Does methyl jasmonate modify the oxidative stress response in Phaseolus coccineus treated with Cu?

The contribution of methyl jasmonate (MJ) as a signal molecule able to take part in the defense mechanism against copper (Cu)-imposed oxidative stress was studied in the leaves and roots of runner bean (Phaseolus coccineus) plants. Roots of plants cultivated hydroponically were preincubated in MJ (10µM) for 1h or 24h and subsequently exposed to Cu (50µM) for 5h (short-term experiment) or 5 days (long-term experiment). Enzymatic (activity of superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX; guaiacol peroxidase, POX) and non-enzymatic (accumulation of malondialdehyde, MDA; homoglutathione, hGSH; proline; anthocyanins; low molecular weight organic acids, LMWOAs) responses were determined in the leaves and roots. The antioxidative defense mechanism was significantly activated after Cu supplementation. In most cases, activities of ROS (reactive oxygen species) scavenging enzymes like SOD, CAT, APX, POX, as well as MDA, hGSH and proline concentrations increased following Cu exposure. MJ showed a time-dependent effect on antioxidative enzymes activity. In the short-term experiment, MJ elevated CAT, APX and POX activities in the roots, and POX activity in the leaves of non-Cu-treated plants. In the long-term experiment, MJ not only decreased POX and partially CAT activity in the roots, but also increased the MDA level and partially CAT activity in the leaves of the control plants. In Cu-treated plants, MJ reduced APX, but elevated POX activity in the leaves after 5-h exposure. After 5-day-Cu treatment, MJ inhibited POX activity in the leaves and mainly reduced SOD and CAT activities in the roots. Moreover, in the long-term experiment, MJ reduced tartrate and pyruvate in the leaves of Cu-stressed plants, but mostly elevated tartrate and malate in the roots comparing with Cu alone treatment. MJ alone and under Cu excess did not alter accumulation of MDA, hGSH and proline comparing with Cu alone, but partially elevated anthocyanin concentration. The results indicated that MJ was both partially potent in modifying the antioxidative enzymes activity and metabolites accumulation in non-stress and Cu-stress conditions.

Molecular architecture of plant thylakoids under physiological and light stress conditions: a study of lipid-light-harvesting complex II model membranes.

In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation.

Organization and functionality of chlorophyll-protein complexes in thylakoid membranes isolated from Pb-treated Secale cereale.

In this study, Secale cereale seedlings cultivated under 0 (control), 2 or 5mM Pb(NO3)2 concentrations were used to examine alterations in the organization and functionality of chlorophyll-protein complexes in thylakoid membranes under Pb ion stress. The studies were conducted on whole leaves of rye seedlings or thylakoid membranes isolated from Pb-treated and control plants. Using non-denaturing electrophoresis, it was assessed that increasing Pb concentrations resulted in an increase in the value of the ratio of the content of LHCII oligomers (mainly trimers) to the content of LHCII monomers. The parameters of chlorophyll fluorescence induction (q(p) and q(n)) indicated that the change in the LHCII supramolecular organization in the presence of Pb ions was connected with an increase in non-photochemical fluorescence quenching. Quantification of photosynthetic pigments showed that both Pb concentrations decreased the content of chlorophyll a, chlorophyll b, and carotenoids. The changes in the pigment content led to a significant reduction in light absorption by antenna complexes. However, the absorption spectra showed that red light was preferentially absorbed by antenna complexes in thylakoid membranes isolated from the Pb-treated plants. Examination of fluorescence emission spectra revealed that Pb ions decreased the fluorescence quantum yield of PSII. The emission spectra of thylakoids indicated a relative increase in the intensity of fluorescence emission from the trimeric and aggregated forms of the LHCII complexes in comparison to the intensity of fluorescence emission from PSI antenna complexes under excitation at 440 nm. Simultaneously, under excitation at 470 nm, we observed a rise in fluorescence intensity from the LHCII trimer after addition of 5mM Pb as well as a decrease in fluorescence intensity from the LHCII aggregates and PSI core and LHCI antenna complexes under both Pb concentrations. Pb treatments also reduced excitation energy absorbed by chlorophyll b and carotenoids within antenna complexes and transferred to chlorophyll a species emitting at 680 nm.

Strong light-induced reorganization of pigment-protein complexes of thylakoid membranes in rye (spectroscopic study).

