Fight against cold: photosynthetic and antioxidant responses of different bell pepper cultivars (Capsicum annuum L.) to cold stress.

The special metabolites of bell pepper (Capsicum annuum L.) leaves can protect the plant under possibly damaging circumstances, such as high light, UV, unfavorable temperatures, or other environmental effects. In this study, we examined the cold stress tolerance of three different Hungarian pepper varieties (Darina, Édesalma, Rekord), focusing on the antioxidant and photosynthetic responses. The plants were developed in growth chambers under optimal temperature conditions (day/night 25 °C/20 °C) until the leaves on the fourth node became fully developed, then half of the plants received a cold treatment (day/night 15 °C/10 °C). Via a detailed pigment analysis, the PS II chlorophyll fluorescence responses, gas exchange parameters and total antioxidant capacities, leaf acclimation to low temperatures has been characterized. Our results display some of the developing physiological and antioxidant properties, which are among the main factors in monitoring the damaging effects of cold temperatures. Nevertheless, despite their differences, the tested pepper varieties did not show different cold responses.

In vivo detection of tobacco mosaic virus-induced local and systemic oxidative burst by electron paramagnetic resonance spectroscopy.

This is the first demonstration that tobacco mosaic virus-induced oxidative stress in a necrotic host plant is signalled by an elevated level of monodehydroascorbate (MDA) radicals detected by electron paramagnetic resonance spectroscopy. Furthermore, systemic acquired resistance induced in remote leaves of Xanthi-nc tobacco is also associated with stimulated MDA signals indicative of a microoxidative burst.

Singlet oxygen imaging in Arabidopsis thaliana leaves under photoinhibition by excess photosynthetically active radiation.

Arabidopsis thaliana leaves were infiltrated with DanePy (3-(N-diethylaminoethyl)-N-dansyl)aminomethyl-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrole), a double, fluorescent and spin sensor of singlet oxygen. DanePy fluorescence was imaged by laser scanning microscopy. We found that DanePy penetrated into chloroplasts but did not alter the functioning of the photosynthetic electron transport as assessed by chlorophyll fluorescence induction. In imaging, DanePy fluorescence was well distinct from chlorophyll fluorescence. Photoinhibition by excess photosynthetically active radiation caused quenching of DanePy fluorescence in the chloroplasts but not in other cell compartments. When leaves were infiltrated with dansyl, the fluorescent group in DanePy, there was no fluorescence quenching during photoinhibition. This shows that the fluorescence quenching of DanePy is caused by the conversion of its pyrrol group into nitroxide, i.e. it was caused by the reaction of singlet oxygen with the double sensor and not by artifacts. These data provide direct experimental evidence for the localization of singlet oxygen production to chloroplasts in vivo.

Photoinhibition of carotenoidless reaction centers from Rhodobacter sphaeroides by visible light. Effects on protein structure and electron transport.

Inhibition of electron transport and damage to the protein subunits by visible light has been studied in isolated reaction centers of the non-sulfur purple bacterium Rhodobacter sphaeroides. Illumination by 1100 muEm(-2) s(-1) light induced only a slight effect in wild type, carotenoid containing 2.4.1. reaction centers. In contrast, illumination of reaction centers isolated from the carotenoidless R26 strain resulted in the inhibition of charge separation as detected by the loss of the initial amplitude of absorbance change at 430 nm arising from the P(+)Q(B) (-) --> PQ(B) recombination. In addition to this effect, the L, M and H protein subunits of the R26 reaction center were damaged as shown by their loss on Coomassie stained gels, which was however not accompanied by specific degradation products. Both the loss of photochemical activity and of protein subunits were suppressed in the absence of oxygen. By applying EPR spin trapping with 2,2,6,6-tetramethylpiperidine we could detect light-induced generation of singlet oxygen in the R26, but not in the 2.4.1. reaction centers. Moreover, artificial generation of singlet oxygen, also led to the loss of the L, M and H subunits. Our results provide evidence for the common hypothesis that strong illumination by visible light damages the carotenoidless reaction center via formation of singlet oxygen. This mechanism most likely proceeds through the interaction of the triplet state of reaction center chlorophyll with the ground state triplet oxygen in a similar way as occurs in Photosystem II.

Do oxidative stress conditions impairing photosynthesis in the light manifest as photoinhibition?

