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.     

Enhanced sensitivity and characterization of photosystem II in transgenic tobacco plants with decreased chloroplast glutathione reductase under chilling stress

Chloroplast glutathione reductase (GR) plays an important role in protecting photosynthesis against oxidative stress. We used transgenic tobacco (Nicotiana tabacum) plants with severely decreased GR activities by using a gene encoding tobacco chloroplast GR for the RNAi construct to investigate the possible mechanisms of chloroplast GR in protecting photosynthesis against chilling stress. Transgenic plants were highly sensitive to chilling stress and accumulated high levels of H?O? in chloroplasts. Spectroscopic analysis and electron transport measurements show that PSII activity was significantly reduced in transgenic plants. Flash-induced fluorescence relaxation and thermoluminescence measurements demonstrate that there was a slow electron transfer between Q(A) and Q(B) and decreased redox potential of Q(B) in transgenic plants, whereas the donor side function of PSII was not affected. Immunoblot and blue native gel analyses illustrate that PSII protein accumulation was decreased greatly in transgenic plants. Our results suggest that chloroplast GR plays an important role in protecting PSII function by maintaining the electron transport in PSII acceptor side and stabilizing PSII complexes under chilling stress. Our results also suggest that the recycling of ascorbate from dehydroascorbate in the ascorbate-glutathione cycle in the chloroplast plays an essential role in protecting PSII against chilling stress.     

Protective role of nitric oxide during hydrogen peroxide-induced oxidative stress in tobacco plants

Nitric oxide, produced from exogenous NO donor, sodium nitroprusside, and hydrogen peroxide exerted antagonistic effects on tobacco leaves at micromolar concentrations but induced synergistic effects at millimolar concentrations. During H₂O₂-induced oxidative stress, low concentrations of NO inhibited lipid peroxidation, counteracted the fragmentation of total DNA, and prevented accumulation of soluble proteins in Nicotiana plumbaginifolia cells. When applied at high concentrations, NO induced the caspase-like activity, promoted degradation of DNA and soluble proteins, and reduced ATP synthesis. The results are consistent with the hypothesis that NO performs a dual role in plants, acting as antioxidant (scavenger of reactive oxygen species) and as a signaling messenger. There are grounds to believe that, irrespective of the mechanism involved, nitric oxide performs a protective role during oxidative stress in tobacco leaves, because even high concentrations of NO exerted no immediate toxic effect but induced the programmed cell death through the activation of caspase-like proteases.     

Accumulation of overproduced ferritin in the chloroplast provides protection against photoinhibition induced by low temperature in tobacco plants

Wild-type tobacco plants (Nicotiana tabacum L. cv. Petit Havanna line SR1) and plants transformed with full-length alfalfa ferritin cDNA with the chloroplast transit peptide under the control of a Rubisco small subunit gene promoter (C3 and C8) were cold-treated at 0 degrees C with continuous light (250mumolm(-2)s(-1)). These transgenic plants had higher chlorophyll content and higher F(v)/F(m) chlorophyll-a fluorescence induction parameters than wild-type plants after 2 or 3d of cold treatment in C3 and C8 transgenic plants, respectively. Thermoluminescence studies on the high-temperature bands suggest that these plants suffered less oxidative damage in comparison to the wild-type genotype. The present experiments provide evidence that transgenic tobacco lines overexpressing alfalfa ferritin, which is accumulated in the chloroplasts, may show higher tolerance to various stress factors, generating ROS including low temperature-induced photoinhibition.     