The supramolecular reorganization of LHCII complexes within the thylakoid membrane in Secale cereale leaves under low and high light condition was examined. Rye seedlings were germinated hydroponically in a climate chamber with a 16 h daylight photoperiod, photosynthetic photon flux density (PPFD) of 150 µmo lm(-2)s(-1) and 24/16°C day/night temperature. The influence of pre-illumination of the plants with high light intensity on the PSII antenna complexes was studied by comparison of the structure and function of the LHCII complexes and organization of thylakoid membranes isolated from 10-day-old plants illuminated with low (150 µmo lm(-2)s(-1)) or high (1200 µmo lm(-2)s(-1)) light intensity. Aggregated and trimeric with monomeric forms of LHCII complexes were separated from the whole thylakoid membranes using non-denaturing electrophoresis. Analyses of fluorescence emission spectra of these different LHCII forms showed that the monomer was the most effective aggregating antenna form. Moreover, photoprotection connected with LHCII aggregation was more effective upon LHCII monomers in comparison to trimer aggregation. Light stress induced specific organization of neighboring LHCII complexes, causing an increase in fluorescence yield of the long-wavelength bands (centered at 701 and 734 nm). The changes in the organization of the thylakoid membrane under light stress, observed by analysis of absorbance spectra obtained by Fourier transform infrared spectroscopy, also indicated light-induced LHCII aggregation.

Structural and functional modifications of the major light-harvesting complex II in cadmium- or copper-treated Secale cereale.

The effects of 50 microM cadmium (Cd) or copper (Cu) ions on the supramolecular conformation of the light-harvesting pigment-protein complex of PSII (LHCII) isolated from rye seedlings were studied. It was found that the action of these two metal ions on the LHCII structure and organization is dissimilar. The Fourier transform infrared (FTIR) measurements indicated inhibition or stimulation of formation of parallel beta-structures and aggregates in the presence of Cd or Cu ions, respectively. The Chl a fluorescence excitation spectra of LHCII extracted from Cd-treated plants showed that the decreased aggregation of complexes was correlated with a decline in efficiency of quenching of excitation energy. From the results of mass spectrometry, changes in LHCII aggregation in the presence of Cd ions might be based on decreases in the molecular mass of Lhcb1 and Lhcb2 proteins. An increase in the content of LHCII aggregates under Cu ion excess was associated with changes in the LHCII xanthophyll pigment pool. In the complexes isolated from Cu-treated plants, all-trans violaxanthin and 9'-cis neoxanthin content declined and the simultaneous appearance of the fraction of 9-cis violaxanthin was observed. 9-cis violaxanthin formation under Cu ion excess might facilitate LHCII inter-trimer interaction and, therefore, aggregation of complexes. RLS (resonance light scattering) spectra indicated that the excitonic interaction between Chl molecules and between Chls and xanthophylls was responsible for the effective dissipation of excitation energy in LHCII isolated from Cu-treated plants. Also, changes in singlet excitation energy transfer between carotenoids and Chls under the action of heavy metals were observed.

The photoprotective mechanisms in Secale cereale leaves under Cu and high light stress condition.

The influence of excess Cu ions and high light treatment on the function of photosystem II was investigated in order to examine how this heavy metal modifies the photoprotective mechanisms operating at the molecular level in Secale cereale plants. Thus, non-treated plants and those treated with 5 or 50 microM Cu, simultaneously illuminated with 150 micromol m(-2) s(-1) or 1200 micromol m(-2) s(-1) light intensity, were studied. To analyze the PSII reaction to the stress conditions, Chl a fluorescence induction was applied. An increase in the value of Phi(PSII) and R(fd) parameters indicated that the photosynthetic apparatus adapted to the high light condition by effective utilization of excitation energy in the light and dark phases of photosynthesis. This phenomenon was accompanied by dissipation of excitation energy within the antenna complexes. The xanthophyll cycle pigments in Secale cereale leaves were separated and quantified by the HPLC technique. The results showed that, under high light irradiance, both 5 and 50 microM Cu induced the process of violaxanthin de-epoxidation and zeaxanthin accumulation. The significant zeaxanthin accumulation was found to be involved in photoprotective energy dissipation as heat, which was supported by correlation between the rate of violaxanthin de-epoxidation and the value of SV parameters. Interestingly, Cu treatment caused violaxanthin isomerization from its trans to 15-, 13- and 9-cis forms in proportional correlation to the metal concentration. This phenomenon was confirmed by a study of Cu-induced violaxanthin isomerization in vitro, which suggests a direct metal-pigment molecule interaction. We also observed that the violaxanthin trans-cis isomerization increased simultaneously with anteraxanthin content. On the basis of these findings, it can be speculated that violaxanthin isomerization is the basic process responsible for the xanthophyll cycle operation.

