Effect of cytokinins on oxidative stress in tobacco plants under nitrogen deficiency

Wild type and transgenic tobacco plants expressing isopentenyltransferase, a gene coding the rate-limiting step in cytokinin synthesis, were grown under limited nitrogen (N) conditions. Our results indicated that the WT plants subjected to N deficiency displayed reduced biomass and relative growth rates, increased levels of oxidative damage and reduced foliar concentrations of the different N forms. However, the transgenic plants expressing P_SARK∷IPT, in spite of showing a significant decline in all the N forms in the leaf, avoided the alteration of the oxidative metabolism and maintained biomass and the relative growth rates at control levels, under suboptimal N conditions. These results suggest that the increased cytokinin synthesis in the transgenic plants is an effective mechanism to improve N-use efficiency.     

Comparative study of the influence of the metal centres: Fe(III), Cu(II) and Zn(II), on the ring opening and oxidative dehydrogenation reactions occurring in a coordinated imidazolidine ligand

Condensation of 2-pyridinecarboxaldehyde and 1,9-bis-(2′-pyridyl)-2,5,8-triazanonane, L¹, yields 1-[3-aza-4-(2-pyridyl)butyl]-2-(2-pyridyl)-3-[(2-pyridyl)methyl]imidazolidine, L², as proven by NMR solution spectra. When L² is reacted with Fe(III) in different alcohols, an imidazolidine ring opening and an oxidative dehydrogenation reaction occur resulting in new complexes of the type: [Fe^IIL^n′]²⁺. Compound 1 with a coordinated L^3′ ligand was obtained in n-propanol as a solvent. Compounds 2, 3 and 4 were obtained with L^4′, L^5′ and L^6′ when iso-propanol, n-butanol and iso-butanol were used as solvent, respectively. The structures for 1, 2, 3 and 4 were determined by NMR solution spectra and additionally by X-ray crystallography in the case of the n-butoxy derivative 3. When Cu(II) was used, the hexadentate ligand L² undergoes also an imidazolidine ring opening reaction on complex formation, however, now generating the well-known pentadentate ligand L¹ that is coordinated to the metal ion, 7. Evidence is again provided by the corresponding X-ray structure. With Zn(II) the initial structure of L² is maintained and in this case L² functions as a tetradentate, 5, or bis-tridentate ligand, 6, depending on whether the stoichiometric ratio M:L was 1:1 or 2:1, respectively. This has been proven by a solid-state X-ray structure analysis as well as by NMR solution spectra. The ring opening reaction in the presence of Fe(III) can be explained as a result of a higher Lewis acidity of this metal centre, which decreases the electronic density on the nitrogen atom of the imidazolidinic cycle, thus weakening the nitrogen-carbon bond, favouring the nucleophilic attack on the carbon atom by alcohols and producing a more stable hexacoordinated species. Electrochemical evidence is provided in order to support this reaction mechanism.     

Polypeptide profiles of chlorophyll · protein complexes and thylakoid membranes of spinach chloroplasts

In addition to the major chlorophyll · protein complexes I and II, two minor chlorophyll proteins have been observed in sodium dodecyl sulfate (SDS)-polyacrylamide gels of spinach chloroplast membranes. These minor pigmented zones appeared to be derived from the light-harvesting chlorophyll $\text{a}\text{b}$ · protein and from the reaction centre complex of Photosystem II.Data are presented on the polypeptide profiles of purified digitonin-subchloroplast particles, with special regard to the effect of solubilization temperature and extraction of lipids. The results are compared with the SDS-polypeptide pattern of spinach thylakoids obtained under exactly the same conditions with respect to electrophoresis technique, solubilization method and presence of lipid. In addition, the effects of temperature and lipid extraction on the distinct chlorophyll · protein complexes appearing in SDS gel electrophoretograms of chloroplast membranes were studied by slicing the chlorophyll-containing regions and subjecting them to a second run with or without heating or extraction with acetone. By supplementing these data with an examination of the polypeptide composition of cytochrome f and coupling factor, it has been possible to identify most of the major chloroplast membrane polypeptides.     

