Non-Vectorial Light-Induced H+ Release from CF1-Depleted Thylakoid Membranes. Absence of Correlation to Stoichiometric H+ Production and Consumption Coupled to Electron Transfer

Light-induced H⁺ release from CF₁-depleted thylakoid membraneswas examined by monitoring the pH in the intra- and extrathylakoidalspace and by analyzing the consumption and production of O₂that accompany the electron transfer. Our results indicatedthat the H⁺ release was not due to the H⁺ transport across themembrane or to the net H⁺ production in PS II as previouslysuggested. (Received April 14, 1988; Accepted June 13, 1988)     

Bound Ribosomes of Pea Chloroplast Thylakoid Membranes: Location and Release in Vitro by High Salt, Puromycin, and RNase

The mode of attachment of 70S ribosomes to thylakoid membranes from pea leaves was studied by determining the proportion of the bound RNA which was released by various incubation conditions. The results supported a model in which several classes of bound ribosomes could be distinguished: (a) very tightly bound, not released by any conditions yet tested (20% of the total); (b) monomeric ribosomes attached by electrostatic interaction with the membranes (30 to 40% of the total) and released by high salt; and (c) polysomes, with some of the ribosomes attached by a combination of electrostatic interactions and insertion of the nascent polypeptide chain into the membrane. These required a combination of puromycin and high salt for release. Other ("hanging") ribosomes of the polysomes were inferred to be attached through mRNA but not actually attached to the membranes directly; they could be released by RNase under low salt conditions, as well as by puromycin plus high salt.To obtain these results, chloroplasts had to be prepared in media containing 0.2 molar Tris at pH 8.5. Using Tricine buffers at pH 7.5 yielded thylakoid membranes whose ribosomes were removed almost completely by high salt alone; these showed no response to puromycin. However, pH 7.5 had to be used in all cases for ribosome dissociation in high salt media, as the ribosome structure appeared to be degraded by high salt at pH 8.5, and release then occurred without the need for puromycin.The kinetics of ribosome release by high salt showed a rapid initial phase with a half-life of 20 seconds. The extent of release by high salt was very dependent on the temperature of the incubation. Plotting the data according to the Arrhenius interpretation shows a significant break at about 15 C, with apparent activation energy of 20 kilocalories per mole below that temperature and 5 kilocalories per mole above that temperature. This result suggests that membrane fluidity might be an important factor permitting release of ribosomes under high salt conditions.Electron microscope pictures of the washed thylakoids showed polysomes closely associated with the outer membranes of grana stacks, and with the stroma lamellae. Following digitonin treatment of the membranes and centrifugation, fractions enriched in Photosystem I and presumed stroma lamellae were also enriched in bound RNA.     

Cycloprodigiosin Uncouples H+-pyrophosphatase of Plant Vacuolar Membranes in the Presence of Chloride Ion

Cycloprodigiosin hydrochloride (cPrG-HCl) at 10 nM uncoupledboth H⁺-ATPase and H⁺-translocating pyrophosphatase in vacuolarmembrane vesicles of mung bean, but did not inhibit the substratehydrolysis. cPrG-HCl destroyed the transmembrane pH gradientonly in the presence of Cl^–. The observations indicatethat cPrG-HCl functions as a H+/Cl^– symporter. Vesicleacidification was restored by SO^2–₄ even in the presenceof cPrG-HCl. (Received November 13, 1998; Accepted January 21, 1999)     

抽薹期叶绿素缺乏油菜突变体类囊体膜的研究

以抽薹期野生型油菜和黄化突变体叶片为材料,分析了叶片和类囊体膜的不合色素组成、色素与蛋白相对含量;比较了类囊体膜光谱特性（室温吸收光谱、荧光光谱和圆二色谱）;用温和电泳、ＳＤＳ－ＰＡＧＥ分析了类囊体色素蛋白和多肽组成。结果显示：与野生型相比,突变体叶片的蛋白质含量不变,而Ｃhaa和Ｃhlb的含量均减少;突变体类囊体膜的Ｃhla/Chlb比值较高,Ｃhl/蛋白质比值较低,ＬＨＣⅡ色素蛋白复合物的单体和三聚体含量明显减少。突变体的天线系统相对较小、捕光效率较低。     

The Effects of Illumination on the Xanthophyll Composition of the Photosystem II Light-Harvesting Complexes of Spinach Thylakoid Membranes

