Spermine and putrescine enhance oxidative stress tolerance in maize leaves

The protective effects of spermine (SPM) and putrescine (PUT) against paraquat (PQ), a herbicide in agriculture and oxidative stress inducer, were investigated in the leaves of maize. Maize leaves were pretreated to SPM and PUT at concentrations of 0.2 and 1 mM and treated with PQ afterwards. Pretreatment with 1 mM of SPM and PUT significantly prevented the losses in chlorophyll and carotenoid levels induced by PQ. Ascorbic acid content in the leaves pretreated with both polyamines was found to be higher than those of the leaves pretreated with water. Also, pretreatment with SPM and PUT was determined to have some effects on the activities of superoxide dismutase (SOD) and peroxidase (POD). 1 mM of SPM increased SOD activity, but PUT has no significant effect on SOD activity. On the other hand, POD activity was recorded to increase slightly in response to both concentrations of SPM and 1 mM of PUT. The results showed that such polyamine pretreated plants may become more tolerant to oxidative stress due to increases in the antioxidative enzymes and antioxidants.     

Nitric oxide improves chilling tolerance of maize by affecting apoplastic antioxidative enzymes in leaves

The effects of nitric oxide (NO) on chilling tolerance (freezing injury, ice nucleation activity, contents of hydrogen peroxide and superoxide anion, and lipid peroxidation level) and the activities of apoplastic antioxidant enzymes (peroxidase and superoxide dismutase) were investigated in the leaves of maize (Zea mays) exposed to short-term chilling. NO treatment was carried out through spraying of sodium nitroprusside (SNP), which is a donor of NO, in concentrations of 0.0, 0.1 and 1 μM on the leaves of 10-day plants. The plants then were transferred into the chilling condition (10/7 °C) 2 days before the harvesting of leaves (14th and 21th days). Application of 0.1 μM NO had more effect on the alleviation by decreasing the freezing injury in maize at least for 11 days after the application. Both concentrations of NO generally increased ice nucleation activity of apoplastic proteins extracted from leaves. The SNP applications decreased the contents of reactive oxygen species such as hydrogen peroxide and superoxide anion and the level of lipid peroxidation, while further increasing the activities of the apoplastic antioxidant enzymes studied. The results show that exogenous NO treatment provides important contributions to increasing the chilling tolerance of maize by regulating the biochemical mechanisms of chilling response, including apoplastic antioxidant enzymes. It can be seen that the NO treatment can play positive roles in alleviating chilling-induced damage in maize. Therefore, it is suggested that NO treatments may contribute to research studies related to diminishing chilling-induced damage in agricultural applications.     

Nitric oxide alleviates manganese toxicity by preventing oxidative stress in excised rice leaves

In the present study, we have investigated the effects of nitric oxide (NO) on alleviating manganese (Mn)-induced oxidative stress in rice leaves. Exogenous MnCl₂ treatment to excised rice leaves for 24 and 48 h resulted in increased production of H₂O₂ and lipid peroxides, decline in the levels of antioxidants, glutathione and ascorbic acid, and increased activities of antioxidative enzymes, superoxide dismutase, guaiacol peroxidase, catalase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. Treatment of rice leaves with 100 μM sodium nitroprusside (SNP), a NO donor, was effective in reducing Mn-induced increased levels of H₂O₂, lipid peroxides and increased activities of antioxidative enzymes. The levels of reduced ascorbate and glutathione were considerably recovered due to SNP treatment. The effect of SNP was reversed by the addition of NO scavenger, 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (c-PTIO) suggesting that ameliorating effect of SNP is due to release of NO. The results indicate that MnCl₂ induces oxidative stress in excised rice leaves, lowers the levels of reduced ascorbate and glutathione, and elevates activities of the key antioxidative enzymes. NO appears to provide a protection to the rice leaves against Mn-induced oxidative stress and that exogenous NO application could be advantageous in combating the deleterious effects of Mn-toxicity in rice plants.     

