A novel cold-inducible gene from Arabidopsis,RCI3, encodes a peroxidase that constitutes a component for stress tolerance

Myrothamnus flabellifolia, a short woody shrub from southern Africa, can survive severe desiccation of its vegetative organs. We studied mechanisms protecting this plant from oxidative damage during desiccation for 2 weeks, 4 and 8 months, and also during subsequent rehydration. This plant retains high concentrations of chlorophyll during desiccation, and these chlorophyll molecules are probably a source for potentially harmful singlet oxygen production. Desiccation triggered substantial increases in zeaxanthin and redox shifts of the antioxidants glutathione and ascorbate towards their oxidised forms. Simultaneously, the concentrations of violaxanthin, beta-carotene, ascorbate, alpha-tocopherol, and glutathione reductase activity progressively decreased. Antheraxanthin, gamma-tocopherol, lutein, neoxanthin and glucose-6-phosphate dehydrogenase displayed less pronounced changes in response to desiccation. Even after 4 months of desiccation, Myrothamnus flabellifolia recovered rapidly upon rehydration. Re-watering induced formation of ascorbate and glutathione, simultaneous reduction of their oxidised forms, and rapid production of alpha-tocopherol and of various carotenoids. Only after 8 months of desiccation did the antioxidant system of M. flabellifolia break down; 3 weeks after the onset of rehydration, these plants abscised their leaves, but even then they were still able to recover and develop new ones. Ascorbate, beta-carotene and alpha-tocopherol were totally depleted after 8 months of desiccation and did not recover upon rehydration; glutathione was partly maintained, but only in the oxidised form. We present a model demonstrating which parts of antioxidant pathways break down as oxidative stress becomes detrimental and we discuss some potential implications of our results for the genetic modification of crop plants to improve their drought tolerance.     

The role of alpha-tocopherol in plant stress tolerance

Environmental stresses trigger a wide variety of plant responses, ranging from altered gene expression to changes in cellular metabolism and growth. A plethora of plant reactions exist to circumvent the potentially harmful effects caused by light, drought, salinity, extreme temperatures, pathogen infections and other stresses. Alpha-tocopherol is the major vitamin E compound found in leaf chloroplasts, where it is located in the chloroplast envelope, thylakoid membranes and plastoglobuli. This antioxidant deactivates photosynthesis-derived reactive oxygen species (mainly 1O2 and OH), and prevents the propagation of lipid peroxidation by scavenging lipid peroxyl radicals in thylakoid membranes. Alpha-tocopherol levels change differentially in response to environmental constraints, depending on the magnitude of the stress and species-sensitivity to stress. Changes in alpha-tocopherol levels result from altered expression of pathway-related genes, degradation and recycling, and it is generally assumed that increases of alpha-tocopherol contribute to plant stress tolerance, while decreased levels favor oxidative damage. Recent studies indicate that compensatory mechanisms exist to afford adequate protection to the photosynthetic apparatus in the absence of alpha-tocopherol, and provide further evidence that it is the whole set of antioxidant defenses (ascorbate, glutathione, carotenoids, tocopherols and other isoprenoids, flavonoids and enzymatic antioxidants) rather than a single antioxidant, which helps plants to withstand environmental stress.     

Interaction of salicylic acid and ethylene and their effects on some physiological and biochemical parameters in canola plants (Brassica napus L.)

Environmental stresses, such as cold, heat, salinity, and drought, induce ethylene production and oxidative stress and cause damage in plants. On the other hand, studies have shown that salicylic acid (SA) induced resistance to environmental stresses in plants. In this research, the effects of ethylene on chlorophyll (Chl), carotenoid (Car), anthocyanin, flavonoids, ascorbic acid, dehydroascorbic acid, total ascorbate, lipid peroxidation, and ethylene production in leaves of canola pretreated with SA were studied. The plants were grown in pots until they have four leaves. Leaves were sprayed for two days with three different concentrations of SA (0, 0.5, and 1 mM). The plants were treated for three days with three concentrations of ethylene (0, 50, and 100 ppm). At the end of the ethylene treatments, all examined parameters were measured. The results showed that the ethylene treatments induced lipid peroxidation, while SA mitigated this effect. The ethylene treatment lowered significantly Chl and Car contents and anthocyanin accumulation, but SA alleviated these effects. SA induced an increase in ascorbic acid content in canola plants after the ethylene treatments. Therefore, we concluded that SA played an important role in the alleviation of damages caused by stress conditions.     

