The capacity of antioxidant protection during modulated ageing of bean (Phaseolus vulgaris L.) cotyledons. 1. The antioxidant enzyme activities

Reactive oxygen species are known to increase in plant senescence. We investigated the participation of antioxidative enzymes in initiation of cotyledon senescence. Senescence of bean (Phaseolus vulgaris L.) cotyledons was modulated by UV C irradiation and by the decapitation of plant apices. Senescence was accompanied by a decrease of protein content and by a decrease of photochemical efficiency. A drop in activity of antioxidative enzymes preceded the onset of senescence in control plants. In cotyledons with prolonged life span, the decrease of antioxidant activities and the markers of senescence onset appeared at a similar age as in controls. Thus we presumed that the period from senescence initiation to cotyledon abscission was extended. On the other hand, in UV C irradiated plants we did not observe actual senescence initiation, and antioxidant enzymes although elevated, did not effectively play their role. The decrease of antioxidant enzymes activity and the markers of senescence appeared at a similar age both in control and in decapitated (D) plants, so we can presume that we prolonged mainly the period from senescence onset to cotyledon abscission in D plants. In UV C irradiated plants the antioxidative enzymes were probably destroyed before the process of senescence could begin.     

The capacity of antioxidant protection during modulated ageing of bean (Phaseolus vulgaris L.) cotyledons. 2. The low-molecular weight antioxidants

The aim of the present work was to evaluate non-enzymic antioxidants during natural and artificially modulated senescence. Senescence of bean (Phaseolus vulgaris L. cv. Jantar) cotyledons was modulated by UV C irradiation or by the decapitation of plants apices. The content of beta-carotene and zeaxanthin decreased in control and decapitated plants but in UV C irradiated plants these contents increased. The degree of de-epoxidation increased in all cultivations with age. The content of total glutathione (sum of reduced and oxidized) sharply decreased in bean cotyledons grown in all conditions. Interestingly, the content of total ascorbate increased at the end of cotyledon life span of control and decapitated plants but decreased in UV plants. Decrease of reduced/oxidized ratio of ascorbate and glutathione during cotyledon ageing confirmed increasing oxidative stress during senescence in all cultivations.     

Changes in French bean cotyledon composition associated with modulated life-span

The onset of Phaseolus vulgaris L. cotyledon senescence and its characteristics were modulated by irradiance (higher or lower than standard) and by epicotyl decapitation. The cotyledon life-span of 16 d was not influenced by irradiance while decapitation prolonged the life-span to 28 d. The fresh mass of cotyledons, an indicator of organ viability, decreased in a similar manner in all non-decapitated plants, though it was relatively slower in plants grown under a low irradiance (LI). Three days after decapitation the fresh mass of cotyledons increased by one third, a slight decrease was observed on the 21st d, and it lasted until the end of the life span. Deducing from the fall of chlorophyll (Chl) concentration expressed per unit protein, senescence started after the 10th day in non-decapitated plants. Decapitation postponed the onset of senescence until the 21st day. Expression of Chl amount per unit dry mass did not detect any changes in LI plants, hence this parameter can not be used for the assessment of senescence. The measurements of Chl a and b concentrations indicated that the light-harvesting complexes (LHCs) proliferated during ageing and were rapidly destroyed at the onset of senescence. Changes of the concentrations of carotenoids supported the hypothesis of free radicals involvement in senescence. The bean cotyledons responded to free radical production induced under higher irradiance by increased -carotene synthesis. Oxidative damage to galactolipids during senescence was documented by fluorescence measurements. The changes in cotyledon composition were correlated to morphologic changes observed by electron microscopy.     

Age-dependence of the antioxidative system in tobacco with enhanced glutathione reductase activity or senescence-induced production of cytokinins