We compared the effect of photoinhibition by excess photosynthetically active radiation (PAR), UV-B irradiation combined with PAR, low temperature stress and paraquat treatment on photosystem (PS) II. Although the experimental conditions ensured that the four studied stress conditions resulted in approximately the same extent of PS II inactivation, they clearly followed different molecular mechanisms. Our results show that singlet oxygen production in inactivated PS II reaction centres is a unique characteristic of photoinhibition by excess PAR. Neither the accumulation of inactive PS II reaction centres (as in UV-B or chilling stress), nor photo-oxidative damage of PS II (as in paraquat stress) is able to produce the special oxidizing conditions characteristic of acceptor-side-induced photoinhibition.

Relationship between activity, D1 loss, and Mn binding in photoinhibition of photosystem II.

Photoinhibition of photosystem II (PSII) activity and loss of the D1 reaction center protein were studied in PSII-enriched membrane fragments in which the water-splitting complex was inhibited by depletion of either calcium or chloride or by removing manganese. The Ca2+-depleted PSII was found to be the least susceptible to inhibition by light as reported previously (Krieger, A., and Rutherford, A. W. (1997) Biochim. Biophys. Acta 1319, 91-98). This different susceptibility to light was not reflected in the extent of D1 protein loss. In Mn-depleted PSII the loss of activity and the loss of the D1 protein were correlated, while in Cl-- and Ca2+-depleted PSII, there was very little loss of the D1 protein. The production of free radicals and singlet oxygen was measured by EPR spin-trapping techniques in the different samples. 1O2 and carbon-centered radicals could be detected after photoinhibition of active PSII, while hydroxyl radical formation dominated in all of the other samples. In addition, photoinhibition of PSII was investigated in which the functional Mn cluster was reconstituted (i. e., photoactivated). As expected this led to a protection against photoinhibition. When the photoactivation procedure was done in the absence of Ca2+ no activity was obtained although a nonfunctional Mn cluster was formed. Despite the lack of activity the binding of Mn partially protected against the loss of D1. These data demonstrate that, during photoinhibition, the extent of D1 loss is neither affected by the water-splitting activity of the sample nor correlated to the kinetics of PSII activity loss. D1 loss seems to be independent of the chemical nature of the reactive oxygen species formed during photoinhibition and seems to occur only in the absence of Mn. It is proposed that Mn binding protects against D1 loss by maintaining a protein structure which is not accessible to cleavage.

Photoinhibition of photosynthesis in vivo results in singlet oxygen production detection via nitroxide-induced fluorescence quenching in broad bean leaves.

In plants experiencing environmental stress, the formation of reactive oxygen is often presumed. In this study, singlet oxygen was detected in broad bean (Vicia faba) leaves that were photoinhibited in vivo. Detection was based on the reaction of singlet oxygen with DanePy (dansyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole) yielding a nitroxide radical (DanePyO) which is EPR active and also features lower fluorescence compared to DanePy. The two (fluorescent and spin) sensor fuctions of DanePy are commensurate, which makes detecting singlet oxygen possible with a spectrofluorimeter in samples hard to measure with EPR spectroscopy [Kálai, T., Hideg, E., Vass, I., and Hideg, K. (1998) Free Radical Biol. Med. 24, 649-652]. We found that in leaves saturated with DanePy, the fluorescence of this double sensor was decreased when the leaves were photoinhibited by 1500 micromol m-2 s-1 photosynthetically active radiation. This fluorescence quenching is the first direct experimental evidence that photoinhibition of photosynthesis in vivo is accompanied by 1O2 production and is, at least partly, governed by the process characterized as acceptor side-induced photoinhibition in vitro.

Double (fluorescent and spin) sensors for detection of reactive oxygen species in the thylakoid membrane.

A series of dansylated sterically hindered amines designed to trapping reactive oxygen species, were synthesized. Compounds were tested in isolated thylakoid membranes subjected to photoinhibition by excess photosynthetically active radiation (400-700 nm). DanePy showed good selectivity for singlet oxygen and the formation of nitroxide was detected by appearance of ESR signal and quenching fluorescence.

