László Sass

Plants ectopically expressing the iron-binding protein, ferritin, are tolerant to oxidative damage and pathogens

Transgenic tobacco plants that synthesize alfalfa ferritin in vegetative tissues—either in its processed form in chloroplasts or in the cytoplasmic nonprocessed form—retained photosynthetic function upon free radical toxicity generated by iron excess or paraquat treatment. Progeny of transgenic plants accumulating ferritin in their leaves exhibited tolerance to necrotic damage caused by viral (tobacco necrosis virus) and fungal (Alternaria alternata, Botrytis cinerea) infections. These transformants exhibited normal photosynthetic function and chlorophyll content under greenhouse conditions. We propose that by sequestering intracellular iron involved in generation of the very reactive hydroxyl radicals through a Fenton reaction, ferritin protects plant cells from oxidative damage induced by a wide range of stresses.

Repair of UV-B induced damage of Photosystem II via de novo synthesis of the D1 and D2 reaction centre subunits in Synechocystis sp. PCC 6803

The repair of ultraviolet-B radiation induced damage to the structure and function of Photosystem II was studied in the cyanobacterium Synechocystis sp. PCC 6803. UV-B irradiation of intact Synechocystis cells results in the loss of steady-state oxygen evolution, an effect accompanied by a parallel loss of both D1 and D2 protein subunits of the Photosystem II reaction centre. Transfer of the UV-irradiated cells to normal growth conditions under visible light results in partial recovery of the inhibited oxygen evolving activity and restoration of the lost D1 and D2 proteins. The extent of recovery decreases with increasing degree of damage: after 50% inhibition, the original activity is completely restored within 2 hours. In contrast, after 90–95% inhibition less than half of the original activity is regained during a 4 hour recovery period. The translation inhibitor lincomycin completely blocks the recovery process if added after the UV-B treatment, and accelerates the kinetics of activity loss if added before the onset of UV-B irradiation. Substantial retardation of recovery and acceleration of activity loss is also observed if the very low intensity short wavelength contribution (λ<290 nm) is not filtered out from the UV-B light source. It is concluded that in intact cells UV-B induced damage of the Photosystem II complex can be repaired. This process is the first example of simultaneous D1 and D2 protein repair in Photosystem II, and considered to function as an important defence mechanism against detrimental UV-B effects in oxygenic photosynthetic organisms. De novo synthesis of the D1 and D2 reaction centre subunits is a key step of the repair process, which itself can also be inhibited by ultraviolet light, especially by the short wavelength UV-C components, or by high doses of UV-B.

UV-B-induced differential transcription of psbA genes encoding the D1 protein of photosystem II in the Cyanobacterium synechocystis 6803

UV-B irradiation of intactSynechocystis sp. PCC 6803 cells results in the loss of photosystem II activity, which can be repaired via de novosynthesis of the D1 (and D2) reaction center subunits. In this study, we investigated the effect of UV-B irradiation on the transcription of the psbA2 and psbA3 genes encoding identical D1 proteins. We show that UV-B irradiation increases the level ofpsbA2 mRNA 2–3-fold and, more dramatically, it induces a 20–30-fold increase in the accumulation of the psbA3mRNA even at levels of irradiation too low to produce losses of either photosystem II activity or D1 protein. The induction ofpsbA3 transcript accumulation is specific for UV-B light (290–330 nm). Low intensity UV-A emission (330–390 nm) and white light induce only a small, at most, 2–3-fold enhancement, whereas no effect of blue light was observed. Expression patterns of chimeric genes containing the promoter regions of the psbA2,psbA3 genes fused to the firefly luciferase (luc) reporter gene indicate that (i) transcription ofpsbA2/luc and psbA3/luc transgenes was elevated, similarly to that of the endogenous psbA genes, by UV-B irradiation, and that (ii) a short, 80-base pairpsbA3 promoter fragment is sufficient to maintain UV-B-induced transcription of the luc reporter gene. Furthermore, our findings indicate that UV-B-induced expression of thepsbA2 and psbA3 genes is a defense response against UV-B stress, which is regulated, at least, partially at the level of transcription and does not require active electron transport.