Tobacco leaves of plants with enhanced glutathione reductase activity (GR46-27, Nicotiana tabacum L. cv. Samsun) or with autoregulated senescence-induced production of cytokinins (P_SAG12-IPT, N. tabacum L. cv. Wisconsin) were studied during the course of leaf development and senescence by measuring photosynthesis, chlorophyll and protein content, the antioxidants ascorbate, glutathione and α -tocopherol as well as the antioxidative enzymes ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC 1.6.4.2) and superoxide dismutase (SOD, EC 1.15.1.1). The photosynthetic rate, as well as the chlorophyll and protein content, dropped with increasing leaf age after having reached a maximum at the end of the exponential growth phase. The concentrations of the water-soluble antioxidants ascorbate and glutathione fell continuously with age, whereas the concentration of the lipophilic α -tocopherol increased. The activities of the antioxidative enzymes APX, GR and SOD reached their maximum at the beginning of leaf development, but were reduced in senescing leaves. The age-dependent course of the measured leaf parameters in GR46-27 leaves was similar to the one in wild-type leaves, with the exception of an overall enhanced GR activity. In contrast, in old leaves of P_SAG12-IPT plants, which possess a much higher life span, the chlorophyll and protein content, the photosynthetic rate, the antioxidant concentrations of ascorbate and glutathione as well as the activities of the antioxidative enzymes were higher than in wild-type leaves. The results show that the capacity of the antioxidative system to scavenge radicals is sufficiently balanced with the plant metabolism, and its decline with increasing age is not the cause, but a consequence of senescence and ageing in plants.     

The Role of Cytokinins and Ethylene in Bean Cotyledon Senescence. The Effect of Free Radicals

During ageing of bean (Phaseolus vulgaris L.) cotyledons in plants with modified life span the time-course of four cytokinins, ethylene, and the end products of free radical attack, lipofuscin-like pigments (LFP), were studied. UV irradiation shortened cotyledon life span, while epicotyl decapitation prolonged it. In controls, LFP increased at the senescence onset but at the end of life span it returned to the initial level. Ethylene increased more than 3-fold at the time of abscission. The content of individual cytokinins (zeatin, zeatin riboside, isopentenyl adenine, isopentenyl adenine riboside) varied differently during ageing but they did not decreased in any case under level observed in young cotyledons at the time of abscission. UV irradiation resulted in 14-fold increase in LFP concentration at the end. Ethylene increased 8-fold 2 h after irradiation. Individual cytokinins increased after UV irradiation to a different extent and time-course, nevertheless cotyledon life span was shortened. Decapitation induced LFP decrease. On day 13, LFP abruptly increased and than decreased and stayed lowered until abscission. Ethylene was maximum on day 24, at the time of abscission, it was above 200 % of control. Decapitation produced transient decrease in some cytokinins namely zeatin and isopentenyl adenine riboside.     

Changes in French bean cotyledon composition associated with modulated life-span

The onset of Phaseolus vulgaris L. cotyledon senescence and its characteristics were modulated by irradiance (higher or lower than standard) and by epicotyl decapitation. The cotyledon life-span of 16 d was not influenced by irradiance while decapitation prolonged the life-span to 28 d. The fresh mass of cotyledons, an indicator of organ viability, decreased in a similar manner in all non-decapitated plants, though it was relatively slower in plants grown under a low irradiance (LI). Three days after decapitation the fresh mass of cotyledons increased by one third, a slight decrease was observed on the 21st d, and it lasted until the end of the life span. Deducing from the fall of chlorophyll (Chl) concentration expressed per unit protein, senescence started after the 10th day in non-decapitated plants. Decapitation postponed the onset of senescence until the 21st day. Expression of Chl amount per unit dry mass did not detect any changes in LI plants, hence this parameter can not be used for the assessment of senescence. The measurements of Chl a and b concentrations indicated that the light-harvesting complexes (LHCs) proliferated during ageing and were rapidly destroyed at the onset of senescence. Changes of the concentrations of carotenoids supported the hypothesis of free radicals involvement in senescence. The bean cotyledons responded to free radical production induced under higher irradiance by increased β-carotene synthesis. Oxidative damage to galactolipids during senescence was documented by fluorescence measurements. The changes in cotyledon composition were correlated to morphologic changes observed by electron microscopy.     

Changes in Ultrastructure of Phaseolus Vulgaris L. Cotyledons Associated with their Modulated Life Span

French bean (Phaseolus vulgaris L.) cotyledons lost most of their reserve substances during several early days of germination and turned green. In cotyledon mesophyll cells of one-week-old seedlings, plastids were represented predominantly by amyloplasts (starch grains) and chloroamyloplasts, and the cells appeared to be metabolically highly active. Cell heterogeneity associated with distance of the cells from cotyledon vascular bundles was evident. Only mesophyll cells near to the bundles were rich in plastids. In two-weeks-old intact bean plants, the cotyledons were yellow and shrunken, and their cells were nearly "empty". The plastids in them were represented by senescent plastids (gerontoplasts) only. In the gerontoplasts as well as freely in cytosol, fluorescent lipoid inclusions were accumulated. This cotyledon development was more or less independent of irradiance. In "decapitated" bean plants, senescence of mesophyll cells and plastids was slowed down considerably, and the life span of the cotyledons was prolonged.     