Sustained accumulation of methyl salicylate alters antioxidant protection and reduces tolerance of holm oak to heat stress

Methyl salicylate (MeSA) is thought to have a major role in biotic and abiotic stresses by acting as a signal to trigger the oxidative burst, which is needed to activate gene expression in plant stress responses. To assess the potential effects of sustained foliar accumulation of MeSA on plant stress tolerance, the extent of photo- and antioxidant protection, lipid peroxidation and visual leaf area damage were evaluated in MeSA-treated ( c. 60 nl l⁻¹ in air) and control holm oak ( Quercus ilex L.) plants exposed to heat stress. Control plants showed an increase in foliar MeSA levels up to 1.8 nmol [gDW]⁻¹ as temperature increased and they displayed tolerance to temperatures as high as 45°C, which might be attributed, at least in part, to enhanced xanthophyll de-epoxidation and increases in ascorbate and α-tocopherol. MeSA-treated plants showed a sustained foliar accumulation of this compound, with values ranging from 10 to 23 nmol [gDW]⁻¹ throughout the experiment. These plants showed lower ascorbate and tocopherol levels and higher oxidative damage at 50°C than controls, as indicated by enhanced malondialdehyde levels and leaf area damage and lower maximum efficiency of PSII photochemistry ( F _v/ F _m ratio). These results demonstrate that a sustained foliar accumulation of MeSA is detrimental to plant function and that it can reduce thermotolerance in holm oak by altering antioxidant defences.     

Plant responses to water stress: Role of reactive oxygen species

Terrestrial plants most often encounter drought stress because of erratic rainfall which has become compounded due to present climatic changes.Responses of plants to water stress may be assigned as either injurious change or tolerance index. One of the primary and cardinal changes in response to drought stress is the generation of reactive oxygen species (ROS), which is being considered as the cause of cellular damage. However, recently a signaling role of such ROS in triggering the ROS scavenging system that may confer protection or tolerance against stress is emerging. Such scavenging system consists of antioxidant enzymes like SOD, catalase and peroxidases, and antioxidant compounds like ascorbate, reduced glutathione; a balance between ROS generation and scavenging ultimately determines the oxidative load. As revealed in case of defence against pathogen, signaling via ROS is initiated by NADPH oxidase-catalyzed superoxide generation in the apoplastic space (cell wall) followed by conversion to hydrogen peroxide by the activity of cell wall-localized SOD. Wall peroxidase may also play role in ROS generation for signaling. Hydrogen peroxide may use Ca2+ and MAPK pathway as downstream signaling cascade. Plant hormones associated with stress responses like ABA and ethylene play their role possibly via a cross talk with ROS towards stress tolerance, thus projecting a dual role of ROS under drought stress.     

Effects of Abscisic Acid,Salicylic Acid,Ethylene and Hydrogen Peroxide in Thermotolerance and Recovery for Creeping Bentgrass

Abscisic acid (ABA), salicylic acid (SA), ethylene, and hydrogen peroxide (H₂O₂) may be involved in the regulation of plant responses to heat stress. The objective of this study was to determine whether these signaling molecules are involved in survival at high temperatures in creeping bentgrass (Agrostis stolonifera). We investigated the effects of treatment with ABA, SA, H₂O₂, and ACC (an ethylene precursor) on physiological damage occurring in creeping bentgrass during heat stress (35 °C for 1 month). We also compared the effects of chemical application and the induction of thermotolerance using moderate heat stress (30 °C for 24 h). All of the pre-treatments (heat or chemical) resulted in increased tolerance to prolonged heat stress (1 month) compared to control plants. All treated samples showed more green leaves, decreased membrane leakage and reduced oxidative damage compared to control plants. We then measured changes in the endogenous concentration of these chemical components during heat stress (35 °C) and during recovery after a stress treatment (cooling back to 20 °C). An oxidative burst was detected 5 min after the initiation of heat treatment, with the increase in H₂O₂ being detected primarily in the apoplast of the cells in both leaf and root tissues. Free SA was detected only an hour after the initiation of heat stress, and concentration remained low subsequently. Neither ABA nor ethylene concentrations rose during heat stress, but the concentration of both increased during subsequent cooling. These results suggest that the signaling components of interest are involved in thermotolerance in creeping bentgrass, but that the different chemicals are likely to be involved in separate signaling pathways. An oxidative burst and SA may be bona fide heat stress signals, but ABA and ethylene appear to be involved in signaling pathways in response to recovery from heat stress in this species.     