Nuclear localization of a hypoxia-inducible novel non-symbiotic hemoglobin in cultured alfalfa cells1

We have isolated a 483‐bp‐long full‐length cDNA clone encoding a non‐symbiotic hemoglobin called Mhb1, the first one found in alfalfa. This non‐symbiotic hemoglobin is a single copy gene localized in linkage group 4 in diploid Medicago genome. The Mhb1 mRNA was found only in the roots of alfalfa plants. The Mhb1 gene was inducible by hypoxia and showed no induction by cold stress treatment. The Mhb1 transcript level increased at the G2/M boundary in a synchronized alfalfa cell suspension culture. The majority of Mhb1 protein was shown to be localized in the nucleus and smaller amounts were detected in the cytoplasm. A potential link to the nitric oxide signalling pathway is also discussed.

Inactivation of photosynthetic oxygen evolution by UV-B irradiation: A thermoluminescence study

The influence of UV-B irradiation on photosynthetic oxygen evolution by isolated spinach thylakoids has been investigated using thermoluminescence measurements. The thermoluminescence bands arising from the S2QB- (B band) and S2QA− (Q band) charge recombination disappeared with increasing UV-B irradiation time. In contrast, the C band at 50°C, arising from the recombination of QA- with an accessory donor of Photosystem II, was transiently enhanced by the UV-B irradiation. The efficiency of DCMU to block QA to QB electron transfer decreased after irradiation as detected by the incomplete suppression of the B band by DCMU. The flash-induced oscillatory pattern of the B band was modified in the UV-B irradiated samples, indicating a decrease in the number of centers with reduced QB. Based on the results of this study, UV-B irradiation is suggested to damage both the donor and acceptor sides of Photosystem II. The damage of the water-oxidizing complex does not affect a specific S-state transition. Instead, charge stabilization is enhanced on an accessory donor. The acceptor-side modifications decrease the affinity of DCMU binding. This effect is assumed to reflect a structural change in the QB/DCMU binding site. The preferential loss of dark stable QB- may be related to the same structural change or could be caused by the specific destruction of reduced quinones by the UV-B light.

Characterization of singlet oxygen production and its involvement in photodamage of Photosystem II in the cyanobacterium Synechocystis PCC 6803 by histidine-mediated chemical trapping

Singlet oxygen production in intact cells of the cynobacterium Synechocystis 6803 was studied using chemical trapping by histidine, which leads to O2 uptake during illumination. The rate of O2 uptake, measured by a standard Clark-type electrode, is enhanced in the presence of D2O, which increases the lifetime of (1)O2, and suppressed by the (1)O2 quencher NaN3. Due to the limited mobility of (1)O2 these data demonstrate that exogenous histidine reaches close vicinity of (1)O2 production sites inside the cells. Flash induced chlorophyll fluorescence measurements showed that histidine does not inhibit Photosystem II activity up to 5mM concentration. By applying the histidine-mediated O2 uptake method we showed that (1)O2 production linearly increases with light intensity even above the saturation of photosynthesis. We also studied (1)O2 production in site directed mutants in which the Gln residue at the 130th position of the D1 reaction center subunit was changed to either Glu or Leu, which affect the efficiency of nonradiative charge recombination from the primary radical pair (Rappaport et al. 2002, Biochemistry 41: 8518-8527; Cser and Vass 2007, BBA 1767:233-243). We found that the D1-Gln130Glu mutant showed decreased (1)O2 production concomitant with decreased rate of photodamage relative to the WT, whereas both (1)O2 production and photodamage were enhanced in the D1-Gln130Leu mutant. The data are discussed in the framework of the model of photoinhibition in which (3)P680 mediated (1)O2 production plays a key role in PSII photodamage, and nonradiative charge recombination of the primary charge separated state provides a photoprotective pathway.

Photoinactivation of photosystem II by flashing light.

Inhibition of Photosystem II (PS II) activity by single turnover visible light flashes was studied in thylakoid membranes isolated form spinach. Flash illumination results in decreased oxygen evolving activity of PS II, which effect is most pronounced when the water-oxidizing complex is in the S2 and S3 states, and increases with increasing time delay between the subsequent flashes. By applying the fluorescent spin-trap DanePy, we detected the production of singlet oxygen, whose amount was increasing with increasing flash spacing. These findings were explained in the framework of a model, which assumes that recombination of the S2QB− and S3QB− states generate the triplet state of the reaction center chlorophyll and lead to the production of singlet oxygen.