Involvement of Lipid Peroxidation in Water Stress-Promoted Senescence of Detached Rice Leaves

Role of lipid peroxidation and antioxidative enzymes (catalase, peroxidase, superoxide dismutase, ascorbate peroxidase and glutathione reductase) in water stress-promoted senescence of detached rice leaves was investigated. The senescence was followed by measuring the decrease in protein content. Increased lipid peroxidation was closely correlated with senescence in water stressed leaves. Decrease in superoxide dismutase activity was evident 8 h after beginning of water stress. However, decreased catalase, peroxidase, and ascorbate peroxidase activity was observed only when senescence was observed. Glutathione reductase was not affected by water stress. Free radical scavengers retarded water stress-enhanced senescence.     

Developmental changes of antioxidative systems in tobacco leaves as affected by limited sucrose export in transgenic plants expressing yeast-invertase in the apoplastic space

It is generally believed that a restricted export of carbohydrates from source leaves causes oxidative stress because of an enhanced utilisation of O₂ instead of NADP⁺ as electron acceptor in photosynthesis. To test this hypothesis, developmental changes of antioxidative systems were investigated in wild-type and transgenic tobacco (Nicotiana tabacum L.) suffering from disturbed sink-source relations by expression of yeast invertase in the apoplastic space. Young expanding leaves of the wild type contained higher activities of Superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11), catalase (EC 1.11.1.6), dehydroascorbate reductase (EC 1.8.5.1), glutathione reductase (EC 1.6.4.2) and a higher glutathione content than mature source leaves. The activity of monodehydroascorbate-radical reductase (EC 1.1.5.4) and the ascorbate content remained unaffected by the developmental stage in the wild type. In young expanding leaves of the transgenic plants the capacity of the antioxidative systems was similar to or higher than in corresponding leaves from the wild type. Source leaves of transgenic tobacco with an increased carbohydrate content showed a small chlorophyll loss, an increased malondialdehyde content, a selective loss of the activities of Cu/Zn-superoxide dismutase isoenzymes and a fourfold decrease in ascorbate compared with the wild type. There was no evidence that the protection from H₂O₂ was insufficient since source leaves of transgenic tobacco contained increased activities of catalase, ascorbate peroxidase, and monodehydroascorbate-radical reductase and an increased ascorbate-to-dehydroascorbate ratio compared with source leaves of the wild type. In severely chlorotic leaf sections of the transgenic plants, most components of the antioxidative system were lower than in green leaf sections, but the ascorbate-to-dehydroascorbate ratio was increased. These results suggest that carbohydrate-accumulating cells have an increased availability of reductant, which can increase the degree of reduction of the ascorbate system via glutathione-related systems or via the activity of monodehydroascorbate-radical reductase. At the same time, transgenic tobacco leaves seem to suffer from an increased oxidative stress, presumably as a result of a decreased consumption of O ₂ ^.- by Cu/Zn-superoxide dismutases in the chloroplasts. There was no evidence that carbohydrate-accumulating leaves acclimated to enhanced O ₂ ^.- production rates in the chloroplasts.     

Identification of nucleases related to nutrient mobilization in senescing cotyledons from French bean