The xanthophyll composition of the light-harvesting chlorophyll a/b proteins of photosystem II (LHCII) has been determined for spinach (Spinacia oleracea L.) leaves after dark adaptation and following illumination under conditions optimized for conversion of violaxanthin into zeaxanthin. Each of the four LHCII components was found to have a unique xanthophyll composition. The major carotenoid was lutein, comprising 60% of carotenoid in the bulk LHCIIb and 35 to 50% in the minor LHCII components LHCIIa, LHCIIc, and LHCIId. The percent of carotenoid found in the xanthophyll cycle pigments was approximately 10 to 15% in LHCIIb and 30 to 40% in LHCIIa, LHCIIc, and LHCIId. The xanthophyll cycle was active for the pigments bound to all of the LHCII components. The extent of deepoxidation for complexes prepared from light-treated leaves was 27, 65, 69, and 43% for LHCIIa, -b, -c, and -d, respectively. These levels of conversion of violaxanthin to zeaxanthin were found in LHCII prepared by three different isolation procedures. It was estimated that approximately 50% of the zeaxanthin associated with photosystem II is in LHCIIb and 30% is associated with the minor LHCII components.     

Origin of Thylakoid Membranes in Chlamydomonas reinhardtii y-1 at 38 degrees C

The origin of thylakoid membranes was studied in Chlamydomonas reinhardtii y-1 cells during greening at 38°C. Previous studies showed that, when dark-grown cells are exposed to light under these conditions, the initial rates of accumulation of chlorophyll and the chlorophyll a/b-binding proteins in membranes are maximal (MA Maloney JK Hoober, DB Marks [1989] Plant Physiol 91: 1100-1106; JK Hoober MA Maloney, LR Asbury, DB Marks [1990] Plant Physiol 92: 419-426). As shown in this paper, photosystem II activity, which was nearly absent in dark-grown cells, also increased at a linear rate in parallel with chlorophyll. As compared with those made at 25°C, photosystem II units assembled during greening at 38°C were photochemically more efficient, as judged by saturation at a lower fluence of light and a negligible loss of excitation energy as fluorescence. Electron microscopy of cells in light for 5 or 15 minutes at 38°C showed that these initial, functional thylakoid membranes developed in association with the chloroplast envelope.     

Glucolipid Synthesis Activities in Cytoplasmic and Thylakoid Membranes from the Cyanobacterium Anacystis nidulans

Cytoplasmic membranes (plasma membranes), thylakoid membranesand cell walls prepared from the cyanobacterium, Anacystis nidulans,were compared for UDP-glucose: l,2-diacylglycerol glucosyltransferaseactivity. When 1,2-dipalmitoylglycerol was added as a glucosylacceptor, both cytoplasmic membranes and thylakoid membranesincorporated glucose from UDP-glucose into monoglucosyl diacylglycerol,but the cell walls containing the outer membranes did not. Thecytoplasmic membranes incorporated about twice as much glucoseas the thylakoid membranes on a protein basis. These observationssuggest that in A. nidulans the UDP-glucose: 1,2-diacylglycerolglucosyltransferase participating in glucolipid biosynthesisis located in both cytoplasmic and thylakoid membranes, butnot in the outer membrane. ¹Solar Energy Research Group, The Institute of Physical andChemical Research (RIKEN), Wako-shi, Saitama 351-01, Japan. (Received November 21, 1985; Accepted January 27, 1986)     

Specific Degradation of D1 Protein during Exposure of Thylakoid Membranes to High Temperature in the Dark

Exposure of thylakoid membranes to high temperature in dark leads to the degradation of D1 protein. Maximum degradation of D1 protein occurred at 45 °C. Using N-terminal specific D1 antibody, a 23 kDa fragment of D1 protein was detected. The degradation of D1 protein could be prevented both by radical scavengers and inhibitors of serine protease and metallo-protease. These results suggest that degradation of D1 protein during exposure of thylakoid membranes to high temperature in dark is catalyzed by protease. This revised version was published online in June 2006 with corrections to the Cover Date.     