Comparative response of maize and rice genotypes to heat stress: status of oxidative stress and antioxidants

In the present study, two genotypes each of maize and rice were compared for their response to varying degrees of temperature stress (35/30, 40/35, 45/40°C) with controls growing at 30/25°C. At elevated temperatures of 40/35 and 45/40°C, the rice genotypes were inhibited to a significantly higher extent, especially for their shoot growth compared to maize genotypes. The stress injury measured as damage to membranes, loss of chlorophyll and reduction in leaf water status was significantly higher in rice plants, especially at 45/40°C. The components of oxidative stress particularly the level of malondialdehyde was significantly greater in rice plants while the differences for hydrogen peroxide concentrations were small at 40/35 and 45/40°C. The expression of enzymatic antioxidants like catalase, ascorbate peroxidase and glutathione reductase was found to be higher in maize plants compared to rice plants while no variations existed for superoxide dismutase at 45/40°C. In addition, the non-enzymatic antioxidants like ascorbic acid, glutathione and proline were maintained at significantly greater levels at 45/40°C in maize than in rice genotypes. These findings suggested that maize genotypes were able to retain their growth under high-temperature conditions partly due to their superior ability to cope up with oxidative damage by heat stress compared to rice genotypes. Since, maize and rice belong to C₄ and C₃ plant groups, respectively, these observations may also reflect the relative sensitivity of these plant groups to heat stress.     

Redox regulation of water stress responses in field-grown plants. Role of hydrogen peroxide and ascorbate

Abiotic stresses, such as drought, can increase the production of reactive oxygen species (ROS) in plants. An increase in ROS levels can provoke a partial or severe oxidation of cellular components inducing redox status changes, so continuous control of ROS and therefore of their metabolism is decisive under stress conditions. The present work focuses on the contribution of one pro-oxidant, hydrogen peroxide (H₂O₂) and one antioxidant, ascorbate (AA) and its redox status, in the control of plant responses to drought-oxidative stress in resistant plants growing in field conditions. After a general introduction to the concept of drought and oxidative stress and its relationship, we describe the role of H₂O₂ in drought stress responses, emphasizing the importance of studies in H₂O₂ subcellular localization, needed for a better understanding of its role in plant responses to stress. Although more studies are needed in the study of changes of redox status in plants subjected to stress, the AA pools and its redox status can be indicative of its involvement as a part of cellular mechanisms by which the plant respond to drought-induced oxidative stress. The mechanism of resistance and/or tolerance to drought-oxidative stress is complex, especially when studies are carried out in plants growing in field conditions, where an interaction of stresses occurs. This study sheds light on the mechanisms of plant responses to water-oxidative stress in plants growing in the field.     

Activity of Stress-related Antioxidative Enzymes in the Invasive Plant Crofton Weed （Eupatorium adenophorum）

Crofton weed is an invasive weed in southwestern China. The activities of several antioxidative enzymes involved in plant protection against oxidative stress were assayed to determine physiological aspects of the crofton weed that might render the plant vulnerable to environmental stress. Stresses imposed on crofton weed were heat （progressively increasing temperatures： 25 ℃, 30 ℃, 35 ℃, 38℃ and 42 ℃ at 24 h intervals）, cold （progressively decreasing temperatures： 25 ℃, 20 ℃, 15℃, 10 ℃ and 5℃ at 24h intervals）, and drought （without watering up to 4days）. The three stresses induced oxidative damage as evidenced by an increase in lipid peroxidation. The effect varied with the stress imposed and the length of exposure. The activity of superoxide dismutase （SOD） increased in response to all stresses but was not significantly different from the controls （P 〈 0.05） when exposed to cold stress. Catalase （CAT） activity decreased in response to heat and drought stress but increased when exposed to cold conditions. Guaiacol peroxidase （POD） and glutathione reductase （GR） activities increased in response to cold and drought but decreased in response to heat stress. The activity of ascorbata peroxidase （APX） responded differently to all three stresses. Monodehydroascorbate reductase （MDHAR） activity decreased in response to heat and drought, and slightly increased in response to the cold stress but was not significantly different from the controls （P 〈 0.05）. The activity of dehydroascorbata reductase （DHAR） increased in response to all three stresses. Taken together, the co-ordinate increase of the oxygen-detoxifying enzymes might be more effective to protect crofton weed from the accumulation of oxygen radicals at low temperatures rather than at high temperatures.     