The knowledge about the physiological function of plant nucleases is scarce besides that they have been involved in nucleic acid degradation related with programmed cell death processes. Cotyledons provide a suitable system to investigate this process and the changes associated to nutrient mobilization. Nuclease activities have been determined in French bean seedlings. The total nuclease activity in French bean cotyledons is lower than in embryonic axes; however, several nucleases were detected by in-gel nuclease activity assays with extracts from cotyledons of French bean and ssDNA as substrate. The nuclease activities induced during cotyledon senescence showed higher activity at neutral than at acidic pH. Five different nuclease genes belonging to S1/P1 family have been identified in French bean genome database named PVN1 to PVN5. Their relative expression in cotyledons has been determined from the start of imbibition to senescence, and three genes from this family showed expression in cotyledons. PVN1 was expressed during early stages of seedlings development, whereas PVN4 and PVN5 were expressed during cotyledons senescence. The removal of epicotyl in French bean seedlings resulted in a decrease in the activity and in the expression of the genes associated with the cotyledons senescence process, i.e. PVN4 and PVN5. At the same time, the mobilization of reserves in those cotyledons was slowed down. In the same way, the deficit in phosphate and nitrate during seedlings development led to an acceleration of induction of these genes at the same time that reserves were utilized early on the time. Therefore, the induction of PVN4 and PVN5, the two S1 nuclease genes involved in the process of cotyledon senescence, is related to nutrient mobilization, supporting a possible role for nucleic acids in nutrient recycling during cotyledon senescence.     

Selenium – an antioxidative protectant in soybean during senescence

Selenium (Se) is regarded as an antioxidant in animals and plants, even though considered as non-essential element in plants. To test its ability to counteract senescence related oxidative stress in soybean a pot culture experiment was conducted. The soybean plant was sprayed with sodium selenate (50ppm) at 78days after sowing (DAS). Soybean leaves were harvested at 80 and 90 DAS for analysis of oxidant production and antioxidative enzymes activity. Se positively promoted growth and acted as antioxidant by inhibiting lipid peroxidation and per cent injury of cell membrane. The antioxidative effect was associated with an increase in superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzymes activity. Significant increase in antioxidant enzyme activity was positively related to Se content. The decrease in antioxidative enzymes at 90 DAS was much faster in control plants than Se-sprayed plants. The reduction in SOD and GSH-Px may be associated with senescence-induced oxidative burst.     

Alleviation of salt stress by low dose γ-irradiation in rice

The effects of salt stress on the growth, photosynthesis, and antioxidative ability of the rice (Oryza sativa L.) plants raising from -irradiated seeds were investigated using two cultivars, Ilpumbyeo and Sanghaehyanghyella. The 50 and 100 mM NaCl solutions caused a remarkable decrease of the early germination rate and seedling growth. However, the salt stress-induced inhibition of the growth was significantly alleviated in the -irradiated plants. The chlorophyll contents and the effective quantum yield of photosystem 2 ( _{PS 2}) were lower in the NaCl-treated plants than in the control ones, while the non-photochemical quenching was higher in the former ones. Activities of the antioxidant enzymes such as superoxide dismutase (SOD) and ascorbate peroxidase (APX) increased with increasing NaCl concentrations, and the irradiated groups had even higher SOD and APX activities than the non-irradiated ones. These alleviation effects were observed similarly in both the cultivars tested.     

Antioxidative response of Lemna minor plants exposed to thallium(I)-acetate

The physiological effects of thallium(I)-acetate on the duckweed Lemna minor after 1-, 4-, 7- and 14-d exposure were analyzed. High bioaccumulation of Tl (221 mg kg⁻¹ dry wt at 2.0 μM Tl-acetate) caused an inhibition of plant growth. After 14-d exposure, 0.2, 0.5, 1.0 and 2.0 μM Tl-acetate reduced the frond-number growth rate by 21.1%, 39.4%, 66% and 83.1%, respectively. Tl-acetate also induced a modulation of the antioxidative response by depleting the ascorbate content and affecting the antioxidative enzymes activities. Superoxide dismutase showed a continuous increase of activity (31–67%) after Tl-acetate exposure. Other antioxidative enzymes displayed a biphasic response to both the concentration and the exposure period. Exposure up to 7 d decreased the catalase activity (up to 40%) in plants treated with higher Tl-acetate concentrations. In contrast, 14-d exposure increased the activity of the enzyme (≥90%). Short-term exposure increased ascorbate peroxidase activity (13–41%), except in plants exposed to the highest Tl-acetate concentration. However, 14-d exposure decreased the enzyme activity at all concentrations tested (38–60%). Although pyrogallol peroxidase activity increased (up to 26%) during 4-d exposure, longer exposures to the highest two concentrations decreased the activity of the enzyme (25–48%). In general, short-term exposure to Tl-acetate activated the antioxidant capacity, which resulted in recovery of the frond-number growth rates in Tl-treated plants. In spite of the activation of the antioxidative response during short-term exposure, higher Tl-acetate concentrations increased the hydrogen peroxide level (up to 45%) and induced marked oxidative damage to lipids, proteins and DNA. Longer exposure induced a decline of the antioxidative response, and plants showed the symptoms of oxidative damage even at lower Tl-acetate concentrations. The genotoxic effect was evaluated by an alkaline version of the cellular and acellular Comet assay, which revealed an indirect genotoxic effect of Tl-acetate, suggesting oxidatively induced damage to DNA.     

