Cytokinins in plant senescence: From spray and pray to clone and play

Three approaches have been used to investigate the inhibitory role of the cytokinin class of phytohormones in plant senescence: external application of cytokinins, measurement of endogenous cytokinin levels before and during senescence, and manipulation of endogenous cytokinin production in transgenic plants. In transgenic plant studies, endogenous cytokinin levels are manipulated by expression of IPT, a gene encoding isopentenyl transferase. Transgenic plants expressing IPT from a variety of promoters exhibit developmental and morphological alterations and often display retarded leaf senescence. A recently developed autoregulatory senescence-inhibition system targets cytokinin production quantitatively, spatially and temporally, and results in transgenic plants that exhibit significantly delayed senescence without abnormalities. These transgenic studies not only confirm the regulatory role of cytokinins in plant senescence, but also provide a way to manipulate senescence for potential agricultural applications.     

The effects of decapitation on the distribution of cytokinins and growth of Phaseolus vulgaris plants

The growth of the primary leaves of Phaseolus vulgaris L. was enhanced greatly by decapitation of the rest of the shoot. This increased growth was manifested by an increase in leaf area, leaf weight, and in a higher synthesis of chlorophyll and soluble proteins. Within the roots and stems decapitation resulted in a detectable increase in the endogenous cytokinins within 2 days after the surgical treatment. In the primary leaves increased cytokinin levels were only detected after 16 days. At this time most of the recorded activity co-chromatographed with the cytokinin glucosides. When plants which were decapitated were left under normal growing conditions for 16 days and then transferred to continuous darkness for 8 days the senescence of the primary leaves of the decapitated plants, in which the cytokinins had increased, was delayed significantly when compared with that of the primary leaves of the intact plants. the significance of these findings is discussed.     

Cytokinin biochemistry in relation to leaf senescence. VII. Endogenous cytokinin levels and exogenous applications of cytokinins in relation to sequential leaf senescence of tobacco

The cytokinin complex in tobacco leaves of various maturities was characterized by radioimmunoassay and mass spectrometry. Zeatin was the major base, whereas zeatin riboside was identified as the main riboside. in leaves of all maturities studied. Relative to upper younger leaves, the basal yellow leaves had reduced levels of both cytokinin bases and ribosides. Exogenous applications of dihydrozeatin and zeatin to detached tobacco leaves in amounts sufficient to delay senescence, elevated cytokinin base and riboside levels 2–5 fold. Presenescent and senescent leaves of intact plants showed quantitatively similar changes in cytokinin content. which therefore appear to be of significance in control of senescence. When supplied exogenously, the principal cytokinin bases found to occur in tobacco leaves (zeatin and dihydrozeatin) were markedly more effective than auxins and gibberellic acid in retarding senescence. Localised application of cytokinins to leaf blades of detopped plants was much less effective than application to intact plants. The cytokinin induced senescence retardation in tobacco leaves was independent of effects on directed metabolite transport. Evidence that endogenous levels of active cytokinins in intact tobacco leaves are involved in control of sequential leaf senescence is discussed.     

Influence of nitrogen supply and sink strength on changes in leaf nitrogen compounds during senescence in two wheat cultivars

Changes in various nitrogen compounds during senescence of the fourth leaf were studied in two cultivars of spring wheat (Triticum aestivum L.). One of the cultivars (Yecora) was supplied with two N levels; the other (Tauro) was grown with the high N level and pruned above the fourth leaf, whereas the control was left intact. In both cultivars grown with high N supply, net nitrogen export from the fourth leaf did not occur until 35 days after sowing (DAS). Loss of leaf soluble proteins started earlier than that of chlorophylis, and coincided initially with an increase in insoluble protein. In N deficient plants the level of total N, soluble protein, and the activity of nitrate reductase (NRA. EC 1.6.6.1) started to decrease about 5 days earlier, and along with chlorophyll, continued to decrease at a faster rate, than in high N plants. Also, with low N supply, the large subunit (LSU, 58 kDa) of ribulose-1.5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) decreased in greater proportion than other soluble proteins, while with high N supply the decrease in Rubisco LSU was similar to that of other soluble proteins. Nitrogen deficiency caused a greater decrease in soluble proteins than in insoluble proteins, and NRA relative to soluble proteins. The faster senescing Tauro cultivar had lower levels of most parameters, especially NRA, soluble protein and, after 35 DAS. Rubisco LSU as a proportion of soluble protein. The decrease in sink strength due to shoot pruning did generally not affect the level of the various nitrogenous compounds until 35 DAS; thereafter the levels of most parameters, especially soluble protein, Rubisco LSU and, at late stages of senescence, insoluble protein, were higher in pruned than in control shoots. Thus, shoot pruning slows down senescence. The 56- and 78-kDa polypeptides increased, rather than decreased, with leaf age; the level of these two polypeptides showed a negative relationship with Rubisco LSU (r = -0.933 and r = -0.758, respectively).     