Nitric oxide induced by hydrogen peroxide mediates abscisic acid-induced activation of the mitogen-activated protein kinase cascade involved in antioxidant defense in maize leaves

* The role of nitric oxide (NO) and the relationship between NO, hydrogen peroxide (H(2)O(2)) and mitogen-activated protein kinase (MAPK) in abscisic acid (ABA)-induced antioxidant defense in leaves of maize (Zea mays) plants were investigated. * Both ABA and H(2)O(2) induced increases in the generation of NO in mesophyll cells of maize leaves, and H(2)O(2) was required for the ABA-induced generation of NO. Pretreatment with NO scavenger and nitric oxide synthase (NOS) inhibitor substantially reduced the ABA-induced production of NO, and partly blocked the activation of a 46 kDa MAPK and the expression and the activities of several antioxidant enzymes induced by ABA. Treatment with the NO donor sodium nitroprusside (SNP) also induced the activation of the MAPK, and enhanced the antioxidant defense systems. * Conversely, SNP treatment did not induce the production of H(2)O(2), and pretreatments with NO scavenger and NOS inhibitor did not affect ABA-induced H(2)O(2) production. * Our results suggest that ABA-induced H(2)O(2) production mediates NO generation, which, in turn, activates MAPK and results in the upregulation in the expression and the activities of antioxidant enzymes in ABA signaling.     

Nitric oxide and nitrosative stress tolerance in yeast

The opportunistic human fungal pathogen Candida albicans encounters diverse environmental stresses when it is in contact with its host. When colonizing and invading human tissues, C. albicans is exposed to ROS (reactive oxygen species) and RNIs (reactive nitrogen intermediates). ROS and RNIs are generated in the first line of host defence by phagocytic cells such as macrophages and neutrophils. In order to escape these host-induced oxidative and nitrosative stresses, C. albicans has developed various detoxification mechanisms. One such mechanism is the detoxification of NO (nitric oxide) to nitrate by the flavohaemoglobin enzyme CaYhb1. Members of the haemoglobin superfamily are highly conserved and are found in archaea, eukaryotes and bacteria. Flavohaemoglobins have a dioxygenase activity [NOD (NO dioxygenase domain)] and contain three domains: a globin domain, an FAD-binding domain and an NAD(P)-binding domain. In the present paper, we examine the nitrosative stress response in three fungal models: the pathogenic yeast C. albicans, the benign budding yeast Saccharomyces cerevisiae and the benign fission yeast Schizosaccharomyces pombe. We compare their enzymatic and non-enzymatic NO and RNI detoxification mechanisms and summarize fungal responses to nitrosative stress.     

Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis

Plant steroid hormones, brassinosteroids (BRs), are perceived by a cell surface receptor kinase, BRI1, but how BR binding leads to regulation of gene expression in the nucleus is unknown. Here we describe the identification of BZR1 as a nuclear component of the BR signal transduction pathway. A dominant mutation bzr1-1D suppresses BR-deficient and BR-insensitive (bri1) phenotypes and enhances feedback inhibition of BR biosynthesis. BZR1 protein accumulates in the nucleus of elongating cells of dark-grown hypocotyls and is stabilized by BR signaling and the bzr1-1D mutation. Our results demonstrate that BZR1 is a positive regulator of the BR signaling pathway that mediates both downstream BR responses and feedback regulation of BR biosynthesis.     