Enhanced alpha-tocopherol quinone levels and xanthophyll cycle de-epoxidation in rosemary plants exposed to water deficit during a Mediterranean winter

Photosynthesis operates in a constantly shifting balance between efficient capture of solar energy and its rapid dissipation when captured in excess. In an attempt to better understand the role of alpha-tocopherol in plant photoprotection, we examined the changes in alpha-tocopherol quinone (alpha-TQ), in parallel with those of other low-molecular-weight antioxidants, in rosemary plants exposed to water deficit during a Mediterranean winter. Relative leaf water content (RWC) decreased from about 85% to approximately 65% in drought, but plants did not show symptoms of oxidative damage, as indicated by constant Fv/Fm ratios and malondialdehyde (MDA) levels. alpha-TQ was present at concentrations of 20 mmol per 100 mol of chlorophyll, and represented less than 1% of total tocopherol content in non-stressed leaves. Although alpha-tocopherol levels were not significantly altered, alpha-TQ reached up to 36 mmol per 100 mol of chlorophyll under stress (under both high light and after exposure to increasing water deficit at lower light intensities). Furthermore, both alpha-TQ and xanthophyll cycle de-epoxidation were strongly negatively correlated with the relative efficiency of photosystem II photochemistry (phiPSII) at midday. The biological significance of alpha-tocopherol and alpha-TQ in the network of photo- and antioxidative protection mechanisms evolved by plants to withstand stress is discussed.     

Photo- and antioxidative protection,and a role for salicylic acid during drought and recovery in field-grown<Emphasis Type="Italic"> Phillyrea angustifolia</Emphasis> plants

Mechanisms of photo- and antioxidative protection, the extent of oxidative stress, and salicylic acid accumulation in leaves of Phillyrea angustifolia L. (Oleaceae) plants exposed to drought and recovery in Mediterranean field conditions were studied. The amounts of alpha-tocopherol increased up to 4-fold and those of zeaxanthin increased up to 3-fold at relative leaf water contents (RWCs) of ca. 60%, which caused up to 70% increases in the de-epoxidation state of the xanthophyll cycle (DPS). While alpha-tocopherol increased further in severe drought, zeaxanthin levels and DPS remained constant, beta-carotene decreased and malondialdehyde (MDA) levels increased at RWCs below 50%. Though this was associated with significant decreases in the maximum efficiency of photosystem II photochemistry (F(v)/ F(m)), the same leaves that suffered from drought recovered after rainfalls, and similar MDA levels and F(v)/ F(m) ratios to those observed before drought were attained. During recovery (i) the F(v)/ F(m) ratio and beta-carotene levels increased slowly, (ii) alpha-tocopherol levels decreased sharply, to increase again, and (iii) MDA levels in leaves increased to values 35% higher than those observed at maximum drought, and decreased later. Salicylic acid (SA) levels showed a strong negative correlation (r(2)=0.857) with the RWC, and increased progressively up to 5-fold, during drought. During recovery, SA levels decreased, but remained slightly higher than those observed before drought. SA levels were positively correlated with those of alpha-tocopherol during drought (r(2)=0.718), but not during recovery (r(2)=0.221). We conclude that (i) P. angustifolia plants activate several mechanisms of photo- and antioxidative protection to withstand drought stress during a Mediterranean summer, (ii) endogenous SA levels increase in leaves of drought-stressed plants, thus suggesting a role for SA in plant responses to drought, and (iii) plants suffer oxidative stress during recovery, and this stress is more severe as the previous drought is more intense.     

Enhanced tolerance to ozone and drought stresses in transgenic tobacco overexpressing dehydroascorbate reductase in cytosol

Ascorbate (vitamin C) is a potent antioxidant protecting plants against oxidative damage imposed by environmental stresses such as ozone and drought. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) is one of the two important enzymes functioning in the regeneration of ascorbate (AsA). To examine the protective role of DHAR against oxidative stress, we developed transgenic tobacco plants overexpressing cytosolic DHAR gene from Arabidopsis thaliana . Incorporation of the transgene in the genome of tobacco plants was confirmed by polymerase chain reaction and Southern blot analysis, and its expression was confirmed by Northern and Western blot analyses. These transgenic plants exhibited 2.3–3.1 folds higher DHAR activity and 1.9–2.1 folds higher level of reduced AsA compared with non-transformed control plants. The transgenic plants showed maintained redox status of AsA and exhibited an enhanced tolerance to ozone, drought, salt, and polyethylene glycol stresses in terms of higher net photosynthesis. In this study, we report for the first time that the elevation of AsA level by targeting DHAR overexpression in cytosol properly provides a significantly enhanced oxidative stress tolerance imposed by drought and salt.     