Changes in fatty acid composition of lipids in chloroplast membranes of tobacco plants during cold hardening

Changes in fatty acid composition of chloroplast membrane lipids were investigated using tobacco (Nicotiana tabacum L., cv. Samsun) plants subjected to cold hardening for 6 days at 8°C. Under optimal growing temperature (22°C), the lipids of thylakoid membranes were characterized by elevated content of 16:3n-3 and 18:3n-3 fatty acids (FA). Compared to the lipids of chloroplast envelope membranes, the thylakoid lipids were less rich in the content of saturated, mono- and diunsaturated FA. The relative content of unsaturated FA in chloroplast membranes increased substantially during cold hardening, which was mainly due to the accumulation of 18:3n-3 FA. It is concluded that the observed changes in FA composition of chloroplast lipids during cold hardening adjust the fluidity of these membranes to the level sufficient for functioning of tobacco photosynthetic apparatus, which is a prerequisite for accumulation of assimilates and allows the hardened tobacco plants to survive under conditions of hypothermia.     

Changes in ultrastructure and protein metabolism of tobacco plants infected by tomato spotted wilt virus

As the result of electron microscope investigation of ultra-thin sections of the tissues infected by tomato spotted wilt virus it was shown that ultrastructural changes in the cells depend on the virus virulence. The isolate with low virulence induces mostly virus-specific changes (virus particles and virus inclusion bodies); the isolate with high virulence besides the virus-specific changes causes essential non-specific violation of cell organelle structure that could be the consequence of pathological action of the virus. It was determined that severe virus infection results in the decrease of general content of the proteins in the leaves. At the same time it induces formation of at least three pathogenesis-associated proteins (PR-proteins) and two antiviral factors of the types AVF (6) and IVR (7) active towards tobacco mosaic virus.     

Leaf anatomy and chloroplast ultrastructure of Mn-deficient orange plants

Leaf samples of Mn-deficient and Mn-sufficient (control) ‘Navelate’ orange plants grown in a greenhouse were taken to investigate the effects of Mn deficiency in leaf structure and chloroplast ultrastructure. Total leaf chlorophyll concentration was significantly lower in Mn-deficient plants than in control ones. Entire lamina thickness was not altered due to Mn deficiency. However, Mn deficiency resulted in disorganization of mesophyll cells, mainly of palisade parenchyma cells. The number of mesophyll chloroplasts per cellular area and their length were both affected negatively. The membranous system of chloroplasts was also disorganized. The percentages of starch grains and plastoglobuli per chloroplast of Mn-deficient leaves were significantly greater than those of control leaves.     

The chloroplast small heat shock protein undergoes oxidation-dependent conformational changes and may protect plants from oxidative stress

The nuclear-encoded chloroplast-localized Hsp21 is an oligomeric heat shock protein (Hsp), belonging to the protein family of small Hsps and alpha-crystallins. We have investigated the effects of high temperature and oxidation treatments on the structural properties of Hsp21, both in purified recombinant form and in transgenic Arabidopsis thaliana plants engineered to constitutively overexpress Hsp21. A conformational change was observed for the 300 kDa oligomeric Hsp21 protein during moderate heat stress (< or =40 degrees C) of Arabidopsis plants, as judged by a shift to lower mobility in non-denaturing electrophoresis. Similar changes in mobility were observed when purified recombinant Hsp21 protein was subjected to an oxidant. Exposure of Hsp21 protein to temperatures above 70 degrees C led to irreversible aggregation, which was prevented in presence of the reductant dithiothreitol. The transgenic plants that constitutively overexpressed Hsp21 were more resistant to heat stress than were wildtype plants when the heat stress was imposed under high light conditions. These results suggest that the physiological role of Hsp21 involves a response to temperature-dependent oxidative stress.     

Enhancement of oxidative stress tolerance in transgenic tobacco plants overproducing Fe-superoxide dismutase in chloroplasts.