Nitric oxide modulates sensitivity to ABA

Nitric oxide (NO) is a gas with crucial signaling functions in plant defense and development. As demonstrated by generating a triple nia1nia2noa1-2 mutant with extremely low levels of NO (February 2010 issue of Plant Physiology), NO is synthesized in plants through mainly two different pathways involving nitrate reductase (NR/NIA) and NO Associated 1 (AtNOA1) proteins. Depletion of basal NO levels leads to a priming of ABA-triggered responses that causes hypersensitivity to this hormone and results in enhanced seed dormancy and decreased seed germination and seedling establishment in the triple mutant. NO produced under non-stressed conditions represses inhibition of seed developmental transitions by ABA. Moreover, NO plays a positive role in post-germinative vegetative development and also exerts a critical control of ABA-related functions on stomata closure. The triple nia1nia2noa1-2 mutant is hypersensitive to ABA in stomatal closure thus resulting in a extreme phenotype of resistance to drought. In the light of the recent discovery of PYR/PYL/RCAR as a family of potential ABA receptors, regulation of ABA sensitivity by NO may be exerted either directly on ABA receptors or on downstream signalling components; both two aspects that deserve our present and future attention.Key words: nitric oxide, abscisic acid, seed germination, stomata openingPlant development is the result of the succesfull execution of several programs that control the transition between different growth phases. Every developmental transition is regulated through coordinated mechanisms that involved exogenous environmental factors such as light and temperature as well as endogenous cues, including levels of primary and secondary metabolites. Among the latter, hormones such as gibberellins (GA), auxins, citokinins, ethylene and abscisic acid (ABA) participate in the control of most of the developmental transitions.^1,2 During the last years, nitric oxide (NO) has gained an increasing role as an essential player in plant defense responses³ as well as a co-regulator of many developmental processes.⁴ However, studies of NO function as a regulatory molecule in plants have been hampered by the scanty, limited and controversial knowledge on how this gas is synthesized in plants.^5,6 This situation has moved researchers in this area to adopt pharmacological approaches based on chemicals acting as artificial NO donors as well as inhibitors or scavengers of NO action. The lack of specificity and the inherent artificial effects of these chemicals can be overcome by genetic approaches based on the use of mutants with endogenous low levels of NO. In February 2010 issue of Plant Physiology, we report the generation and further characterization of a triple nia1nia2noa1-2 mutant that contains extremely low levels of NO due to the impairment of two NO biosynthetic pathways involving nitrate reductase (NIA/NR) or NO Associated 1 (AtNOA1) proteins.⁷ These findings support that NO is mainly produced through those pathways in Arabidopsis. However, the possible existence of a minor still uncharacterized pathways involved in the residual production of NO can not be ruled out at this time.Further functional characterization of nia1nia2noa1-2 mutant in terms of development has pointed to NO as an overall positive regulator of plant growth, affecting to almost every developmental stage from seed germination to reproductive development. Accordingly, triple mutant plants display a delayed growth resulting in small shoot and root size and they also produce low amounts of viable seeds.⁷Dormancy and seed germination are developmental programs largely regulated by the combined action of GA and ABA.¹ GA promote breaking of dormancy and promote germination whereas ABA acts as a brake in those processes, thus ensuring a timely seed germination. Our data from the characterization of dormancy and seed germination in the nia1nia2noa1-2 mutant suggest that NO’s role in the control of those processes may be exerted through modulation of the sensitivity to ABA (Fig. 1A). Seeds from NO deficient plants have increased dormancy and lower seed germination and seedling establishment rates than wild type seeds due to the enhanced ABA inhibitory action. These effects can be reversed by exogenous application of NO to nia1nia2noa1-2, suggesting that the sensitivity to ABA is actually controlled by the endogenous levels of NO. The recent identification of PYR/PYL/RCAR family of ABA receptors,^8,9 and the further characterization of the essential ABA regulatory module including receptor, protein phosphatases of the 2C class and kinases of the SnRK2 family¹⁰ point to these components as potential targets of NO in regulating sensitivity to ABA (Fig. 1B). This work is in progress in our lab but we already know that some of the PYR/PYL/RCAR receptors and SnRKs are actually regulated by NO and also that this regulation may be exerted at different levels (Lozano-Juste J and León J, unpublished data).Open in a separate windowFigure 1Interactions between NO and ABA results in modulated sensitivity to ABA throughout development. (A) NO synthesized through nitrate reductase (NR/NIA) and NO associatedI (AtNOA1) protein regulate germinative and post-germinative development as well as stomata movements through modulation of the sensitivity to ABA. Arrows and bars represent positive and negative effects, and the thickness of lines are proportional to the magnitud of regulatory effects. (B) Scheme of a minimal ABA signalling module and the potential targets of NO. Dashed lines represent effects still to be demonstrated. (C) ABA signalling in stomata guard cells through Ca²⁺-dependent and -independent pathways and the potential interactions with NO as represented by dashed lines.The enhanced sensitivity to ABA observed in germinative and post-germinative development of nia1nia2noa1-2, is extended throughout plant life cycle and it is actually the cause of the very strong resistance of nia1nia2noa1-2 plants to water deficit conditions.⁷ Stomatal aperture is a fine-tuned process controlled mainly through a balance between the light-promoted opening and the ABA-mediated promotion of closure and inhibition of opening¹¹ (Fig. 1A). It has been previously reported that ABA function on stomata movements involve the participation of NO as well as Ca²⁺ in such a way that Ca²⁺ chelators and NO scavengers block ABA action on stomata movements.¹² Stomata of nia1nia2noa1-2 leaves, despite of being depleted of NO, are not impaired for ABA inhibition of stomata opening but, in turn, they seem to be primed for a more efficient ABA response (Fig. 1A). Contrary to the Ca²⁺ requirements for ABA action on wild type stomata movements, this process is not affected by Ca²⁺ chelators in nia1nia2noa1-2 stomata, and it thus seems to be independent of Ca²⁺ in NO-deficient backgrounds (Fig. 1C). As mentioned above, NO might regulate sensitivity to ABA by acting on ABA receptors or on SnRKs, some of which are Ca²⁺-independent kinases. Both receptors and Ca^2+--independent kinases are likely targets of NO in the modulation of stomata sensitivity to ABA thus explaining the more efficient stomata closure in nia1nia2noa1-2 leaves, and the consequent low rates of evapotranspiration that leads to the extreme resistance of triple mutant plants to drought.The future characterization of the interactions between NO and key components of ABA signaling will be the basis for a better knowledge of the functional interactions between different hormones in plant development and defense, but it will also open up new possibilities of identifying new targets and strategies leading to improved drought resistance.     