Antioxidant enzymatic protection during tobacco leaf ageing is affected by cytokinin depletion

Plant ageing and senescence are associated with increased levels of reactive oxygen species. Level of cytokinins, the apparent inhibitors of plant senescence, is controlled by their irreversible degradation catalysed by cytokinin oxidase/dehydrogenase (CKX). We investigated the CKX activity, cytokinin concentration, and activities of antioxidative enzymes in tobacco (Nicotiana tabacum L. cv. Samsun NN) overexpressing the Arabidopsis gene for AtCKX2, targeted for extracellular secretion pathway. The control and AtCKX2 plants differed substantially in their phenotypes. When the lowest leaves in controls became yellow all leaves in AtCKX2 tobacco still remained green. Activities of antioxidant enzymes decreased with leaf age in both tobacco plants except for ascorbate peroxidase (APX) in the old leaves and glutathione reductase (GR) in young leaves. Enhancement of GR activity at all leaf stages, an increase of superoxide dismutase and a decline of catalase in young leaves, as well as an increase of APX in the oldest leaves were observed in AtCKX2 plant compared to control. Similar changes were detected after determination of isoenzymes on zymograms. It is evident that AtCKX2 plants had postponed onset of senescence despite the significantly lowered level of cytokinins. Enhanced antioxidant protection, especially in the oldest leaves, could subsidise this phenomenon.     

Expression of a nitric oxide degrading enzyme induces a senescence programme in Arabidopsis

Nitric oxide (NO) has been proposed to act as a factor delaying leaf senescence and fruit maturation in plants. Here we show that expression of a NO degrading dioxygenase (NOD) in Arabidopsis thaliana initiates a senescence-like phenotype, an effect that proved to be more pronounced in older than in younger leaves. This senescence phenotype was preceded by a massive switch in gene expression in which photosynthetic genes were down-regulated, whereas many senescence-associated genes (SAGs) and the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene ACS6 involved in ethylene synthesis were up-regulated. External fumigation of NOD plants with NO as well as environmental conditions known to stimulate endogenous NO production attenuated the induced senescence programme. For instance, both high light conditions and nitrate feeding reduced the senescence phenotype and attenuated the down-regulation of photosynthetic genes as well as the up-regulation of SAGs. Treatment of plants with the cytokinin 6-benzylaminopurin (BAP) reduced the down-regulation of photosynthesis, although it had no consistent effect on SAG expression. Metabolic changes during NOD-induced senescence comprehended increases in salicylic acid (SA) levels, accumulation of the phytoalexin camalexin and elevation of leaf gamma-tocopherol contents, all of which occurred during natural senescence in Arabidopsis leaves as well. Moreover, NO fumigation delayed the senescence process induced by darkening individual Arabidopsis Columbia-0 (Col-0) leaves. Our data thus support the notion that NO acts as a negative regulator of leaf senescence.     

Senescence-Inducible Expression of Isopentenyl Transferase Extends Leaf Life, Increases Drought Stress Resistance and Alters Cytokinin Metabolism in Cassava

Cassava (Manihot esculenta Crantz) sheds its leaves during growth, especially within the tropical dry season. With the production of SAG12-IPT transgenic cassava we want to test the level of leaf retention and altered cytokinin metabolism of transgenic plants via the autoregulatory senescence inhibition system. After confirmation of transgene expression by molecular analysis and phenotype examination in greenhouse plants, two transgenic plant lines, 529-28 and 529-48, were chosen for further investigation. Detached mature leaves of 529-28 plants retained high levels of chlorophyll compared with wild-type leaves after dark-induced senescence treatment. Line 529-28 showed significant drought tolerance as indicated by stay-green capacity after drought stress treatment. Field experiments proved that leaf senescence syndrome was significantly delayed in 529-28 plants in comparison with wild-type and 529-48 plants. Physiological and agronomical characterizations of these plants also revealed that the induced expression of IPT had effects on photosynthesis, sugar allocation and nitrogen partitioning. Importantly, the 529-28 plants accumulated a high level of trans-zeatin-type cytokinins particularly of corresponding storage O-glucosides to maintain cytokinin homeostasis. Our study proves the feasibility of prolonging the leaf life of woody cassava and also sheds light on the control of cytokinin homeostasis in cassava leaves.     