Two-dimensional separation of chloroplast membrane proteins by isoelectri focusing and electrophoresis in sodium dodecyl sulphate

Proteins of chloroplast subfragments enriched in Photosystem I and Photosystem II electron flow activity have been analyzed by two-dimensional polyacrylamide gel electrophoresis. In the first dimension, polyacrylamide gel isoelectric focusing (pH 5–7) was used in the presence of Triton X-100, followed at right angle by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Characteristic fingerprints were obtained for the Photosystem I and II fractions and a correlation between the major proteins separated by isoelectric focusing and the major polypeptides separated by undimensional SDS electrophoresis was established. Two dominant spots of 68 000 and 60 000 daltons appeared in the two-dimensional patterns of Photosystem I fractions $\text{p}\text{I}$ values about 5.6; two spots with molecular weights of 33 000 and 23 000 were characteristics for Photosystem II fractions $\text{p}\text{I}$ values about 5.3 and 6.3). Photosystem I fractions were furthermore characteristics by a series of spots in the 44 000–33 000 range $\text{p}\text{I}$ values from about 5.9 to 6.8). The two-dimensional system revealed that (a) several SDS-polypeptides have multiple forms differing in charge only, (b) some proteins separated by isoelectric focusing are resolved in the second dimensional into polypeptides of different size. The two-dimensional method combining Triton X-100 isoelectric focusing' and SDS electrophoresis provides a higher degree of resolution than either of the unidimensional methods thus allowing a detailed analysis of chloroplast membrane proteins.     

Reconstitution of photosynthetic charge accumulation and oxygen evolution in CaCl2-treated PS II particles: II: EPR evidence for reactivation of the S1 → S2 transition in CaCl2-treated PS II particles with the 17-, 23-, and 34-kDa proteins

Extraction of PS II particles with 1 M CaCl₂ caused complete disappearance of the light-induced signal of the possible Kok S₂ state of the water-splitting complex and total loss of the O₂, evolving activity, concomitant with perfect removal of the 17-, 23- and 34-kDa proteins from the particles. The recovery of the multiline signal in the CaCl₂-treated PS II was performed by reinserting the 34-kDa protein, when CI^? was present in the solution for the EPR measurement. However, in the absence of Cl^?, besides the 34-kDa protein, the 17- and 23-kDa proteins were required for the recovery of the signal. These results are compared with the results on the recovery of the O₂, evolution in the reconstituted PS II to examine the role of these three proteins on the water splitting.     

Membrane Injury and PSⅡ Inactivation in Some Subtropical Woody Plants Induced by Photooxidation

Leaf discs from the pot-cultured seedlings of tree species and understory shrub species in a natural subtropical forest were treated with methyl viologen (MV) solution under low light or high light illumination. The degradation of chlorophyll and the leakage rate of electrolytes through membrane were enhanced by the increasing MV concentration and prolonged time of treatment. Membrane injury was more serious than oxidative bleaching of chlorophyll under the same condition. When leaf discs were subjected to short term (1 h) exposure of MV-photooxidation in high light (1 300 μmol·m-2·s-1), a pronounced decrease of Fv/Fm, ФPSⅡ and qp, as well as △A505, △A320 and ratio of qp/qN were observed in all tested species, which were accompanied by the increase of qN, KD and Fo. It demonstrated that photooxidation led to PSⅡ inactivation expressing in the reduction of the number of PS Ⅱ centre undergoing a stable charge separation and efficiency of the primary photochemical efficiency and noncyclic electron transport of PS Ⅱ. Partial excitation energy might dissipate via the useful nonphotochemical fluorescence quenching, but the lowering of △A505 was not consistent with the elevation of qN. Understory shrub Psychotria rubra Poir., Ardisia quinquegona BI. Showed more susceptibility to photooxidation than the upper canopy tree species Schima superba Gardn. et Champ., Castanopsis fissa (Champ.) ex Benth. ) Rend. et Wils. and Syzygium rehderianum Merr. et Perry.     