Selective Photoinhibition of Photosystem I in Isolated Thylakoid Membranes from Cucumber and Spinach

The site of photoinhibition at low temperatures in leaves ofa chilling-sensitive plant, cucumber, is photosystem I [Terashimaet al. (1994) Planta 193: 300]. As described herein, selectivephotoinhibition of PSI can also be induced in isolated thylakoidmembranes in vitro. Inhibition was observed both at chillingtemperatures and at 25°C, and not only in the thylakoidmembranes isolated from cucumber, but also in those isolatedfrom a chilling-tolerant plant, spinach. Comparison of theseobservations in vitro to the earlier results in vivo indicatesthat (1) photoinhibition of PSI is a universal phenomenon; (2)a mechanism exists to protect PSI in vivo; and (3) the protectivemechanism is chilling-sensitive in cucumber. The chilling-sensitivecomponent seems to be lost during the isolation of thylakoidmembranes. Very weak light (10–20µmol m^-2 s^-1) wassufficient to cause the inhibition of PSI. About 80% of theoxygen-evolving activity by PSII was maintained even after theactivity of PSI had decreased by more than 70%. This is thefirst report of the selective photoinhibition of PSI in vitro. (Received March 1, 1995; Accepted April 26, 1995)     

Enzymatic Release of Zn2+-Glycerophosphocholine Cholinephosphodiesterase from Brain Membranes by Glycosylphosphatidylinositol-Specific Phospholipases and its Regulation

Enzymatic conversion of glycosylphosphatidylinositol (GPI)-linked Zn²⁺-glycerophosphocholine phosphodiesterase was investigated. The activity of glycosylphosphatidylinositol-specific phospholipase-D (GPI-PLD), based on the conversion of amphiphilic form of phosphodiesterase into hydrophilic form, showing an optimum pH of about pH 6.6, increased continuously until 60 min. The activity of membrane-bound GPI-PL, based on the formation of hydrophilic form of phosphodiesterase, exhibiting an optimum pH of 7.4, increased up to 30 min, and reached a plateau. Inhibition studies indicate that while GPI-PLD activity was generally sensitive to ionic bio-detergents, it was not inhibited by myristoyl glycerol, a neutal detergent. Meanwhile, the membrane-bound GPI-PL was not affected remarkably by these detergents except that myristoyl glycerol expressed a modest increase of activity of membrane bound GPI-PL. In addition, the membrane-bound GPI-PL appeared to be enhanced by by suramin or oleic acid, which strongly inhibited GPI-PLD. From this results, it is suggested that in brain there may be two phospholipases responsible for the conversion of membrane-bound GPI-anchors to hydrophilic forms, and that this conversion might be regulated by endogenous lipids.     

Transformation of Thylakoid Membranes during Differentiation from Vegetative Cell into Heterocyst Visualized by Microscopic Spectral Imaging