A chimeric gene consisting of the coding sequence for chloroplastic Fe superoxide dismutase (FeSOD) from Arabidopsis thaliana, coupled to the chloroplast targeting sequence from the pea ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, was expressed in Nicotiana tabacum cv Petit Havana SR1. Expression of the transgenic FeSOD protected both the plasmalemma and photosystem II against superoxide generated during illumination of leaf discs impregnated with methyl viologen. By contrast, overproduction of a mitochondrial MnSOD from Nicotiana plumbaginifolia in the chloroplasts of cv SR1 protected only the plasmalemma, but not photosystem II, against methyl viologen (L. Slooten, K. Capiau, W. Van Camp, M. Van Montagu, C. Sybesma, D. Inzé [1995] Plant Physiol 107: 737-750). The difference in effectiveness correlates with different membrane affinities of the transgenic FeSOD and MnSOD. Overproduction of FeSOD does not confer tolerance to H2O2, singlet oxygen, chilling-induced photoinhibition in leaf disc assays, or to salt stress at the whole plant level. In nontransgenic plants, salt stress led to a 2- to 3-fold increase in activity, on a protein basis, of FeSOD, cytosolic and chloroplastic Cu/ZnSOD, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. In FeSOD-overproducing plants under salt stress, the induction of cytosolic and chloroplastic Cu/ZnSOD was suppressed, whereas induction of a water-soluble chloroplastic ascorbate peroxidase isozyme was promoted.     

Elevated glutathione biosynthetic capacity in the chloroplasts of transgenic tobacco plants paradoxically causes increased oxidative stress

Glutathione (GSH), a major antioxidant in most aerobic organisms, is perceived to be particularly important in plant chloroplasts because it helps to protect the photosynthetic apparatus from oxidative damage. In transgenic tobacco plants overexpressing a chloroplast-targeted gamma-glutamylcysteine synthetase (gamma-ECS), foliar levels of GSH were raised threefold. Paradoxically, increased GSH biosynthetic capacity in the chloroplast resulted in greatly enhanced oxidative stress, which was manifested as light intensity-dependent chlorosis or necrosis. This phenotype was associated with foliar pools of both GSH and gamma-glutamylcysteine (the immediate precursor to GSH) being in a more oxidized state. Further manipulations of both the content and redox state of the foliar thiol pools were achieved using hybrid transgenic plants with enhanced glutathione synthetase or glutathione reductase activity in addition to elevated levels of gamma-ECS. Given the results of these experiments, we suggest that gamma-ECS-transformed plants suffered continuous oxidative damage caused by a failure of the redox-sensing process in the chloroplast.     

Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplasts

The effect of simultaneous expression of genes encoding three antioxidant enzymes, copper zinc superoxide dismutase (CuZnSOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), and dehydroascorbate (DHA) reductase (DHAR, EC 1.8.5.1), in the chloroplasts of tobacco plants was investigated under oxidative stress conditions. In previous studies, transgenic tobacco plants expressing both CuZnSOD and APX in chloroplast (CA plants), or DHAR in chloroplast showed enhanced tolerance to oxidative stresses, such as paraquat and salt. In this study, in order to develop transgenic plants that were more resistant to oxidative stress, we introduced the gene encoding DHAR into CA transgenic plants. Mature leaves of transgenic plants expressing all three antioxidant genes (CAD plants) had approximately 1.6–2.1 times higher DHAR activity, and higher ratios of reduced ascorbate (AsA) to DHA, and oxidized glutathione (GSSG) to reduced glutathione (GSH) compared to CA plants. CAD plants were more resistant to paraquat-induced stress, exhibiting only 18.1% reduction in membrane damage relative to CA plants. In addition, seedlings of CAD plants had enhanced tolerance to NaCI (100 mM) compared to CA plants. These results indicate that the simultaneous expression of multiple antioxidant enzymes, such as CuZnSOD, APX, and DHAR, in chloroplasts is more effective than single or double expression for developing transgenic plants with enhanced tolerance to multiple environmental stresses.     