l-Canavanine Transport and Utilization in Developing Jack Bean, Canavalia ensiformis (L.) DC. [Leguminosae

l-Canavanine, the guanidinooxy structural analog of l-arginine, is an important nonprotein amino acid of many leguminous plants with nitrogen storage a major proported role. l-[Guanidinooxy-¹⁴C]canavanine, [¹⁴C] urea, and [¹⁵N]urea were injected separately into the fleshy, green cotyledons of 9-day old jack bean plants, Canavalia ensiformis (L.) DC. [Leguminosae]. There was significant transport of canavanine from the cotyledons to the aboveground portions of the plant, but not to the roots. Within 1.5 hours of isotope administration, the remaining labeled canavanine was divided equally between the cotyledons and the aboveground portions of the plant. During the 48-hour postinjection period, the contribution of l-[guanidinooxy-¹⁴C]canavanine to the total ¹⁴carbon of the cotyledons decreased rapidly while it increased in the aboveground portions of the plant.     

Antioxidative protection in the leaves of dark-senescing intact barley seedlings

In order to elucidate the possibility of in vivo oxidative modification of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) as a triggering mechanism for its preferential degradation early in senescence, some antioxidant compounds, protective enzymes, H₂O₂ and protein carbonylation levels were studied in the leaves during dark-induced senescence of barley (Hordeum vulgare L. cv. “Obzor”) seedlings. Analyses were performed in extracts as well as in purified chloroplasts. Some weakening of the antioxidative protection was detected during the treatment: diminution in the ascorbate and non-protein SH (mainly glutathione) pools, lower activities of superoxide dismutase, guaiacol and ascorbate peroxidases. However, no accumulation of H₂O₂ was found, lower level of protein carbonylation in darkness was measured and the percentage of reduced ascorbate was maintained high. Data concerning antioxidant compounds in chloroplasts revealed some impairment of the ascorbate and glutathione pools under induced senescence - the level of non-protein thiols declined during early senescence whereas the ascorbate pool was not significantly changed. The percentage of reduced ascorbate remained high in the chloroplasts and the activities of superoxide dismutase and of ascorbate peroxidase were conserved. Taken together the results are not in accordance with the possibility of in vivo oxidative modification of Rubisco in the case of dark-induced senescence. Our data bring some support to the view about redox regulation of Rubisco turnover in senescence through the pool of the low-molecular chloroplastic thiols.     

Changes in antioxidative enzymes in cucumber cotyledons during natural senescence: comparison with those during dark-induced senescence

Changes in 7 antioxidative enzymes in naturally senescent cotyledons of cucumber ( Cucumis sativus ) were investigated. The activities of superoxide dismutase (SOD; EC 1.15.1.1), catalase (EC 1.11.1.6), dehydroascorbate reductase (EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) gradually decreased during the progression of senescence, while those of ascorbate peroxidase (APX; EC 1.11.1.11) and guaiacol peroxidase (GPX; EC 1.11.1.7) gradually increased. The activity of monodehydroascorbate reductase (MDAR; EC 1.6.5.4) was not significantly changed. Western blot analysis showed that the protein level of mitochondrial SOD gradually declined. The protein level of catalase transiently decreased and then increased in the later stages of senescence, despite the decrease in its activity. The overall behavior was markedly different from that found in cotyledons of artificially senescing seedlings transferred into darkness; the activities of SOD, catalase, APX, GPX and GR gradually increased.     

Heterochromatin and repetitive DNA frequency variation in regenerated plants of Helianthus annuus L.

