhodella violacea, by performing 77 K fluorescence spectra and fluorescence yield measurements at room temperature in the presence of uncouplers and inhibitors of the electron transfer. We show that transfer of light 1-adapted cells to light 2 (green light) induces a large quenching of photosystem II which is suppressed by subsequent incubation in light 1 (far-red or blue light). The level of the photosystem I-related fluorescence does not change during these transfers. We demonstrate that the large quenching of photosystem II induced by low intensities of green light is completely suppressed by addition of NH4Cl, an uncoupler that inhibits ATP synthesis by canceling the delta pH across the membrane. DCCD, which is an inhibitor of the ATPase that swells the delta pH, maintains the quenched state even under light 1 illumination. The opposite effects of DCMU and DBMIB on state transitions are demonstrated to be due to a suppression (by DCMU) or maintenance (by DBMIB) of the delta pH and not to change in the redox state of the plastoquinone. We conclude that, in R. violacea, the fluorescence change commonly associated with state 2 transition is in fact a delta pH-dependent quenching. This type of quenching has always been associated with near-saturating light intensities. Here, we show that very low intensities of a light that activates only the photosystem II induce a delta pH across the membrane that is not dissipated since the ATPase is not activated. The delta pH is dissipated only under conditions in which the photosystem I turns, confirming that the thioredoxin must be reduced to activate the ATPase. We suggest that the fluorescence changes, induced by various light conditions, in cyanobacteria and red algae could be associated with different phenomena.

A Mutation in the D-de Loop of D1 Modifies the Stability of the S2QA- and S2QB- States in Photosystem II.

Photosystem II electron transfer, charge stabilization, and photoinhibition were studied in three site-specific mutants of the D1 polypeptide of Synechocystis PCC 6803: E243K, E229D, and CA1 (deletion of three glutamates 242-244 and a substitution, glutamine-241 to histidine). The phenotypes of the E229D and E243K mutants were similar to that of the control strain (AR) in all of the studied aspects. The characteristics of CA1 were very different. Formate, which inhibits the QA- to QB- reaction, was severalfold less effective in CA1 than in AR. The S2QA- and S2QB- states were stabilized in CA1. It was previously shown that the electron transfer between QA- and QB was modified in CA1 (P Maenpaa, T. Kallio, P. Mulo, G. Salih, E.-M. Aro, E. Tyystjarvi, C. Jansson [1993] Plant Mol Biol 22: 1-12). A change in the redox potential of the QA/QA- couple, which renders the reoxidation of QA- by back or forward reactions more difficult, could explain the phenotype of CA1. Although the rates of photoinhibition measured as inhibition of oxygen evolution, Chl fluorescence quenching, and decrease of thermoluminescence B and Q bands were similar in AR and CA1, the CA1 strain more quickly reached a state from which the cells were unable to recover their activity. The results described in this paper suggest that a modification in the structure of the D-de loop of D1 could influence the properties of the couple QA/QA- in D2 and the mechanism of recovery from photoinhibition.

Influence of DCMU and ferricyanide on photodamage in photosystem II.

The effect of strong illumination of thylakoid membranes was studied under a range of conditions. Under anaerobic conditions, the relatively small quenching of the maximum fluorescence (Fmax) is accompanied by a large increase of the initial fluorescence (F0), which is partially reversible. Changes in the extent of the QA-Fe2+ and chlorophyll triplet EPR signal during anaerobic photoinhibition were consistent with double reduction of QA [as reported by Vass et al. ((1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1408-1412)]. When illumination was done in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or ferricyanide, no change occurred in F0 while Fmax was quenched. The quenching of Fmax occurred more rapidly than the loss of oxygen evolution, and they were both irreversible. In the presence of ferricyanide, the percentage of inhibition of oxygen evolution was larger than the decrease in the extent of the QA-Fe2+ signal, indicating that damage of the donor side occurred. In the presence of DCMU, a decrease of the QA-Fe2+ EPR signal occurred which corresponded to the inhibition of oxygen evolution and to an increase of the triplet EPR signal, indicating a possible overreduction of QA. However, these changes were less marked in the DCMU-treated samples than in the sample without additions and occurred despite the quenching of Fmax. These results suggest that strong illumination of thylakoids, in the presence of DCMU, results in a slower formation of stable forms of reduced QA, thereby allowing the occurrence of side-path reactions leading to Fmax quenching.(ABSTRACT TRUNCATED AT 250 WORDS)

S1 destabilization and higher sensitivity to light in metribuzin-resistant mutants.

Mutations in the secondary quinone electron acceptor (QB) pocket of the D1 protein conferring a modification on the donor side of photosystem II (PSII) have been characterized by gene cloning and sequencing in two metribuzin-resistant mutants of Synechocystis PCC 6714. The mutations induce different herbicide resistances: in M30, a point mutation at the codon 248, isoleucine to threonine, results in resistance only to metribuzin; in M35, a single mutation, Ala251Val, confers metribuzin, atrazine, and ioxynil resistance. As with other herbicide-resistant mutants, M30 and M35 present modifications in the electron transfer between the primary quinone electron acceptor (QA) and QB. In addition, they have a modified oscillatory pattern of oxygen emission: after dark adaptation, the maximum oscillation is shifted by one flash. Both mutants have a higher concentration of the redox state in the dark-adapted state than the wild type. The mutations render the oxygen-evolving system more accessible to cell reductants. The mutation Ala251Val also confers to PSII an increased sensitivity to high light. We have already demonstrated that under light stress a double mutant, AzV (Ala251Val, Phe211Ser), lost the ability to recover the PSII activity sooner than the wild type. Here, we confirm that the modification of the alanine-251 is responsible for this specific sensitivity to high light. We conclude that specific mutations of the QB pocket modify the behavior of the cells under light stress and have an effect on the structure of the D1 protein in the other side of the membrane.