Changes in chloroplast ultrastructure in leaves of drought-stressed maize inbred lines

Chloroplasts are commonly the site of the earliest abiotic injury visible in plant ultrastructure. In this study, six inbred lines of maize (Zea mays L.) were used to analyze changes in the ultrastructure of chloroplasts and related physiological parameters under conditions of drought stress simulated by 20% polyethylene glycol 6000 (?0.6 MPa) for two days. Chloroplasts of three maize lines proved to be more sensitive. They showed changes in the ultrastructure in response to drought, including damage of thylakoid membranes, an increase in the number and size of plastoglobuli, swelling of thylakoid membranes both stromal and granal, disorganization of the thylakoid membrane system, an obvious increase in the intrathylakoid space, and a decrease in the length-to-width ratio and area of chloroplasts. In addition, the contents of malondialdehyde increased markedly in the sensitive lines. Contrary to the sensitive lines, stable structures and shapes of chloroplasts were observed in the drought-resistant lines; it could be considered as an advantage contributing to drought tolerance in the plants. In addition, the drought index of leaf fresh mass (LMDI) in the drought-sensitive lines was ≤ 0.5, which was also associated with a lower content of leaf chlorophyll. In contrast, drought tolerance coincided with lesser growth reduction, and higher LMDI and leaf chlorophyll content.     

The role of glycine betaine in the protection of plants from stress: clues from transgenic plants

The acclimation of a plant to a constantly changing environment involves the accumulation of certain organic compounds of low molecular mass, known collectively as compatible solutes, in the cytoplasm. The evidence from numerous investigations of the physiology, genetics, biophysics and biochemistry of plants strongly suggests that glycine betaine (GB), an amphoteric quaternary amine, plays an important role as a compatible solute in plants under various types of environmental stress, such as high levels of salts and low temperature. Plant species vary in their capacity to synthesize GB and some plants, such as spinach and barley, accumulate relatively high levels of GB in their chloroplasts while others, such as Arabidopsis and tobacco, do not synthesize this compound. Genetic engineering has allowed the introduction into GB-deficient species of biosynthetic pathways to GB from both micro-organisms and higher plants; this approach has facilitated investigations of the importance of GB in stress protection. In this review, we summarize recent progress in the genetic manipulation of the synthesis of GB, with special emphasis on the relationship between the protective effects of GB in vivo and those documented in vitro.     

Arsenic induced oxidative stress in plants

Arsenic is a highly toxic metalloid for all forms of life including plants. Arsenic enters in the plants through phosphate transporters as a phosphate analogue or through aquaglycoporins. Uptake of arsenic in plant tissues adversely affects the plant metabolism and leads to various physiological and structural disorders. Photosynthetic apparatus, cell division machinery, energy production, and redox status are the major section of plant system that are badly affected by As (V). Similarly As (III) can react with thiol (-SH) groups of enzymes and inhibits various metabolic processes. Arsenic is also known to induce oxidative stress directly by generating reactive oxygen species (ROS) during conversion of its valence forms or indirectly by inactivating antioxidant molecules through binding with their -SH groups. As-mediated oxidative stress causes cellular, molecular and physiological disturbances in various plant species. Activation of enzymatic antioxidants namely, superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR), Glutathione s-transferase, glutathione peroxidase (GPX) as well as non antioxidant compounds such as, ascorbate, glutathione, carotenoids are reported to neutralize arsenic mediated oxidative stress. Understanding of biochemistry of arsenic toxicity would be beneficial for the development of arsenic tolerant crops and other economically important plants.     

Reorganization of chloroplast ultrastructure associated with low-temperature hardening of Arabidopsis plants

When following low-temperature acclimation (5 days at 2°C) of cold-resistant plants of Arabidopsis (Arabidopsis thaliana Heynh. (L.), ecotype Columbia) in relation to the changes in chloroplast ultrastructure, we registered the high efficiency of hardening and the ability of hardened plants to lower a threshold of frost damage by about 3°C. During hardening, the area of grana in the chloroplasts more than doubled, with considerably increased numbers of thylakoids per granum and thylakoids per chloroplast. The rate of apparent photosynthesis decreased to lesser extent than the rate of dark respiration, as a result the content of soluble sugars increased fourfold, ensuring an adaptive reorganization of metabolism, which enabled the hardened plants to survive even at below-zero temperatures (up to ?7°C). The authors conclude that a considerable increase in the number of thylakoids in the chloroplasts helps maintain photosynthesis at low above-zero temperatures and is a prerequisite for the accumulation of soluble sugars in Arabidopsis leaves.     