Plant regeneration from cotyledons of seeds of a single progeny of a pure line of Helianthus annuus was studied in respect of the nuclear DNA contents of control and regenerated plants. Control plants were divided into two groups: those developed from seeds at the periphery of the inflorescence (showing a high basic 4C DNA content) and those from seeds developed in the middle of the inflorescence (showing a low basic 4C DNA content). It was observed that plants from peripheral seeds have a higher morphogenetic potential than those from central seeds. Cytophotometric analyses indicated that plants regenerated from cotyledons of both peripheral and central seeds show the same basic 4C DNA amount, which is higher that that observed in vivo in peripheral seeds. Molecular analysis by slot blotting and hybridization with different DNA families showed that the difference in nuclear DNA content between plants from peripheral and central seeds in vivo are mainly related to differences in the frequency of highly repeated, slow medium repeated (MR2), and ribosomal DNA families; by contrast, the increase in DNA amount in regenerated plants is mainly due to fast medium repeated sequences (MR1). Moreover, the frequency of kinetically isolated unique sequences was higher in peripheral seeds than in central ones and still higher in regenerated plants. Optical-density measurements of interphase nuclei showed an increase of heterochromatin in regenerated plants, suggesting that, whatever DNA is amplified in these plants, it remains condensed and probably inactive.Research supported by National Research Council of Italy, Special Project RAISA, Sub-project No. 2, Paper No. 2069     

Occurrence of phytoalexins and other putative defense-related substances in uninfected parsley plants

Considerable amounts of the following substances were found in uninfected parsley (Petroselinum crispum) cotyledons: furanocoumarins, the putative phytoalexins of this and some related plant species, two enzymes of the furanocoumarin pathway (S-adenosyl-L-methionine: xanthotoxol and S-adenosyl-L-methionine: bergaptol O-methyltransferases), two hydrolytic enzymes (1,3--glucanase, EC 3.2.1.39, and chitinase, EC 3.2.1.14), and pathogenesis-related proteins. The furanocoumarins and the methyltransferase activities reached their highest levels at the onset of cotyledon senescence as the hydrolytic enzymes increased from low to relatively high activity values. The relative amounts of pathogenesis-related proteins 1 and 2, as well as the corresponding mRNAs, also increased markedly. Two enzymes of general phenylpropanoid metabolism, L-phenylalanine ammonia-lyase and 4-coumarate: CoA ligase, decreased in activity in a biphasic fashion during cotyledon development. At all developmental stages, the levels of these putative defense-related agents in total cotyledon extracts were too high to enable detection of, possibly, additional changes upon infection with zoospores of Phytophthora megasperma f. sp. glycinea, a fungal pathogen to which parsley shows a non-host, hypersensitive resistance response.Abbreviations BMT S-adenosyl-L-methionine: bergaptol O-methyltransferase (EC 2.1.1.-) - 4CL 4-coumarate: CoA ligase (EC 6.1.1.12) - CMT S-adenosyl-L-methionine: caffeate O-methyltransferase (EC 2.1.1.-) - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5) - PR pathogenesis-related - XMT S-adenosyl-L-methionine: xanthotoxin O-methyltransferase (EC 2.1.1.-)     

Comparative changes in the antioxidant system in the flag leaf of early and normally senescing near-isogenic lines of wheat (Triticum aestivum L.)

Key message

The antioxidant system was significantly inhibited in the early aging line than the near-isogenic normal aging line during senescence.

Abstract

The antioxidant system plays pivotal roles in removal of reactive oxygen species (ROS) produced during leaf senescence. To explore its roles in leaf senescence of wheat (Triticum aestivum L.), the concentrations of antioxidants, activities, and gene expression of antioxidant enzymes were evaluated in flag leaves of the early aging line (EAL) and the near-isogenic normal aging line (NL) during senescence. The results showed that the total chlorophyll and soluble protein in the EAL declined earlier and faster, while more malondialdehyde and ROS accumulated compared with the NL. The activities of superoxide dismutase, catalase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase were lower in the EAL than in the NL across multiple measuring dates. Additionally, the EAL had less amounts of reduced ascorbate and glutathione as well as lower reduction state with the progression of senescence. Concomitantly, the gene expression of antioxidant enzymes in the EAL was also significantly repressed relative to those in the NL during natural senescence. Taken together, the earlier onset and faster rate of senescence in the EAL could be a result of an imbalance of ROS production and ROS-scavenging antioxidant system, which provided valuable hints toward understanding leaf senescence of wheat.