Variation in oxidative stress and photochemical activity in Arabidopsis thaliana leaves subjected to cadmium and excess copper in the presence or absence of jasmonate and ascorbate.

We have presented changes in the photosynthetic apparatus activity of Arabidopsis thaliana plants occurring within 15-144 h of 100 microM Cu or Cd action with regard to jasmonate (JA) as well as expression of the oxidative stress and non-enzymic defense mechanisms. The inhibitory effect of both heavy metals related to developing dissipative processes and lipid peroxide formation was expressed in dark-adapted state after the longest time as a decrease in potential quantum yield of PSII. In dark- and light-adapted state the heavy metals affected the enzymic phase of photosynthesis already from the 15th hour, which was related to the lipid peroxide formation. Photochemical quenching decrease was induced after 48th hour and did not show a close correlation with the JA pathway. Blockade of endogenously formed JA by propyl gallate decreased the effect of Cu and Cd on both the whole photosynthetic apparatus starting from the 48th hour and on the primary photochemistry of PSII after 144 h. In the case of Cu the effect was related to a lipid peroxidation decrease and to an increase in glutathione and phytochelatin (PC) levels, but in the case of Cd to lipid peroxidation, O.2- and especially to PCs increase. The obtained results indicated that JA after the longest time might enhance the sensitivity of A. thaliana to Cu and Cd stress. Asc enhanced toxic action of Cu and Cd after 15 h, but after a longer time it diminished the influence of Cd (but not Cu) on photosynthetic activity.

Xanthophyll-induced aggregation of LHCII as a switch between light-harvesting and energy dissipation systems.

The xanthophyll cycle pigments, violaxanthin and zeaxanthin, present outside the light-harvesting pigment-protein complexes of Photosystem II (LHCII) considerably enhance specific aggregation of proteins as revealed by analysis of the 77 K chlorophyll a fluorescence emission spectra. Analysis of the infrared absorption spectra in the Amide I region shows that the aggregation is associated with formation of intermolecular hydrogen bonding between the alpha helices of neighboring complexes. The aggregation gives rise to new electronic energy levels, in the Soret region (530 nm) and corresponding to the Q spectral region (691 nm), as revealed by analysis of the resonance light scattering spectra. New electronic energy levels are interpreted in terms of exciton coupling of protein-bound photosynthetic pigments. The energy of the Q excitonic level of chlorophyll is not high enough to drive the light reactions of Photosystem II but better suited to transfer excitation energy to Photosystem I, which creates favourable energetic conditions for the state I-state II transition. The lack of fluorescence emission from this energy level, at physiological temperatures, is indicative of either very high thermal energy conversion rate or efficient excitation quenching by carotenoids. Chlorophyll a fluorescence was quenched up to 61% and 34% in the zeaxanthin- and violaxanthin-containing samples, respectively, as compared to pure LHCII. Enhanced aggregation of LHCII, observed in the presence of the xanthophyll cycle pigments, is discussed in terms of the switch between light-harvesting and energy dissipation systems.

The level of jasmonic acid in Arabidopsis thaliana and Phaseolus coccineus plants under heavy metal stress.

The effect of heavy metal stress as a potent abiotic elicitor for triggering an accumulation of jasmonic acid (JA) was investigated. Copper and cadmium in in vivo conditions induced accumulation of jasmonates in mature leaves of Arabidopsis thaliana and in young and oldest Phaseolus coccineus plants. The dynamics of jasmonate accumulation showed a biphasic character in both plants. In the first phase, after 7 (A. thaliana) or 14h (P. coccineus) of exposure to Cu or Cd, a rapid increase of JA level occurred, followed by a rapid decrease observed during 7 successive hours. In the next phase, a repeated but slow increase of JA content occurred. The heavy metal stress induced in particular a more stable (3R,7R) form of jasmonates. These results indicate that JA is connected with the mechanism of toxic action of both heavy metals in plants, differentially reacting to exogenous JA and possessing variable dynamics depending on the plants studied as well as their growth stage.