Synthesis of alpha-aryl N-adamant-1-yl nitrones and using them for spin trapping of hydroxyl radicals.

alpha-Aryl N-adamant-1-yl nitrones were synthesized and evaluated with respect to the stability of the hydroxyl radical adduct. The polarity and water solubility of nitrones were altered with changing the alpha-aryl groups. Introduction of adamantane ring instead of tert-butyl group resulted in a reasonable good stability of hydroxyl radical adduct for biological measurements.

The role of phospholipids in regulating photosynthetic electron transport activities: Treatment of thylakoids with phospholipase C.

The involvement of phospholipids in the regulation of photosynthetic electron transport activities was studied by incubating isolated pea thylakoids with phospholipase C to remove the head-group of phospholipid molecules. The treatment was effective in eliminating 40-50% of chloroplast phospholipids and resulted in a drastic decrease of photosynthetic electron transport. Measurements of whole electron transport (H2O→methylviologen) and Photosystem II activity (H2O→p-benzoquinone) demonstrated that the decrease of electron flow was due to the inactivation of Photosystem II centers. The variable part of fluorescence induction measured in the absence of electron acceptor was decreased by the progress of phospholipase C hydrolysis and part of the signal could be restored on addition of 3-(3',4'-dicholorophenyl)-1,1-dimethylurea. The B and Q bands of thermoluminescence corresponding to S2S3QB (-) and S2S3QA (-) charge recombination, respectively, was also decreased with a concomitant increase of the C band, which originated from the tyrosine D(+)QA (-) charge recombination. These results suggest that phospholipid molecules play an important role in maintaining the membrane organization and thus maintaining the electron transport activity of Photosystem II complexes.

Superoxide radicals are not the main promoters of acceptor-side-induced photoinhibitory damage in spinach thylakoids.

Superoxide anion radical formation was studied with isolated spinach thylakoid membranes and oxygen evolving Photosystem II sub-thylakoid preparations using the reaction between superoxide and Tiron (1,2-dihydroxybenzene-3,5-disulphonate) which results in the formation of stable, EPR detectable Tiron radicals.We found that superoxide was produced by illuminated thylakoids but not by Photosystem II preparations. The amount of the radicals was about 70% greater under photoinhibitory conditions than under moderate light intensity. Superoxide production was inhibited by DCMU and enhanced 4-5 times by methyl viologen. These observations suggest that the superoxide in illuminated thylakoids is from the Mehler reaction occurring in Photosystem I, and its formation is not primarily due to electron transport modifications brought about by photoinhibition.Artificial generation of superoxide from riboflavin accelerated slightly the photoinduced degradation of the Photosystem II reaction centre protein D1 but did not accelerate the loss of oxygen evolution supported by a Photosystem II electron acceptor. However, analysis of the protein breakdown products demonstrated that this added superoxide did not increase the amount of fragments brought about by photoinhibition but introduced an additional pathway of damage.On the basis of the above observations we propose that superoxide redicals are not the main promoters of acceptor-side-induced photoinhibition of Photosystem II.

The Unsaturation of Membrane Lipids Stabilizes Photosynthesis against Heat Stress.

The effect of the unsaturation of glycerolipids of thylakoid membranes on the heat tolerance of the photosynthetic evolution of oxygen was studied in vivo by mutation and transformation of fatty-acid desaturases in the cyanobacterium Synechocystis PCC6803. The experimental results indicate that elimination of dienoic lipid molecules decreases, to a small but distinct extent, the heat tolerance of photosynthetic oxygen evolution, but that elimination of trienoic lipid molecules has no effect on the heat tolerance. This conclusion contrasts with the previous hypothesis that the heat tolerance of photosynthesis is enhanced upon an increase in the level of saturation of membrane lipids. It is also shown that light does not affect the nature of the effect of lipid unsaturation on the heat tolerance of photosynthesis.

Singlet oxygen production in thylakoid membranes during photoinhibition as detected by EPR spectroscopy.

Exposure of isolated spinach thylakoids to high intensity illumination (photoinhibition) results in the well-characterized impairment of Photosystem II electron transport, followed by degradation of the D1 reaction centre protein. In the present study we demonstrate that this process is accompanied by singlet oxygen production. Singlet oxygen was detected by EPR spectroscopy, following the formation of stable nitroxide radicals from the trapping of singlet oxygen with a sterically hindered amine TEMP (2,2,6,6-tetramethylpiperidine). There was no detectable singlet oxygen production during anaerob photoinhibition or in the presence of sodium-azide. Comparing the kinetics of the loss of PS II function and D1 protein with that of singlet oxygen trapping suggests that singlet oxygen itself or its radical product initiates the degradation of D1.