The role of chloroplast NAD(P)H dehydrogenase in protection of tobacco plant against heat stress

After incubation at 42°C for more than 48 h, brown damages occurred on the stems of tobacco (Nicotiana tabacum L.) ndhC-ndhK-ndhJ deletion mutant (ΔndhCKJ), followed by wilt of the leaves, while less the phenotype was found in its wild type (WT). Analysis of the kinetics of post-illumination rise in chlorophyll fluorescence indicated that the PSI cyclic electron flow and the chlororespiration mediated by NAD(P)H dehydrogenase (NDH) was significantly enhanced in WT under the high temperature. After leaf disks were treated with methyl viologen (MV), photosynthetic apparatus of ΔndhCKJ exhibited more severe photo-oxidative damage, even bleaching of chlorophyll. Analysis of P700 oxidation and reduction showed that the NDH mediated cyclic electron flow probably functioned as an electron competitor with Mehler reaction, to reduce the accumulation of reactive oxygen species (ROS). When leaf disks were heat stressed at 42°C for 6 h, the photochemical activity declined more markedly in ΔndhCKJ than in WT, accompanied with more evident decrease in the amount of soluble Rubisco activase. In addition, the slow phase of millisecond-delayed light emission (ms-DLE) of chlorophyll fluorescence indicated that NDH was involved in the building-up of transthylakoid proton gradient (ΔpH), while the consumption of ΔpH was highly inhibited in ΔndhCKJ after heat stress. Based on the results, we supposed that the cyclic electron flow mediated by NDH could be stimulated under the heat stressed conditions, to divert excess electrons via chlororespiration pathway, and sustain CO₂ assimilation by providing extra ΔpH, thus reducing the photooxidative damage.     

外源亚精胺对NaCl胁迫下毕氏海蓬子光合参数和叶绿体超微结构的影响

在0、100、300、500和700 mmol·L-1NaCl胁迫条件下比较了喷施0.1mmol·L-1亚精胺(Spd)对毕氏海蓬子(Salicomia bigelovii Torr.)幼苗叶绿素含量、净光合速率、气孔导度、胞间CO2浓度和叶绿体超微结构的影响.结果表明:毕氏海蓬子的叶绿素含量、净光合速率和气孔导度均呈低浓度条件下(0、100和300 mmol·L-1NaCl)升高、高浓度条件下(500和700 mmol·L-1NaCl)降低的趋势,在300 mmol·L-1 NaCl胁迫条件下达到最高值:胞间CO2浓度则呈低浓度NaCl胁迫条件下降低、500 mmol·L-1NaCl条件下升高、700 mmol·L-1NaCl条件下略降低的趋势;在0～500 mmol·L-1NaCl胁迫条件下叶绿素a/b值变化不明显,但在700 mmol·L-1NaCl条件下急剧降低.在低浓度NaCl胁迫条件下,叶绿体整体膨胀,类囊体片层结构松散,但叶绿体和类囊体结构仍保持完整;而经500和700mmol·L-1NaCl处理后,叶绿体超微结构被严重破坏,叶绿体膜结构破裂、类囊体结构松散呈放射状、有些叶绿体完全解体.而在相应的NaCl胁迫条件下喷施0.1 mmol·L-1Spd,毕氏海蓬子的叶绿素含量、净光合速率、气孔导度和胞间CO2浓度虽然也呈现出相同的变化趋势,但其数值均显著高于对照(未喷施Spd);且叶绿体超微结构的损伤程度也轻于对照.研究结果说明:喷施外源Spd能够减缓NaCl胁迫对毕氏海蓬子的伤害作用.