Expression of Arabidopsis NDPK2 increases antioxidant enzyme activities and enhances tolerance to multiple environmental stresses in transgenic sweetpotato plants

Transgenic sweetpotato (Ipomoea batatas L. cv. Yulmi) plants expressing the Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) gene under the control of an oxidative stress–inducible peroxidase (SWPA2) promoter (referred to as SN plants) were developed and evaluated for enhanced tolerance of SN plants under various abiotic stress conditions. The level of AtNDPK2 expression and NDPK activity in SN plants following methyl viologen (MV) treatment was positively correlated with the plant’s tolerance to MV. Interestingly, we observed that antioxidant enzyme activities such as peroxidase, ascorbate peroxidase, and catalase increased in MV-treated SN plants. In addition, SN plants showed enhanced tolerance to cold, high salinity, and drought stresses by an increase in the activity of H₂O₂ scavenging enzymes. These results indicate that overexpression of AtNDPK2 in sweetpotato might efficiently modulate oxidative stress from various environmental stresses.     

黄土高原冰草叶片抗坏血酸和谷胱甘肽合成及循环代谢对干旱胁迫的生理响应

通过盆栽实验, 对干旱胁迫下黄土高原地区冰草(Agropyron cristatum)叶片的抗坏血酸和谷胱甘肽合成及循环代谢相关酶及物质含量进行了研究。结果表明: 冰草可以通过增强叶片的抗坏血酸和谷胱甘肽合成及循环代谢酶: 抗坏血酸过氧化物酶、谷胱甘肽还原酶、脱氢抗坏血酸还原酶、单脱氢抗坏血酸还原酶、L-半乳糖酸-1, 4-内酯脱氢酶和γ-谷氨酰半胱氨酸合成酶活性, 维持植物体内抗坏血酸和谷胱甘肽水平及氧化还原状态, 从而抵御干旱造成的氧化胁迫。但叶片抗坏血酸和谷胱甘肽合成及循环代谢对不同水平干旱胁迫的响应, 随胁迫时间的延长而不同。在胁迫24天以前, 严重干旱下叶片的抗坏血酸和谷胱甘肽合成及循环代谢增强较显著; 在胁迫24天后, 由于该胁迫下植物所遭受的氧化胁迫较为严重, 叶片中上述6种酶的活性均呈降低趋势。而在中度干旱下叶片抗坏血酸和谷胱甘肽合成及循环代谢相关的6种酶在整个胁迫过程中均保持较高的活性。这说明, 冰草能够长时间有效地抵御中度干旱所造成的氧化胁迫, 但只能在一定时间范围内有效地抵御严重干旱所造成的氧化胁迫, 胁迫时间延长则会降低其抵御严重干旱的能力。     

Expression of the Nicotiana protein kinase (NPK1) enhanced drought tolerance in transgenic maize

Drought is one of the most important abiotic stresses affecting the productivity of maize. Previous studies have shown that expression of a mitogen-activated protein kinase kinase kinase (MAPKKK) gene activated an oxidative signal cascade and led to the tolerance of freezing, heat, and salinity stress in transgenic tobacco. To analyse the role of activation of oxidative stress signalling in improving drought tolerance in major crops, a tobacco MAPKKK (NPK1) was expressed constitutively in maize. Results show that NPK1 expression enhanced drought tolerance in transgenic maize. Under drought conditions, transgenic maize plants maintained significantly higher photosynthesis rates than did the non-transgenic control, suggesting that NPK1 induced a mechanism that protected photosynthesis machinery from dehydration damage. In addition, drought-stressed transgenic plants produced kernels with weights similar to those under well-watered conditions, while kernel weights of drought-stressed non-transgenic control plants were significantly reduced when compared with their non-stressed counterparts.     

Involvement of cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved tolerance against drought stress

In order to understand the role of cytosolic antioxidant enzymes in drought stress protection, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants overexpressing cytosolic Cu/Zn-superoxide dismutase (cytsod) (EC 1.15.1.1) or ascorbate peroxidase (cytapx) (EC 1.11.1.1) alone, or in combination, were produced and tested for tolerance against mild water stress. The results showed that the simultaneous overexpression of Cu/Znsod and apx or at least apx in the cytosol of transgenic tobacco plants alleviates, to some extent, the damage produced by water stress conditions. This was correlated with higher water use efficiency and better photosynthetic rates. In general, oxidative stress parameters, such as lipid peroxidation, electrolyte leakage, and H(2)O(2) levels, were higher in non-transformed plants than in transgenic lines, suggesting that, at the least, overexpression of cytapx protects tobacco membranes from water stress. In these conditions, the activity of other antioxidant enzymes was induced in transgenic lines at the subcellular level. Moreover, an increase in the activity of some antioxidant enzymes was also observed in the chloroplast of transgenic plants overexpressing cytsod and/or cytapx. These results suggest the positive influence of cytosolic antioxidant metabolism on the chloroplast and underline the complexity of the regulation network of plant antioxidant defences during drought stress.