Further characterization of the psbH locus of Synechocystis sp. PCC 6803: inactivation of psbH impairs QA to QB electron transport in photosystem 2.

The psbH gene encodes a small protein which copurifies with photosystem 2. In the cyanobacterium Synechocystis sp. PCC 6803, psbH is located upstream of the cytochrome b6-f complex genes petC and petA. In striking contrast, in the genomes of plant chloroplasts, psbH is cotranscribed with petB and petD, encoding the other two major subunits of the cytochrome b6-f complex. We report that in Synechocystis sp. PCC 6803 monocistronic psbH and dicistronic petCA transcripts are probably initiated separately, each from DNA regions bearing some similarity to Escherichia coli sigma 70 promoters. Synechocystis sp. PCC 6803 psbH null mutants were generated by cartridge mutagenesis. Studies using a rapid screening procedure involving in situ complementation showed that the PsbH protein is not absolutely required for the assembly of a functionally active photosystem 2 complex since psbH insertion and deletion strains were able to grow photoautotrophically. The rate of photoautotrophic growth was, however, slower than the wild type, and studies of oxygen evolution, chlorophyll fluorescence, and thermoluminescence indicated that this reduction in growth rate is probably due mainly to an impairment in electron flow from QA to QB. We conclude, therefore, that the PsbH protein is not an absolute requirement for photosystem 2 activity but that it functions to optimize electron flow between the two secondary plastoquinone acceptors by interacting with the QB site on the D1 protein.

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.

Dark adapted leaves of paraquat-resistant tobacco plants emit less ultraweak light than susceptible ones.

Long term light emission was compared from leaves of paraquat-resistant and -susceptible tobacco plants. In the minutes time scale, delayed light emission of the two biotypes was similar both in kinetics and in intensity. However, after several hours in the dark, ultraweak light emission from leaves of resistant plants was about one third of the light emitted by susceptible samples. We suggest, that this difference is due to the higher activity of superoxide dismutase in resistant biotypes, earlier reported by Tanaka et al. (1988) (Plant Cell Physiol. 29, 743-746), and propose a model for the mechanism of ultraweak light emission from these samples.

Spectral resolution of long term (0.5-50 s) delayed fluorescence from spinach chloroplasts.

The emission spectrum of room temperature delayed fluorescence from spinach chloroplasts does not change during the period 0.5-50 s after the cessation of illumination. This provides experimental evidence that charge recombination processes originating in various charge pairs of photosystem II, and manifest as various kinetical components of long term delayed fluorescence, result in the excitation of the same emitters, as predicted by the charge recombination hypothesis.

The far red induced slow component of delayed light from chloroplasts is emitted from Photosystem II : Evidence from emission spectroscopy.

In spinach chloroplasts illuminated with far red light, the relative intensity maximum during the decay of delayed light is emitted at 680-690 nm. This finding supports previous models predicting emission from Photosystem II, and contradicts earlier attributions to Photosystem I.Due to self absorption, the emission spectrum of the relative maximum is shifted to longer wavelengths and displays apparent Photosystem I characteristics in chloroplast samples of higher concentration or in leaves. This may have caused earlier investigators to ascribe the emission to Photosystem I.A differences between the spectral width of the emission spectra of delayed fluorescence and the relative maximum indicates that these two phenomena represent emission from different sub-populations of Photosystem II centers.

Ultraweak photoemission from dark-adapted leaves and isolated chloroplasts.

Dark-adapted isolated spinach chloroplasts and leaves, unlike sub-chloroplast fractions, are capable of emitting ultraweak light spontaneously (50-125 counts/s per cm2). The emission of leaves is due to two processes with activation energies of 97 and 25 kJ/mol while in isolated chloroplasts, it is the result of a single process (98 kJ/mol), as indicated by the Arrhenius plots of the intensity. Emission spectra demonstrate that the terminal step of these reactions is the excitation of chlorophyll in both samples. We suggest that the additional component in the ultraweak light emission of leaves may be related to mitochondria.