Synthesis and characterization of a high spin d Fe(II) complex with the tridentate ligand dipyrido(2,3-a:3,2-j)phenazine (dpop)

A new bis-(N-tridentate) Fe(II) complex [Fe(dpop^′)₂](PF₆)₂ (dpop^′=dipyrido(2,3-a:3^′,2^′-j)phenazine) was prepared and studied. The magnetic moment of the solid was determined as μ=5.2-4.9 BM and in CH₃CN solution as μ=4.9 BM and indicate the high spin Fe(II) state. The electronic absorption spectrum displays a broad weak absorption MLCT transition at 602 nm (ε=3.8×10³ M⁻¹ cm⁻¹), consistent with CT absorptions of other Fe(II) HS complexes. The cyclic voltammogram of the complex shows an irreversible Fe^2+/3+ oxidation at +1.55 V and two dpop^′0/−1 centered reductions at −0.20 and −0.59 V versus Ag/AgCl.     

Effect of monochromatic UV-B radiation on electron transfer reactions of Photosystem II

The adverse effect of low intensity, small band UV-B irradiation (λ = 305 ± 5 nm, I = 300 mW m⁻²) on PS II has been studied by comparative measurements of laser flash-induced changes of the absorption at 325 nm, ΔA₃₂₅(t), as an indicator of redox changes in Q_A, and of the relative fluorescence quantum yield, F(t)/F_o, in PS II membrane fragments. The properties of untreated control were compared with those of samples where the oxygen evolution rate under illumination with continuous saturating light was inhibited by up to 95%. The following results were obtained: a) the detectable initial amplitude (at a time resolution of 30 μs) of the 325 nm absorption changes, ΔA₃₂₅, remained virtually invariant whereas the relaxation kinetics exhibit significant changes, b) the 300 μs kinetics of ΔA₃₂₅ dominating the relaxation in UV-B treated samples was largely replaced by a 1.3 ms kinetics after addition of MnCl₂, c) the extent of the flash induced rise of the relative fluorescence quantum yield was severely diminished in UV-B treated PS II membrane fragments but the relaxation kinetics remain virtually unaffected. Based on these results the water oxidizing complex (WOC) is inferred to be the primary target of UV-B impairment of PS II while the formation of the ‘stable’ radical pair P680^+·Q_A ^−● is almost invariant to this UV-B treatment. This revised version was published online in June 2006 with corrections to the Cover Date.     

The physiological significance of photosystem II heterogeneity in chloroplasts

Photosystem II in green plant chloroplasts displays heterogeneity both in the composition of its light-harvesting antenna and in the ability to reduce the plastoquinone pool. These two features are discussed in terms of chloroplast development and in view of a proposed photosystem II repair cycle.     

Redox Properties of Iron in the Binding Site(s) of F1ATPase from Mammalian Mitochondria and Thermophilic Bacterium PS3: A Comparative Study

Iron ions in the two iron centers of beef heart mito-chondrial F, ATPase, which we have been recently characterized (FEBS Letters 1996,379, 231-235), exhibit different redox properties. In fact, the ATP-dependent site is able to maintain iron in the redox state of Fe(II) even in the absence of reducing agents, whereas in the nucleotide-independent site iron is oxidized to Fe(III) upon removal of the reductant. Fe(III) ions in the two sites display different reactivity towards H₂O₂, because only Fe(III) bound in the nucleotide-independent site rapidly reacts with H₂O₂ thus mediating a 30% enzyme inactivation. Thermophilic bacterium PS3 bears one Fe(III) binding site, which takes up Fe(III) either in the absence or presence of nucleotides and is unable to maintain iron in the redox state of Fe(II) in the absence of ascorbate. Fe(III) bound in thermophilic F₁ATPase in a molar ratio 1:1 rapidly reacts with H₂O₂ mediating a 30% enzyme inactivation. These results support the presence in mitochon-drial and thermophilic F₁ATPase of a conserved site involved in iron binding and in oxidative inactivation, in which iron exhibits similar redox properties. On the other hand, at variance with thermophilic F₁ATPase, the mitochondrial enzyme has the possibility of maintaining one equivalent of Fe(II) in its peculiar ATP-dependent site, besides one equivalent of Fe(III) in the conserved nucleotide-independent site. In this case mitochondrial F, ATPase undergoes a higher inactivation (75%) upon exposure to H₂O₂. Under all conditions the inactivation is significantly prevented by PBN and DMSO but not by Cu, Zn superoxide dis-mutase, thus suggesting the formation of OH radicals as mediators of the oxidative damage. No dityrosines, carbonyls or oxidized thiols are formed. In addition, in any cases no protein fragmentation or aggregation is observed upon the treatment with H₂O₂.     