Some filamentous cyanobacteria carry out oxygenic photosynthesis in vegetative cells and nitrogen fixation in specialized cells known as heterocysts. Thylakoid membranes in vegetative cells contain photosystem I (PSI) and PSII, while those in heterocysts contain predominantly PSI. Therefore, the thylakoid membranes change drastically when differentiating from a vegetative cell into a heterocyst. The dynamics of these changes have not been sufficiently characterized in situ. Here, we used time-lapse fluorescence microspectroscopy to analyze cells of Anabaena variabilis under nitrogen deprivation at approximately 295 K. PSII degraded simultaneously with allophycocyanin, which forms the core of the light-harvesting phycobilisome. The other phycobilisome subunits that absorbed shorter wavelengths persisted for a few tens of hours in the heterocysts. The whole-thylakoid average concentration of PSI was similar in heterocysts and nearby vegetative cells. PSI was best quantified by selective excitation at a physiological temperature (approximately 295 K) under 785-nm continuous-wave laser irradiation, and detection of higher energy shifted fluorescence around 730 nm. Polar distribution of thylakoid membranes in the heterocyst was confirmed by PSI-rich fluorescence imaging. The findings and methodology used in this work increased our understanding of how photosynthetic molecular machinery is transformed to adapt to different nutrient environments and provided details of the energetic requirements for diazotrophic growth.The most essential pigment-protein complexes for oxygenic photosynthesis are PSI and PSII, which are embedded in the thylakoid membranes of chloroplasts and cyanobacteria. Cooperation between PSI and PSII achieves light-driven noncyclic electron transport from the oxidative splitting of water to the reduction of ferredoxin and is accompanied by the generation of a proton gradient for ATP synthesis. Phycobilisomes (PBS), another pigment-protein complex, are attached to the stromal side of the thylakoid membrane in cyanobacteria and red algae; they work as light-harvesting antennae to transfer electronic excitation energy mainly to PSII and, in some cases, to PSI (Gantt 1994). The integration of these pigment-protein complexes changes in response to light conditions, nutrient status, and developmental stage (Fujita et al., 1994; Grossman et al., 1994; Wolk et al., 1994).Some cyanobacteria, including Anabaena variabilis, are able to grow diazotrophically using the nitrogen-fixing enzyme nitrogenase. Because nitrogenase is sensitive to oxygen, oxygenic photosynthesis is not readily compatible with diazotrophic growth. When this filamentous cyanobacterium is grown under fixed nitrogen-deficient conditions, approximately 1 in 10 to 20 vegetative cells differentiates into a heterocyst, in which oxygenic photosynthesis is suppressed and nitrogenase becomes operative (Haselkorn, 1978; Wolk et al., 1994). The other vegetative cells continue oxygenic photosynthesis. The differentiation of heterocysts from chains of vegetative cells has been studied extensively (Golden and Yoon, 2003; Toyoshima et al., 2010). The abundances of PSII and PBS decrease during the transition. PSI appears to persist in the heterocyst to produce ATP by cyclic electron transport, because nitrogen fixation demands a large amount of ATP (Wolk et al., 1994). However, the mechanisms by which PBS and PSII are degraded during heterocyst differentiation remain unclear, and whether the amount of PSI per cell changes is unknown.The PBS of A. variabilis contain three types of phycobiliproteins, pigment-protein complexes with distinct absorption and fluorescence spectra. The core PBS contains allophycocyanin (APC), which absorbs around 654 nm (Ying and Xie, 1998); the core is most closely connected to PSII. More peripherally in the PBS, the so-called rod contains phycoerythrocyanin (PEC) and phycocyanin (PC), which absorb maximally around 575 and 604 to 620 nm, respectively (Switalski and Sauer, 1984; Zhang et al., 1998). Photon energy is absorbed by PEC, then transferred downhill through PC and APC and finally to PSII. The structure of PBS is probably optimized not only for efficient energy transfer to PSII and/or PSI but also for transformation and/or degradation under various nutrient conditions. However, the order in which these subunits degrade during heterocyst differentiation remains unknown. One strategy to address this question is to isolate heterocysts at several stages during differentiation and quantify their proteomes via mass spectrometry. However, such isolation procedures work well only when there is a good understanding of the properties of cells at different stages. Ideally, noninvasive methods should be used to understand changes in the integrity of PSII and PBS in intact cells in filaments.In principle, time-lapse microscopic observations can clarify the process of differentiation from a vegetative cell into a mature heterocyst. Spectral microscopy is an ideal tool to analyze physiological state and/or amounts of pigment-protein complexes under various conditions. Acquiring microscopic fluorescence spectra of individual cells is a natural extension of laser scanning confocal fluorescence microscopy, which has been applied to several types of cyanobacterial cells, including heterocysts (Peterson et al., 1981; Ying et al., 2002; Wolf and Schüssler, 2005; Kumazaki et al., 2007; Vermaas et al., 2008; Sukenik et al., 2009; Bordowitz and Montgomery, 2010; Collins et al., 2012, Sugiura and Itoh, 2012). Microscopic fluorescence spectra reflect the concentration of pigment-protein complexes and the energy transfer dynamics between photosynthetic pigments. However, to date, there have been no thorough time-lapse investigations of the fluorescence spectra of heterocysts and vegetative cells during the differentiation process.In this study, we investigated the dynamic changes in thylakoid membranes of A. variabilis during heterocyst differentiation. Our unique microscopic system can acquire fluorescence spectra from an entire linearly illuminated region with about 2-nm wavelength resolution in a single exposure (Kumazaki et al., 2007). Heterocyst formation was induced by transferring vegetative cell filaments from fixed-nitrogen-sufficient incubation medium to nitrogen-deprived medium. We conducted long-term observations (60–96 h) on identical filaments. Another unique feature of our setup is that it uses a near-infrared (NIR) excitation laser source. Our previous microspectroscopic study of chloroplasts of a higher plant, maize (Zea mays), and a green alga (Parachlorella kessleri) showed that continuous wave (CW) laser light emitting at 785 to 820 nm excited PSI with high selectivity under the one-photon excitation (OPE) mode. This enabled us to observe highly PSI-rich fluorescence spectra and images with signals around 710 to 740 nm, even at approximately 295 K (Hasegawa et al., 2010, 2011). We used this technique to quantify PSI in individual heterocysts compared with its parental and contiguous vegetative cells. Pigment fluorescence under OPE qualitatively differed from that under two-photon excitation (TPE) using a pulsed NIR laser (typically achieved with picosecond or femtosecond pulses), because TPE using 800 to 830 nm resulted in spectra with contributions from PBS, PSII, and PSI, as typically observed by visible light excitation (Kumazaki et al., 2007; Hasegawa et al., 2010, 2011). The advantages of our microscopic system are the high wavelength resolution and coverage of the entire fluorescence spectrum, the availability of fluorescence spectra at several differentiation stages, and the multiple excitation modes with different selectivities for pigment-protein complexes. Together, these analyses allowed us to characterize spectral decomposition and to understand the time dependence of different pigment-protein complexes, even at a physiological temperature. Microscopic absorption spectra were also obtained from single cells. These data were tentatively used to estimate the absolute concentrations of PSI and PSII in heterocysts and vegetative cells.     