The role of reactive nitrogen species in secondary spinal cord injury: formation of nitric oxide, peroxynitrite, and nitrated protein

To determine whether reactive nitrogen species contribute to secondary damage in CNS injury, the time courses of nitric oxide, peroxynitrite, and nitrotyrosine production were measured following impact injury to the rat spinal cord. The concentration of nitric oxide measured by a nitric oxide-selective electrode dramatically increased immediately following injury and then quickly declined. Nitro-L-arginine reduced nitric oxide production. The extracellular concentration of peroxynitrite, measured by perfusing tyrosine through a microdialysis fiber into the cord and quantifying nitrotyrosine in the microdialysates, significantly increased after injury to 3.5 times the basal level, and superoxide dismutase and nitro-L-arginine completely blocked peroxynitrite production. Tyrosine nitration examined immunohistochemically significantly increased at 12 and 24 h postinjury, but not in sham-control sections. Mn(III) tetrakis(4-benzoic acid)-porphyrin (a novel cell-permeable superoxide dismutase mimetic) and nitro-L-arginine significantly reduced the numbers of nitrotyrosine-positive cells. Protein-bound nitrotyrosine was significantly higher in the injured tissue than in the sham-operated controls. These results demonstrate that traumatic injury increases nitric oxide and peroxynitrite production, thereby nitrating tyrosine, including protein-bound tyrosine. Together with our previous report that trauma increases superoxide, our results suggest that reactive nitrogen species cause secondary damage by nitrating protein through the pathway superoxide + nitric oxide peroxynitrite protein nitration.     

Influence of mycorrhizae and Rhizobium on cytokinin content in drought-stressed alfalfa

The objective of this research was to study the growth responseto drought of arbuscular mycorrhizal and non-mycorrhizal alfalfa(Medicago sativacv. Aragn) in relation to leaf cytokinin levels.In the experiment, four treatments were used: (a) plants inoculatedwith Clomus fasciculatum (Taxter sensu Gerd.) Gerdemann andTrappe and Rhizobium meliloti 102 F51 strain, (MR); (b) plantsinoculated with only Rhizobium (RP); (c) plants inoculated withonly mycorrhizae (MN); and (d) plants non-inoculated (NP). Non-mycorrhizalplants were supplemented with phosphorus and nonnodulated oneswith nitrogen to achieve similar size in all treatments. Plantswere subjected to drought by withholding irrigation in a cyclicway. The effects of drought on growth, number of stems, degreeof senescence, and leaf cytokinin levels were measured. Results of identification of cytokinins showed that dihydrozeatinriboside (dHZR) and ortno-topolin riboside (oTR) were predominantin alfalfa leaves. Nonsymbiotic plants (NP) showed higher totalcytokinin concentrations (dHZR and oTR). Under drought, NP plantsshowed the largest percentage drop in cytokinins and lower numberof stems as well as increased degree of senescent leaf tissuerelative to control values. By contrast, stressed symbioticplants (RP, MN and MR) showed higher green leaf weight thannonsymbiotic ones (NP) due to delay of leaf senescence and maintenance(RP) or increase (MN, MR) of stem leaf cytokinin levels duringdrought. The relationships between growth and the different cytokininsare discussed, suggesting an important role of mycorrhizal symbiosisin maintaining cytokinin levels under drought. Key words: Alfalfa, arbuscular mycorrhizae, cytokinins, drought, leaf senescence     

Delayed Leaf Senescence in Tobacco Plants Transformed with tmr,a Gene for Cytokinin Production in Agrobacterium