Nitric oxide induces oxidative stress and apoptosis in neuronal cells

Within the central nervous system and under normal conditions, nitric oxide (NO) is an important physiological signaling molecule. When produced in large excess, NO also displays neurotoxicity. In our previous report, we have demonstrated that the exposure of neuronal cells to NO donors induced apoptotic cell death, while pretreatment with free radical scavengers L-ascorbic acid 2-[3, 4-dihydro-2,5,7,8-tetramethyl-2-(4,8, 12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K1) or superoxide dismutase attenuated apoptosis effectively, suggesting that reactive oxygen species (ROS) may be involved in the cascade of events leading to apoptosis. In the present investigation, we directly studied the kinetic generation of ROS in NO-treated neuronal cells by flow cytometry using 2', 7'-dichloro-fluorescein diacetate and dihydrorhodamine 123 as redox-sensitive fluorescence probes. The results indicated that exposure of cerebellar granule cells to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) induced oxidative stress, which was characterized by the accumulation of cytosolic and mitochondrial ROS, the increase in the extracellular hydrogen peroxide level, and the formation of lipid peroxidation products. SNAP treatment also induced apoptotic cell death as confirmed by the formation of cytosolic mono- and oligonucleosomes. Pretreating cells with the novel antioxidant EPC-K1 effectively prevented oxidative stress induced by SNAP, and attenuated cells from apoptosis.     