A study of molecular interactions in light-harvesting complexes LHCIIb, CP29, CP26 and CP24 by Stark effect spectroscopy.

Electric field-induced absorption changes (electrochromism or Stark effect) of the light-harvesting PSII pigment-protein complexes LHCIIb, CP29, CP26 and CP24 were investigated. The results indicate the lack of strong intermolecular interactions in the chlorophyll a (Chl a) pools of all complexes. Characteristic features occur in the electronic spectrum of Chl b, which reflect the increased values of dipole moment and polarizability differences between the ground and excited states of interacting pigment systems. The strong Stark signal recorded for LHCIIb at 650-655 nm is much weaker in CP29, where it is replaced by a unique Stark band at 639 nm. Electrochromism of Chl b in CP26 and CP24 is significantly weaker but increased electrochromic parameters were also noticed for the Chl b transition at 650 nm. The spectra in the blue region are dominated by xanthophylls. The differences in Stark spectra of Chl b are linked to differences in pigment content and organization in individual complexes and point to the possibility of electron exchange interactions between energetically similar and closely spaced Chl b molecules.

Spectral analysis of pigment photobleaching in photosynthetic antenna complex LHCIIb.

Light-induced photooxidation of chlorophyll (Chl) a, b and xanthophylls was investigated in LHCIIb, the antenna pigment-protein complex of photosystem II. Absorption difference spectra at normal and low temperatures show initially (at less than 25% Chl a decay) a selective bleaching of a red-shifted Chl b with absorption bands at 487 and 655 nm, Chl b (460/650 nm) and Chl a (433/670 nm), which changes to a less selective photooxidation pattern at deeper bleaching stages. Difference absorption spectra and HPLC analyses indicate different photooxidation rates of pigments in the order neoxanthin>Chl a>lutein approximately Chl b. Despite significant pigment loss as monitored with absorption spectra, CD spectra indicate an essentially complete persistence of the protein secondary structure. Fluorescence excitation spectra suggest the conversion of a small fraction of Chl a into pheophytin a which acts as a fluorescence quencher, possibly through temporary charge separation process. The strong features in the electroabsorption (Stark effect) spectra due to chlorophyll b at 655 nm and a xanthophyll at 510 nm, and the spectral changes mentioned above are assigned to Chl molecules located at several binding sites in LHCIIb protein and are discussed in the context of spatial configuration and interactions of pigment molecules.

Conformational rearrangements in light-harvesting complex II accompanying light-induced chlorophyll a fluorescence quenching.

Light-induced chlorophyll a (Chl a) fluorescence quenching was studied in light-harvesting complex of photosystem II (LHCII). Fluorescence intensity decreased by ca. 20% in the course of 20 min illumination (412 nm, 36 micromol m(-2) s(-1)) and was totally reversible within 30 min dark adaptation. The pronounced quenching was observed only in LHCII in an aggregated form and exclusively in the presence of molecular oxygen. Structural rearrangement of LHCII correlated to the quenching was monitored by measuring changes in UV-Visible light absorption spectra, and by measuring Fourier-transform infrared spectroscopy (FTIR) in the Amide I region of the protein (1600-1700 cm(-1)). The light-induced structural rearrangement of LHCII was interpreted as a partial disaggregation of the complex based on the decrease in the light scattering signal and the characteristic features observed in the FTIR spectra: the relative increase in the intensity of the band at 1653 cm(-1), corresponding to a protein in the alpha-helical structure at the expense of the band centered at 1621 cm(-1), characteristic of aggregated forms. The fact that the light-driven isomerization of the all-trans violaxanthin to the 13-cis form was not observed under the non-oxygenic conditions coincided with the lack of large-scale conformational reorganization of LHCII. The kinetics of this large-scale structural effect does not correspond to the light-induced fluorescence quenching, in contrast to the kinetics of structural changes in LHCII observable at low oxygen concentrations. Photo-conversion of 5% of the pool of all-trans violaxanthin to 9-cis isomer was observed under such conditions. Possible involvement of the violaxanthin isomerization in the process of structural rearrangements and excitation quenching in LHCII is discussed.