The aim of this study was to investigate whether enhanced levels of endogenous cytokinins could influence plant development, particularly leaf senescence. Tobacco plants were transformed with the Agrobacterium tumefaciens gene tmr, under the control of the soybean heat shock promoter HS6871. This gene encodes the enzyme isopentenyl transferase, which catalyzes the initial step in cytokinin biosynthesis. After heat shock, the cytokinin level increased greatly and the level of tmr mRNA, undetectable at 20[deg]C, rose and remained high for up to 8 hours. The levels of cytokinin and tmr mRNA were substantially lower by 24 hours. Transformed plants grown at 20[deg]C were shorter, had larger side shoots, and remained green for longer than untransformed plants. The differences were more pronounced after several heat shocks of whole plants or defined areas of leaves. Our results demonstrated that plant morphology and leaf senescence can be manipulated by changing the endogenous level of cytokinins.     

Role of roots in regulating the growth rate and cytokinin content in leaves

The role of roots in the enhancement of cytokinin content and leaf growth of Phaseolus vulgaris plants after decapitation and partial defoliation was investigated. Partial excision of the roots of plants which were decapitated above the primary leaf node resulted in a reduction of leaf growth and soluble proteins accumulation in the primary leaves. Roots excision was done at time of decapitation and repeated 8 days later. Endogenous cytokinins, known to be involved in enhancing shoot growth, accumulated in the leaves and stems of decapitated and partially defoliated plants. Lower levels of cytokinins were detected in the leaves of decapitated plants with only a partial root system. The level of cytokinins in the roots of decapitated plants was reduced by partial root excision. The growth and accumulation of cytokinins in leaves were, however, not totally suppressed by removing a large proportion of the roots. At the commencement of the experiment the stem had a higher cytokinin content than both the leaves and roots. This suggests that the stem could be an alternative source of cytokinins to the leaves. The cytokinin complement in the leaves of decapitated plants is not identical to that in the roots. It appears that cytokinins supplied by the roots are metabolized in the leaves, or that alternatively certain cytokinins are synthesized in the leaves themselves.     

Contrasting patterns of cytokinins between years in senescing aspen leaves

Cytokinins are plant hormones that typically block or delay leaf senescence. We profiled 34 different cytokinins/cytokinin metabolites (including precursors, conjugates and degradation products) in leaves of a free‐growing mature aspen (Populus tremula) before and after the initiation of autumnal senescence over three consecutive years. The levels and profiles of individual cytokinin species, or classes/groups, varied greatly between years, despite the fact that the onset of autumn senescence was at the same time each year, and senescence was not associated with depletion of either active or total cytokinin levels. Levels of aromatic cytokinins (topolins) were low and changed little over the autumn period. Diurnal variations and weather‐dependent variations in cytokinin content were relatively limited. We also followed the expression patterns of all aspen genes implicated as having roles in cytokinin metabolism or signalling, but neither the pattern of regulation of any group of genes nor the expression of any particular gene supported the notion that decreased cytokinin signalling could explain the onset of senescence. Based on the results from this tree, we therefore suggest that cytokinin depletion is unlikely to explain the onset of autumn leaf senescence in aspen.     

Extracellular invertase is an essential component of cytokinin-mediated delay of senescence

Leaf senescence is the final stage of leaf development in which the nutrients invested in the leaf are remobilized to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, exogenous application of cytokinins or an increase of the endogenous concentration delays senescence and causes nutrient mobilization. The finding that extracellular invertase and hexose transporters, as the functionally linked enzymes of an apolasmic phloem unloading pathway, are coinduced by cytokinins suggested that delay of senescence is mediated via an effect on source-sink relations. This hypothesis was further substantiated in this study by the finding that delay of senescence in transgenic tobacco (Nicotiana tabacum) plants with autoregulated cytokinin production correlates with an elevated extracellular invertase activity. The finding that the expression of an extracellular invertase under control of the senescence-induced SAG12 promoter results in a delay of senescence demonstrates that effect of cytokinins may be substituted by these metabolic enzymes. The observation that an increase in extracellular invertase is sufficient to delay leaf senescence was further verified by a complementing functional approach. Localized induction of an extracellular invertase under control of a chemically inducible promoter resulted in ectopic delay of senescence, resembling the naturally occurring green islands in autumn leaves. To establish a causal relationship between cytokinins and extracellular invertase for the delay of senescence, transgenic plants were generated that allowed inhibition of extracellular invertase in the presence of cytokinins. For this purpose, an invertase inhibitor was expressed under control of a cytokinin-inducible promoter. It has been shown that senescence is not any more delayed by cytokinin when the expression of the invertase inhibitor is elevated. This finding demonstrates that extracellular invertase is required for the delay of senescence by cytokinins and that it is a key element of the underlying molecular mechanism.     