Strength of winter leaf redness as an indicator of stress vulnerable individuals in Pistacia lentiscus

Leaf anthocyanins are believed to afford protection against photoinhibition, yet the dependence of protection on actual anthocyanin concentrations has not been investigated. To that aim, non-invasive optical methods (spectral reflectance and chlorophyll fluorescence) were used to assess the levels of anthocyanins and chlorophylls as well as photosystem II photochemical efficiency in hundreds of leaves from the mastic tree (Pistacia lentiscus), which displays in the field a continuum of leaf tints during winter from fully green to fully red. Contrary to expectations based on the photoprotective hypothesis, the strength of leaf redness was positively correlated to the extent of mid-winter chronic photoinhibition and negatively correlated to leaf chlorophyll contents. Hence, a photoprotective role for anthocyanins is not substantiated. Instead, we argue that winter leaf redness may be used reliably, quickly and non-invasively to locate vulnerable individuals in the field.     

On the significance of photoinhibition of photosynthesis in the field and its generality among species

Photoinhibition was examined in naturally exposed willow leaves in the field. In the afternoon on clear and warm days, the quantum yield of electron transport, derived from gas exchange data, was decreased by 28%. Besides this photoinhibition, decreases in the photosynthetic capacity and in the stomatal conductance were also observed. Of these three limitations of carbon assimilation, photoinhibition was the major one at limiting light only.To investigate the generality of photoinhibition, shade- and sun-acclimated leaves of fourteen different species were compared in a laboratory study. Photoinhibition was quantified by fluorescence measurements following exposure to moderate and high light for 30 min. The extent of photoinhibition was inversely related to the photochemical quenching, q_p, during exposure (the proportion of open PS II traps). This relationship was the same independent of the species, the light-acclimation state of the leaf and the light intensity. However, sun- and shade-acclimated leaves occupied opposite sides of the relationship: the sun-leaves showed lower photoinhibition and higher q_p. The sun-leaves were also more competent than shade-leaves by showing faster recovery from a given level of photoinhibition.Abbreviations F₀, F_V, F_M, F_S minimal, variable, maximal and steady-state fluorescence - q_N, q_i total and photoinhibitory non-photochemical quenching of variable fluorescence - q_p photochemical quenching     

Fe Resupply to Fe-deficient Sugar Beet Plants Leads to Rapid Changes in the Violaxanthin Cycle and other Photosynthetic Characteristics without Significant de novo Chlorophyll Synthesis

The effects of Fe resupply to Fe-deficient plants have been investigated in hydroponically-grown sugar beet. In the short-term (24 h) after Fe resupply, major changes were observed, although de novo chlorophyll (Chl) synthesis had not begun yet. Approximately 50% of the zeaxanthin was converted into violaxanthin, whereas the actual Photosystem II (PS II) efficiency increased by 69% and non-photochemical quenching (NPQ) and the amount of thermally dissipated energy decreased markedly (by 47% and 40%, respectively). At the same time, photosynthetic rate increased approximately by 50%. From one to two days after Fe resupply, there was a gradual increase in the leaf concentrations of Chl and other photosynthetic pigments, accompanied by a further conversion of zeaxanthin into violaxanthin, increases in actual PS II efficiency and photosynthetic rates and decreases in NPQ and the amount of thermally dissipated energy. At 72-96 h after Fe resupply, leaf pigment concentrations, photosynthetic rates and actual PS II efficiency had increased further, although both photosynthetic rate and leaf pigment concentrations were still lower than those found in Fe-sufficient leaves. Good correlations were observed between the amount of light thermally dissipated by the PS II antenna, NPQ and the antheraxanthin + zeaxanthin concentration after Fe resupply, confirming the photoprotective role of the xanthophyll cycle in Fe-deficient sugar beet leaves. Similar correlations were observed for lutein, suggesting a possible role of this pigment in photoprotection.