The primary structure of D1 near the QB pocket influences oxygen evolution.

Photosystem II (PS II) is the site of oxygen evolution. Activation of dark adapted samples by a train of saturating flashes produces oxygen with a yield per flash which oscillates with a periodicity of four. Damping of the oxygen oscillations is accounted for by misses and double hits. The mechanisms hidden behind these parameters are not yet fully understood. The components which participate in charge transfer and storage in PS II are believed to be anchored to the heterodimer formed by the D1 and D2 proteins. The secondary plastoquinone acceptor QB binds on D1 in a loop connecting the fourth and fifth helices (the QB pocket). Several D1 mutants, mutated in the QB binding region, have been studied over the past ten years.In the present report, our results on nine D1 mutants of Synechocystis PCC 6714 and 6803 are analyzed. When oxygen evolution is modified, it can be due to a change in the electron transfer kinetics at the level of the acceptor side of PS II and also in some specific mutants to a long ranging effect on the donor side of PS II. The different properties of the mutants enable us to propose a classification in three categories. Our results can fit in a model in which misses are substantially determined by the fraction of centers which have QA (-) before each flash due to the reversibility of the electron transfer reactions. This idea is not new but was more thoroughly studied in a recent paper by Shinkarev and Wraight (1993). However, we will show in the discussion that some doubts remain as to the true origin of misses and double hits.

Oxygen-evolving photosystem II preparation from wild type and photosystem II mutants of Synechocystis sp. PCC 6803.

We present here a simple and rapid method which allows relatively large quantities of oxygen-evolving photosystem II- (PS-II-) enriched particles to be obtained from wild-type and mutants of the cyanobacterium Synechocystis 6803. This method is based on that of Burnap et al. [Burnap, R., Koike, H., Sotiropoulou, G., Sherman, L. A., & Inoue, Y. (1989) Photosynth. Res. 22, 123-130] but is modified so that the whole preparation, from cells to PS-II particles, is achieved in 10 h and involves only one purification step. The purified preparation exhibits a 5-6-fold increase of O2-evolution activity on a chlorophyll basis over the thylakoids. The ratio of PS-I to PS-II is about 0.14:1 in the preparation. The secondary quinone electron acceptor, QB, is present in this preparation as demonstrated by thermoluminescence studies. These PS-II particles are well-suited to spectroscopic studies as demonstrated by the range of EPR signals arising from components of PS-II that are easily detectable. Among the EPR signals presented are those from a formal S3-state, attributed to an oxidized amino acid interacting magnetically with the Mn complex in Ca(2+)-deficient PS-II particles, and from S2 modified by the replacement of Ca2+ by Sr2+. Neither of these signals has been previously reported in cyanobacteria. Their detection under these conditions indicates a similar lesion caused by Ca2+ depletion in both plants and cyanobacteria. The protocol has also been applied to mutants which have site-specific changes in PS-II. Data are presented on mutants having changes on the electron donor (Y160F) and electron acceptor (G215W) side of the D2 polypeptide.

Changes in the photosynthetic apparatus in the cyanobacterium Synechocystis sp. PCC 6714 following light-to-dark and dark-to-light transitions.

The photosynthetic apparatus of Synechocystis sp. PCC 6714 cells grown chemoheterotrophically (dark with glucose as a carbon source) and photoautotrophically (light in a mineral medium) were compared. Dark-grown cells show a decrease in phycocyanin content and an even greater decrease in chlorophyll content with respect to light-grown cells. Analysis of fluorescence emission spectra at 77 K and at 20 °C, of dark- and light-grown cells, and of phycobilisomes isolated from both types of cells, indicated that in darkness the phycobiliproteins were assembled in functional phycobilisomes (PBS). The dark synthesized PBS, however, were unable to transfer their excitation energy to PS II chlorophyll. Upon illumination of dark-grown cells, recovery of photosynthetic activity, pigment content and energy transfer between PBS and PS II was achieved in 24-48 h according to various steps. For O2 evolution the initial step was independent of protein synthesis, but the later steps needed de novo synthesis. Concerning recovery of PBS to PS II energy transfer, light seems to be necessary, but neither PS II functioning nor de novo protein synthesis were required. Similarly, light, rather than functional PS II, was important for the recovery of an efficient energy transfer in nitrate-starved cells upon readdition of nitrate. In addition, it has been shown that normal phycobilisomes could accumulate in a Synechocystis sp. PCC 6803 mutant deficient in Photosystem II activity.

Protection of reaction center II from photodamage by low temperature and anaerobiosis in spinach chloroplasts.

The rate of the irreversible damage to the reaction center II, caused by exposure of spinach thylakoids to high light was slowed down by anaerobic conditions and by lowering the temperature. The protective mechanisms of these conditions were different. In both cases Fmax decreased more slowly than in control photoinhibition. A reversible intermediate step was only observed under anaerobic conditions. This state was inactive for oxygen evolution and it was characterized by an increase of Fo.