Localization of Cytochrome b-559 in the Chloroplast Thylakoid Membranes in Spinach

Cytochrome b-559 was purified from spinach leaves and antibodies were made against it in rabbit. Using affinity-purified, monospecific antibodies, we have found that cytochrome b-559, which is closely associated with the primary photochemical activity of photosystem II, is localized exclusively in the grana thylakoids.     

Phase Transition of Thylakoid Membranes Modulates Photoinhibition in the Cyanobacterium Anabaena siamensis

A shift of the growth temperature from 40 degrees C to 18 degrees C promoted an increase in the degree of fatty acids unsaturation and a decrease, from 26 degrees C to 0 degrees C, of the phase transition temperature of thylakoid membranes in Anabaena siamensis. The pattern of photoinhibition of photosynthesis at distinct temperatures varied as a function of the phase transition temperature. In the absence of streptomycin, a pronounced photoinhibition at temperatures near the phase transition (26 degrees C) was observed in cells grown at 40 degrees C, while protection from photodamage was observed at chilling temperatures (15 degrees C to 5 degrees C). In this same range of temperature, such a protection was not verified if cells were grown at 18 degrees C. In both types of cells, however, the rate of photoinactivation in the presence of streptomycin was progressively decreased by lowering the temperature of photoinhibition. When recovery from photoinhibition was followed at the respective temperature in which cells were grown, the restoration profile of the photosynthetic O(2) evolution to initial levels was essentially the same in both types of cells. The protective effect of low temperatures against photoinhibition was attributed to a decreased solubility and diffusion of oxygen in the thylakoid membranes due to an increase of the membrane viscosity that would avoid the photogeneration of reactive oxygen species around PS II.     

Ascorbate-mediated Iron Release from Ferritin in the Presence of Alloxan

Release of iron from ferritin requires reduction of ferric to ferrous iron. The iron can participate in the diabetogenic action of alloxan. We investigated the ability of ascorbate to catalyze the release of iron from ferritin in the presence of alloxan. Incubation of ferritin with ascorbate alone elicited iron release (33 nmol/10 min) and the generation of ascorbate free radical, suggesting a direct role for ascorbate in iron reduction. Iron release by ascorbate significantly increased in the presence of alloxan, but alloxan alone was unable to release measurable amounts of iron from ferritin. Superoxide dismutase significantly inhibited ascorbate-mediated iron release in the presence of alloxan, whereas catalase did not. The amount of alloxan radical (A·⁻) generated in reaction systems containing both ascorbate and alloxan decreased significantly upon addition of ferritin, suggesting that A·⁻ is directly involved in iron reduction. Although release of iron from ferritin and generation of A·⁻ were also observed in reactions containing GSH and alloxan, the amount of iron released in these reactions was not totally dependent on the amount of A·⁻ present, suggesting that other reductants in addition to A·⁻ (such as dialuric acid) may be involved in iron release mediated by GSH and alloxan. These results suggest that A·⁻ is the main reductant involved in ascorbate-mediated iron release from ferritin in the presence of alloxan and that both dialuric acid and A·⁻ contribute to GSH/alloxan-mediated iron release.     