Nitric oxide in plants. To NO or not to NO

The current knowledge on the occurrence and activity of NO in plants is reviewed. The multiplicity of nitrogen monoxide species and implications for differentiated reactivity are indicated. Possible sources of NO are evaluated, and the evidence for the presence of nitric oxide synthase in plants is summarised. The regulatory role of NO. in plant development and in plant interactions with microorganisms, involving an interplay with other molecules, like ethylene or reactive oxygen species is demonstrated. Finally, some other suggestions on potential functions of NO. in plants are indicated.     

Cirrhotic patients with minimal hepatic encephalopathy have increased capacity to eliminate superoxide and peroxynitrite in lymphocytes,associated with cognitive impairment

Patients with minimal hepatic encephalopathy (MHE) show increased oxidative stress in blood. We aimed to assess whether MHE patients show alterations in different types of blood cells in (a) basal reactive oxygen and nitrogen species levels; (b) capacity to metabolise these species. To assess the mechanisms involved in the altered capacity to metabolise these species we also analysed: (c) peroxynitrite formation and d) peroxynitrite reaction with biological molecules. Levels of reactive oxygen and nitrogen species were measured by flow cytometry in blood cell populations from cirrhotic patients with and without MHE and controls, under basal conditions and after adding generators of superoxide (plumbagin) or nitric oxide (NOR-1) to assess the capacity to eliminate them. Under basal conditions, MHE patients show reduced superoxide and peroxynitrite levels and increased nitric oxide (NO) and nitrotyrosine levels. In patients without MHE plumbagin strongly increases cellular superoxide, moderately peroxynitrite and reduces NO levels. In MHE patients, plumbagin increases slightly superoxide and strongly peroxynitrite levels and affects slightly NO levels. NOR-1 increases NO levels much less in patients with than without MHE. These data show that the mechanisms and the capacity to eliminate cellular superoxide, NO and peroxynitrite are enhanced in MHE patients. Superoxide elimination is enhanced through reaction with NO to form peroxynitrite which, in turn, is eliminated by enhanced reaction with biological molecules, which could contribute to cognitive impairment in MHE. The data show that basal free radical levels do not reflect the oxidative stress status in MHE.     

Increased cytokinin levels in transgenic PSAG12–IPT tobacco plants have large direct and indirect effects on leaf senescence, photosynthesis and N partitioning

We studied the impact of delayed leaf senescence on the functioning of plants growing under conditions of nitrogen remobilization. Interactions between cytokinin metabolism, Rubisco and protein levels, photosynthesis and plant nitrogen partitioning were studied in transgenic tobacco (Nicotiana tabacum L.) plants showing delayed leaf senescence through a novel type of enhanced cytokinin syn‐thesis, i.e. targeted to senescing leaves and negatively auto‐regulated (P_SAG12–IPT), thus preventing developmental abnormalities. Plants were grown with growth‐limiting nitrogen supply. Compared to the wild‐type, endogenous levels of free zeatin (Z)‐ and Z riboside (ZR)‐type cytokinins were increased up to 15‐fold (total ZR up to 100‐fold) in senescing leaves, and twofold in younger leaves of P_SAG12–IPT. In these plants, the senescence‐associated declines in N, protein and Rubisco levels and photosynthesis rates were delayed. Senescing leaves accumulated more (¹⁵N‐labelled) N than younger leaves, associated with reduced shoot N accumulation (–60%) and a partially inverted canopy N profile in P_SAG12–IPT plants. While root N accumulation was not affected, N translocation to non‐senescing leaves was progressively reduced. We discuss potential consequences of these modified sink–source relations, associated with delayed leaf senescence, for plant productivity and the efficiency of utilization of light and minerals.     