Nitric oxide, induced by wounding, mediates redox regulation in pelargonium leaves

The subject of this study was the participation of nitric oxide (NO) in plant responses to wounding, promoted by nicking of pelargonium ( Pelargonium peltatum L.) leaves. Bio-imaging with the fluorochrome 4,5-diaminofluorescein diacetate (DAF-2DA) and electrochemical in situ measurement of NO showed early (within minutes) and transient (2 h) NO generation after wounding restricted to the site of injury. In order to clarify the functional role of NO in relation to modulation of the redox balance during wounding, a pharmacological approach was used. A positive correlation was found between NO generation and regulation of the redox state. NO caused a slight restriction of post-wounded O₂⁻ production, in contrast to the periodic and marked increase in H₂O₂ level. The observed changes were accompanied by time-dependent inhibition of catalase (CAT) and ascorbate peroxidase (APX) activity. The effect was specific to NO, since the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) reversed the inhibition of CAT and APX, as well as temporarily enhancing H₂O₂ synthesis. Finally, cooperation of NO/H₂O₂ restricted the depletion of the low-molecular weight antioxidant pool ( i.e . ascorbic acid and thiols) was positively correlated with sealing and reconstruction changes in injured pelargonium leaves ( i.e . lignin formation and callose deposition). The above results clearly suggest that NO may promote restoration of wounded tissue through stabilisation of the cell redox state and stimulation of the wound scarring processes.     

Nitric oxide enhances catechol estrogen-induced oxidative stress in LNCaP cells

Catechol estrogens (CEs), such as 4-hydroxyestradiol (4-OHE2), undergo redox cycling during which reactive oxygen species (ROS) such as superoxide (O2*-) and the chemically reactive estrogen semiquinone (CE-SQ) and quinone (CE-Q) intermediates are produced. The quinone's putative mutagenicity may be enhanced by ROS and/or reactive nitrogen species. High concentrations of nitric oxide (NO) present during inflammatory conditions may react with (O2*-) to form peroxynitrite (ONOO-), a potent oxidant implicated in many pathological conditions. In this study, the possible generation of peroxynitrite from the interaction of CEs and NO and its effect on plasmid DNA and intact cells were investigated. A combination of 4-OHE2 and NO increased the level of single strand breaks (SSB) in plasmid DNA by more than 60% compared to vehicle controls in a metal-free buffer system. 4-OHE2 alone or NO alone had no effect. Results obtained from use of different antioxidants and ROS scavengers suggested a role of peroxynitrite in oxidative stress. In cells, 4-OHE2 or NO alone induced dose-dependent DNA damage as assessed by single cell gel electrophoresis. Co-treatment with 4-OHE2 and NO had an additive effect at lower doses. Generation of intracellular ROS was measured by the oxidation of carboxy-2',7'-dichlorofluorescein diacetate to the fluorescent compound carboxy-2',7'-dichlorofluorescein. NO alone, in oxygenated media, generated little ROS whereas 4-OHE2 produced approximately 70% increase in fluorescence. When added together 4-OHE2 and NO, produced a 2-fold increase in ROS. The generation and involvement ofperoxynitrite to this increase was implied since uric acid inhibited it. Generation ofperoxynitrite was also observed by use of dihydrorhodamine 123. Therefore, we conclude that combined treatments with 4-OHE2 and NO generated peroxynitrite seen from increased fluorescence and its inhibition by uric acid or combined SOD and catalase treatments. Results reported here suggest a role of peroxynitrite in causing damage to biomolecules when CEs and NO are present simultaneously. This may have biological relevance as high concentrations of NO formed during inflammatory conditions may exacerbate cancers due to estrogens.     