Thermoluminescence studies on the function of photosystem II in the desiccation tolerant lichen Cladonia convoluta

The effect of desiccation and rehydration on the function of Photosystem II has been studied in the desiccation tolerant lichen Cladonia convoluta by thermoluminescence. We have shown that in functional fully hydrated thalli thermoluminescence signals can be observed from the recombination of the S2(3)QB− (B band), S2QA− (Q band), Tyr-D+QA− (C band) and Tyr-Z+(His+)QA− (A band) charge stabilization states. These thermoluminescence signals are completely absent in desiccated thalli, but rapidly reappear on rehydration. Flash-induced oscillation in the amplitude of the thermoluminescence band from the S2(3)QB− recombination shows the usual pattern with maxima after 2 and 6 flashes when rehydration takes place in light. However, after rehydration in complete darkness, there is no thermoluminescence emission after the 1 st flash, and the maxima of the subsequent oscillation are shifted to the 3rd and 7th flashes. It is concluded that desiccation of Cladonia convoluta converts PS II into a nonfunctional state. This state is characterized by the lack of stable charge separation and recombination, as well as by a one-electron reduction of the water-oxidizing complex. Restoration of PS II function during rehydration can proceed both in the light and in darkness. After rehydration in the dark, the first charge separation act is utilized in restoring the usual oxidation state of the water-oxidizing comples.

Potato Annexin STANN1 Promotes Drought Tolerance and Mitigates Light Stress in Transgenic Solanum tuberosum L. Plants.

Annexins are a family of calcium- and membrane-binding proteins that are important for plant tolerance to adverse environmental conditions. Annexins function to counteract oxidative stress, maintain cell redox homeostasis, and enhance drought tolerance. In the present study, an endogenous annexin, STANN1, was overexpressed to determine whether crop yields could be improved in potato (Solanum tuberosum L.) during drought. Nine potential potato annexins were identified and their expression characterized in response to drought treatment. STANN1 mRNA was constitutively expressed at a high level and drought treatment strongly increased transcription levels. Therefore, STANN1 was selected for overexpression analysis. Under drought conditions, transgenic potato plants ectopically expressing STANN1 were more tolerant to water deficit in the root zone, preserved more water in green tissues, maintained chloroplast functions, and had higher accumulation of chlorophyll b and xanthophylls (especially zeaxanthin) than wild type (WT). Drought-induced reductions in the maximum efficiency and the electron transport rate of photosystem II (PSII), as well as the quantum yield of photosynthesis, were less pronounced in transgenic plants overexpressing STANN1 than in the WT. This conferred more efficient non-photochemical energy dissipation in the outer antennae of PSII and probably more efficient protection of reaction centers against photooxidative damage in transgenic plants under drought conditions. Consequently, these plants were able to maintain effective photosynthesis during drought, which resulted in greater productivity than WT plants despite water scarcity. Although the mechanisms underlying this stress protection are not yet clear, annexin-mediated photoprotection is probably linked to protection against light-induced oxidative stress.

Symbiodinium sp cells produce light-induced intra- and extracellular singlet oxygen, which mediates photodamage of the photosynthetic apparatus and has the potential to interact with the animal host in coral symbiosis

Coral bleaching is an important environmental phenomenon, whose mechanism has not yet been clarified. The involvement of reactive oxygen species (ROS) has been implicated, but direct evidence of what species are involved, their location and their mechanisms of production remains unknown. Histidine-mediated chemical trapping and singlet oxygen sensor green (SOSG) were used to detect intra- and extracellular singlet oxygen ((1) O2 ) in Symbiodinium cultures. Inhibition of the Calvin-Benson cycle by thermal stress or high light promotes intracellular (1) O2 formation. Histidine addition, which decreases the amount of intracellular (1) O2 , provides partial protection against photosystem II photoinactivation and chlorophyll (Chl) bleaching. (1) O2 production also occurs in cell-free medium of Symbiodinium cultures, an effect that is enhanced under heat and light stress and can be attributed to the excretion of (1) O2 -sensitizing metabolites from the cells. Confocal microscopy imaging using SOSG showed most extracellular (1) O2 around the cell surface, but it is also produced across the medium distant from the cells. We demonstrate, for the first time, both intra- and extracellular (1) O2 production in Symbiodinium cultures. Intracellular (1) O2 is associated with photosystem II photodamage and pigment bleaching, whereas extracellular (1) O2 has the potential to mediate the breakdown of symbiotic interaction between zooxanthellae and their animal host during coral bleaching.