Ethylene and Cytokinin in the Control of Senescence in Detached Leaves of Arabidopsis thaliana eti-5Mutant and Wild-Type Plants

We studied the effects of cytokinin benzyladenine (BA) and ethylene on the senescence in the dark of detached leaves of Arabidopsis thaliana(L.) Heynh wild-type plants and theeti-5mutant, which was described in the literature as the ethylene-insensitive one. Leaf senescence was assessed from a decrease in the chlorophyll content. The content of endogenous cytokinins (zeatin and zeatin riboside) was estimated by the ELISA technique. We demonstrated that the content of endogenous cytokinins in the leaves of the three-week-old eti-5mutants exceeded that of the wild-type leaves by an order of magnitude; in the five-week-old mutants, by several times; and in the seven-week-old plants, the difference became insignificant. Due to the excess of endogenous cytokinins in the three–five-week-old mutant leaves, their senescence in the dark was retarded and exogenous cytokinin affected these leaves to a lesser extent. The seven-week-old mutant and the wild-type leaves, which contained practically similar amounts of endogenous cytokinins, did not differ in these indices. Thus, the level of endogenous cytokinins determined the rate of senescence and the leaf response to cytokinin treatment. Ethylene accelerated the senescence of detached wild-type leaves. Ethylene action increased with increasing its concentration from 0.1 to 100 l/l. BA (10^–6M) suppressed ethylene action. Similar data were obtained for the eti-5mutant leaves. We therefore suggest that the mutant leaves comprised the pathways of the ethylene signal reception and transduction, which provided for the acceleration of their senescence.     

Stem girdling influences concentrations of endogenous cytokinins and abscisic acid in relation to leaf senescence in cotton

Many studies have shown that root–shoot imbalance influences vegetative growth and development of cotton (Gossypium hirsutum L.), but few have examined changes in leaf senescence and endogenous hormones due to stem girdling. The objective of this study was to determine the correlation between some endogenous phytohormones, particularly cytokinins and abscisic acid (ABA), and leaf senescence following stem girdling. Field-grown cotton plants were girdled on the main stem 5 days after squaring (DAS), while the non-girdled plants served as control. Plant biomass, seed cotton yield, main-stem leaf photosynthetic (Pn) rate, chlorophyll (Chl) and malondialdehyde (MDA) concentrations, as well as levels of cytokinins and ABA in main-stem leaves and xylem sap were determined after girdling or at harvest. Main-stem girdling decreased the dry root weight and root/shoot ratio from 5 to 70 days after girdling (DAG) and reduced seed cotton yield at harvest. Main-stem leaf Pn and Chl concentration in girdled plants were significantly lower than in control plants. Much higher levels of MDA were observed in main-stem leaves from 5 to 70 DAG, suggesting that stem girdling accelerated leaf senescence. Girdled plants contained less trans-zeatin and its riboside (t-Z + t-ZR), dihydrozeatin and its riboside (DHZ + DHZR), and isopentenyladenine and its riboside (iP + iPA), but more ABA than control plants in both main-stem leaves and xylem sap. These results suggested that main-stem girdling accelerated leaf senescence due to reduced levels of cytokinin and/or increased ABA. Cytokinin and ABA are involved in leaf senescence following main-stem girdling.     

Augmentation of Nitric Oxide,Superoxide, and Peroxynitrite Production During Cerebral Ischemia and Reperfusion in the Rat

The effect of ischemia produced by bilateral occlusion of the common carotid arteries (30 min) followed by 4 hours of reperfusion on total and inducible nitric oxide synthase (NOS) activity and the production of nitric oxide (NO), superoxide and peroxynitrite in the cerebral hemispheres was determined in the rat. Compared to sham-operated controls, cerebral ischemia-reperfusion resulted in a significant increase in total and inducible NOS activity and a significant increase in the production of NO and superoxide in the cerebral hemispheres. The level of NO in the plasma and the peripheral leukocyte count were also significantly increased. Immunohistochemical staining for nitrotyrosine (a marker of peroxynitrite production) showed that ischemia-reperfusion resulted in increased synthesis of cerebral peroxynitrite. Administration of the irreversible NOS inhibitor, N-nitro-L-arginine (L-NA), increased superoxide levels in the brain and significantly reduced plasma NO. Total and inducible NOS activity as well as NO and immunoreactive nitrotyrosine, in the cerebral hemispheres were reduced with L-NA administration. The number of leukocytes in the plasma was unaffected by administration of L-NA. These findings suggest that cerebral ischemia-reperfusion causes increased production of reactive oxygen species in the cerebral hemispheres and that the production of peroxynitrite, and not superoxide, may be dependent upon the availability of NO.