Isolation of Plasma and Thylakoid Membranes from the Heterocystous Cyanobacterium Anabaena sp. PCC 7120

具异型胞蓝细菌 Anabaena sp.PCC 7120质膜和类囊体膜的分离纯化 李斌 徐冬一 赵进东*     

Selected Pseudomonas putida Strains Able To Grow in the Presence of High Butanol Concentrations

Adapted Pseudomonas putida strains grew in the presence of up to 6% (vol/vol) butanol, the highest reported butanol concentration tolerated by a microbe. P. putida might be an alternative host for biobutanol production, overcoming the primary limitation of currently used strains—insufficient product titers due to low butanol tolerance.The focus of biofuel production research has recently shifted from ethanol to bioenergy carriers that are more compatible with existing infrastructure (e.g., refineries, transport, and car engines). At the forefront is n-butanol (hereafter referred to as butanol) for which large-scale production processes have been implemented (16, 35). Existing fermentations, however, are limited in energetically attractive butanol titers, because butanol inhibits microbial growth at concentrations above 16 g/liter (2, 10). As reported for other organic solvents with low logarithm of the partition coefficient in a two-phase octanol/water system (log P_ow), this toxicity is due primarily to accumulation of butanol (log P_ow, 0.8) in the cell membrane and subsequent impairment (4, 17, 30, 33). With the maximum aqueous solubility of 0.97 M (8.8% [vol/vol]), the maximum membrane concentration of butanol was calculated to be 1.59 M (17), spotlighting its potential toxicity. The low achievable butanol titers have necessitated large reactor volumes, resulting in high purification costs (8, 15). Recent metabolic engineering strategies for improving biobutanol fermentation have focused on maximization of butanol production rates (10, 19), reducing the levels of by-products (20), finding alternative substrates (20), or finding alternative hosts (2, 12, 21, 31). However, recently engineered microbial strains (1, 14) have not overcome butanol toxicity.High organic solvent concentrations are tolerated by strains of the bacterial species Pseudomonas putida reported to grow in a second phase of octanol (25), toluene (13), or styrene (32). This suggests that solvent-tolerant P. putida strains withstand high butanol titers and therefore warrant exploitation as host for butanol production. Indeed, viable solvent-tolerant P. putida S12 cells were observed at butanol concentrations of up to 10% (vol/vol) by live-dead staining and fluorescence microscopy (5) (see supplemental material). We used growth as the parameter of interest, because growth in the presence of butanol directly indicates the potential of selected P. putida strains as hosts for recombinant butanol production.Three solvent-tolerant P. putida strains, DOT-T1E (23), S12 (32), and Pseudomonas sp. strain VLB120 (18), and the solvent-sensitive P. putida reference strain KT2440 (24) were examined for their ability to grow in the presence of butanol. Toxicity assays were performed in 96-well microtiter plates (System Duetz [7]) at 30°C and 300 rpm using glucose-supplemented LB and M9 media (with 10 and 5 g/liter glucose, respectively) (26). Higher glucose concentrations in LB medium did not increase butanol tolerance (data not shown). Butanol was added in all experiments to cells in the mid-exponential phase. Cell growth was monitored by changes in optical density, and substrate and butanol concentrations were analyzed by high-pressure liquid chromatography (Trentec 308R-Gel.H; VWR Hitachi). Comparable low butanol concentrations were withstood by all P. putida strains, with butanol tolerance highly dependent on the medium composition (Table ​(Table1).1). Growth was observed at butanol concentrations up to 3% (vol/vol), occurring in a culture of Pseudomonas sp. strain VLB120 using glucose-supplemented LB medium.

TABLE 1.

Tolerated butanol concentrations in different growth media

Stimulation of Antioxidant Enzymes and Lipid Peroxidation by UV-B Irradiation in Thylakoid Membranes of Wheat

In wheat seedlings (Triticum aestivum L. cv. 2329) oxidative stress caused by UV-B radiation led to lipid peroxidation of thylakoid membrane; it was expressed in term of malondialdehyde (MDA) formation. The peroxidation of lipids of thylakoid membrane in isolated chloroplasts was prevented when flavonoids quercetin and rutin were supplied into the incubation medium. The activities of superoxide dismutase, ascorbate peroxidase, and catalase increased during the first hours of UV-B exposure. A comparative study of UV-B and temperature effects showed different profiles of the antioxidant enzymes and MDA, suggesting that these two stresses have distinct sites of action. In addition to quantitative increase in flavonoids, qualitative change in flavonoid composition was also marked during UV-B stress, and a new peak at 330 nm was found as compared to control. This revised version was published online in July 2006 with corrections to the Cover Date.