Nitric oxide is involved in the regulation of ascorbate and glutathione metabolism in Agropyron cristatum leaves under water stress

This study investigated the regulation of ascorbate and glutathione metabolism by nitric oxide in Agropyron cristatum leaves under water stress. The activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), L-galactono-1,4-lactone dehydrogenase (GalLDH) and γ-glutamylcysteine synthetase (γ-ECS), and the contents of NO, reduced ascorbic acid (AsA), reduced glutathione (GSH), total ascorbate and total glutathione increased under water stress. These increases were suppressed by pretreatments with NO synthesis inhibitors N ^G-nitro-L-arginine methyl ester (L-NAME) and 4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). However, application of L-NAME and cPTIO to plants sufficiently supplied with water did not affect the activities of above mentioned enzymes and the contents of NO and above mentioned antioxidants. Pretreatments with L-NAME and cPTIO increased the malondialdehyde (MDA) content and electrolyte leakage of plants under water stress. Our results suggested that water stress-induced NO is a signal that leads to the upregulation of ascorbate and glutathione metabolism and has important role for acquisition of water stress tolerance.     

Nitric oxide alleviates oxidative damage induced by high temperature stress in wheat

Effect of sodium nitroprusside (SNP), a donor of nitric oxide (NO) was examined in two wheat (Triticum aestivum L.) cultivars, C 306 (heat tolerant) and PBW 550 (comparatively heat susceptible) to study the extent of oxidative injury and activities of antioxidant enzyme in relation to high temperature (HT) stress. HT stress resulted in a marked decrease in membrane thermostability (MTS) and 2, 3, 5-triphenyl tetrazolium chloride (TTC) cell viability whereas content of lipid peroxide increased in both the cultivars. The tolerant cultivar C 306 registered less damage to cellular membranes compared to PBW 550 under HT stress. Activities of antioxidant enzymes viz, superoxide dismutase, catalase, ascorbate peroxidase, guaicol peroxidase and glutathione reductase increased with HT in both the cultivars. Following treatment with SNP, activities of all antioxidant enzymes further increased in correspondence with an increase in MTS and TTC. Apparently, lipid peroxide content was reduced by SNP more in shoots of heat tolerant cultivar C 306 indicating better protection over roots under HT stress. The up-regulation of the antioxidant system by NO possibly contributed to better tolerance against HT induced oxidative damage in wheat.     

Aluminum-induced oxidative stress in maize

The relation between Al-toxicity and oxidative stress was studied for two inbred lines of maize (Zea mays L.), Cat100-6 (Al-tolerant) and S1587-17 (Al-sensitive). Peroxidase (PX), catalase (CAT) and superoxide dismutase (SOD) activities were determined in root tips of both lines, exposed to different Al(3+) concentrations and times of exposure. No increases were observed in CAT activities in either line, although SOD and PX were found to be 1.7 and 2.0 times greater than initial levels, respectively, in sensitive maize treated with 36 microM of Al(3+) for 48 h. The results indicate that Al(3+) induces the dose- and time dependent formation of reactive oxygen species (ROS) and subsequent protein oxidation in S1587-17, although not in Cat100-6. After exposure to 36 microM of Al(3+) for 48 h, the formation of 20+/-2 nmol of carbonyls per mg of protein was observed in S1587-17. The onset of protein oxidation took place after the drop of the relative root growth observed in the sensitive line, indicating that oxidative stress is not the primary cause of root growth inhibition. The presence of Al(3+) did not induce lipid peroxidation in either lines, contrasting with the observations in other species. These results, in conjunction with the data presented in the literature, indicate that oxidative stress caused by Al may harm several components of the cell, depending on the plant species. Moreover, Al(3+) treatment and oxidative stress in the sensitive maize line induced cell death in root tip cells, an event revealed by the high chromatin fragmentation detected by TUNEL analysis.     

Isolation and characterization of maize PMP3 genes involved in salt stress tolerance

Plasma membrane protein 3 (PMP3), a class of small hydrophobic polypeptides with high sequence similarity, is responsible for salt, drought, cold, and abscisic acid. These small hydrophobic ploypeptides play important roles in maintenance of ion homeostasis. In this study, eight ZmPMP3 genes were cloned from maize and responsive to salt, drought, cold and abscisic acid. The eight ZmPMP3s were membrane proteins and their sequences in trans-membrane regions were highly conserved. Phylogenetic analysis showed that they were categorized into three groups. All members of group II were responsive to ABA. Functional complementation showed that with the exception of ZmPMP3-6, all were capable of maintaining membrane potential, which in turn allows for regulation of intracellular ion homeostasis. This process was independent of the presence of Ca(2+). Lastly, over-expression of ZmPMP3-1 enhanced growth of transgenic Arabidopsis under salt condition. Through expression analysis of deduced downstream genes in transgenic plants, expression levels of three ion transporter genes and four important antioxidant genes in ROS scavenging system were increased significantly in transgenic plants during salt stress. This tolerance was likely achieved through diminishing oxidative stress due to the possibility of ZmPMP3-1's involvement in regulation of ion homeostasis, and suggests that the modulation of these conserved small hydrophobic polypeptides could be an effective way to improve salt tolerance in plants.     

Differential expression of candidate genes for lignin biosynthesis under drought stress in maize leaves

In order to provide information for the development of molecular selection markers for drought tolerance improvement, the methods of prometric analysis, quantitative real-time PCR and field evaluation were employed for the identification of the differential expression of candidate genes under drought stress in maize. At seventeen, twenty-four and forty-eight hours of polyethylene glycol-simulated drought stress at the seventh leaf stage, leaf samples were collected from two drought-tolerant inbred lines for prometric analysis by two-dimensional electrophoresis and peptide mass fingerprinting. Fifty-eight proteins out of more than 500 were found in response to drought stress. Three drought-induced spots 2506, 3507 and 4506 showed sequence similarity with cinnamyl alcohol dehydrogenase, cytochrome protein 96A8 and S-adenosyl-L-methionine synthase, respectively. The expression of two key enzymes to lignin biosynthesis was quantified by quantitative real-time PCR among three drought-tolerant and one drought-sensitive inbred lines under drought stress and well-watered control conditions. After a decrease at the beginning of drought stress, the expression of cinnamyl alcohol dehydrogenase and caffeateO-methyltransferase recovered at twenty-four hours of the drought stress in the three drought-tolerant lines, but not in the drought-sensitive lines. Leaf lignin content, anthesis-silking interval and grain weight per plant were investigated with six inbred lines of varying drought tolerance under drought stress and well-watered control. Drought tolerance coefficients of these three characters were calculated and the correlation coefficients among these drought tolerance coefficients were estimated. Significant difference in leaf lignin content was found among the inbred lines and in response to drought stress. Close correlations were observed between the drought tolerant coefficients for leaf lignin content and grain weight per plant, and between the drought tolerant coefficients for leaf lignin content and anthesis-silking interval. These results indicate that leaf lignin content is a useful index for evaluation of drought tolerance in maize. Molecular selection markers can be developed on the basis of differential expression of the candidate genes and applied to maize improvement for drought tolerance.     

Isoprene and nitric oxide reduce damages in leaves exposed to oxidative stress

Isoprene and nitric oxide (NO) are two volatile molecules that are produced in leaves. Both compounds were suggested to have an important protective role against stresses. We tested, in two isoprene-emitting species, Populus nigra and Phragmites australis, whether: (1) NO emission outside leaves is measurable and is affected by oxidative stresses; and (2) isoprene and NO protect leaves against oxidative stresses, both singularly and in combination. The emission of NO was undetectable, and the compensation point was very low in control poplar leaves. Both emission and compensation point increased dramatically in stressed leaves. NO emission was inversely associated with stomatal conductance. More NO was emitted in leaves that were isoprene-inhibited, and more isoprene was emitted when NO was reduced by NO scavenger c-PTIO. Both isoprene and NO reduced oxidative damages. Isoprene-emitting leaves which were also fumigated with NO, or treated with NO donor, showed low damage to photosynthesis, a reduced accumulation of H(2)O(2) and a reduced membrane denaturation. We conclude that measurable amounts of NO are only produced and emitted by stressed leaves, that both isoprene and NO are effective antioxidant molecules and that an additional protection is achieved when both molecules are released.