Over-expression of ascorbate oxidase in the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox states and increased sensitivity to ozone

Transgenic tobacco ( Nicotiana tabacum L. cv. Xanthi) plants expressing cucumber ascorbate oxidase (EC.1.10.3.3) were used to examine the role of extracellular ascorbic acid in mediating tolerance to the ubiquitous air pollutant, ozone (O(3)). Three homozygous transgenic lines, chosen on the basis of a preliminary screen of AO activity in the leaves of 29 lines, revealed up to a 380-fold increase in AO activity, with expression predominantly associated with leaf cell walls. Over-expression of AO resulted in no change in the total ascorbate content recovered in apoplast washing fluid, but the redox state of ascorbate was reduced from 30% in wild-type leaves to below the threshold for detection in transgenic plants. Levels of ascorbic acid and glutathione in the symplast were not affected by AO over-expression, but the redox state of ascorbate was reduced, while that of glutathione was increased. AO over-expressing plants exposed to 100 nmol mol(-1) ozone for 7 h day(-1) exhibited a substantial increase in foliar injury, and a greater pollutant-induced reduction in both the light-saturated rate of CO(2) assimilation and the maximum in vivo rate of ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation, compared with wild-type plants. Transgenic plants also exhibited a greater decline in CO(2) assimilation rate when exposed to a brief ozone episode (300 nmol mol(-1) for 8 h). Stomatal conductance, hence O(3) uptake, was unaffected by AO over-expression. Our findings illustrate the important role played by ascorbate redox state and sub-cellular compartmentation in mediating the tolerance of plants to ozone-induced oxidative stress.     

Apoplastic ascorbate metabolism and its role in the regulation of cell signalling

The apoplast has crucial functions in plant biology. It comprises all the compartments beyond the plasmalemma, including the cell wall. As the reservoir of information on the biotic and abiotic environment surrounding the cell and a major conduit of information between cells, the apoplast has functions in stress perception and the subsequent appropriate control of growth and defence. The oxidative burst phenomenon, caused by environmental challenges and pathogen attack in particular, oxidises the apoplast. Ascorbic acid (AA), the major and probably the only antioxidant buffer in the apoplast, becomes oxidised in these conditions. The apoplastic enzyme ascorbate oxidase (AO) also regulates the reduction/oxidation (redox) state of the apoplastic ascorbate pool. We propose that a key function of the oxidative burst and of AO is to modify the apoplastic redox state in such a way as to modify receptor activity and signal transduction to regulate defence and growth.     

Altered stomatal dynamics in ascorbate oxidase over-expressing tobacco plants suggest a role for dehydroascorbate signalling

Control of stomatal aperture is of paramount importance forplant adaptation to the surrounding environment. Here, we reporton several parameters related to stomatal dynamics and performancein transgenic tobacco plants (Nicotiana tabacum L., cv. Xanthi)over-expressing cucumber ascorbate oxidase (AO), a cell wall-localizedenzyme of uncertain biological function that oxidizes ascorbicacid (AA) to monodehydroascorbic acid which dismutates yieldingAA and dehydroascorbic acid (DHA). In comparison to WT plants,leaves of AO over-expressing plants exhibited reduced stomatalconductance (due to partial stomatal closure), higher watercontent, and reduced rates of water loss on detachment. Transgenicplants also exhibited elevated levels of hydrogen peroxide anda decline in hydrogen peroxide-scavenging enzyme activity. LeafABA content was also higher in AO over-expressing plants. Treatmentof epidermal strips with either 1 mM DHA or 100 µM hydrogenperoxide resulted in rapid stomatal closure in WT plants, butnot in AO-over-expressing plants. This suggests that signalperception and/or transduction associated with stomatal closureis altered by AO over-expression. These data support a specificrole for cell wall-localized AA in the perception of environmentalcues, and suggest that DHA acts as a regulator of stomatal dynamics. Key words: ABA, apoplast, ascorbic acid, ascorbate oxidase, dehydroascorbic acid (DHA), hydrogen peroxide, Nicotiana tabacum L., cv. Xanthi, stomata, transgenic plants, water stress Received 26 September 2007; Revised 11 December 2007 Accepted 12 December 2007     

Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state

To investigate the possible mechanisms of glutathione reductase (GR) in protecting against oxidative stress, we obtained transgenic tobacco (Nicotiana tabacum) plants with 30–70% decreased GR activity by using a gene encoding tobacco chloroplastic GR for the RNAi construct. We investigated the responses of wild type and transgenic plants to oxidative stress induced by application of methyl viologen in vivo. Analyses of CO₂ assimilation, maximal efficiency of photosystem II photochemistry, leaf bleaching, and oxidative damage to lipids demonstrated that transgenic plants exhibited enhanced sensitivity to oxidative stress. Under oxidative stress, there was a greater decrease in reduced to oxidized glutathione ratio but a greater increase in reduced glutathione in transgenic plants than in wild type plants. In addition, transgenic plants showed a greater decrease in reduced ascorbate and reduced to oxidized ascorbate ratio than wild type plants. However, there were neither differences in the levels of NADP and NADPH and in the total foliar activities of monodehydroascorbate reductase and dehydroascorbate reductase between wild type and transgenic plant. MV treatment induced an increase in the activities of GR, ascorbate peroxidase, superoxide dismutase, and catalase. Furthermore, accumulation of H₂O₂ in chloroplasts was observed in transgenic plants but not in wild type plants. Our results suggest that capacity for regeneration of glutathione by GR plays an important role in protecting against oxidative stress by maintaining ascorbate pool and ascorbate redox state.     

An apoplastic h-type thioredoxin is involved in the stress response through regulation of the apoplastic reactive oxygen species in rice

Thioredoxins (Trxs) are a multigenic family of proteins in plants that play a critical role in redox balance regulation through thiol-disulfide exchange reactions. There are 10 members of the h-type Trxs in rice (Oryza sativa), and none of them has been clearly characterized. Here, we demonstrate that OsTRXh1, a subgroup I h-type Trx in rice, possesses reduction activity in vitro and complements the hydrogen peroxide sensitivity of Trx-deficient yeast mutants. OsTRXh1 is ubiquitously expressed in rice, and its expression is induced by salt and abscisic acid treatments. Intriguingly, OsTRXh1 is secreted into the extracellular space, and salt stress in the apoplast of rice induces its expression at the protein level. The knockdown of OsTRXh1 results in dwarf plants with fewer tillers, whereas the overexpression of OsTRXh1 leads to a salt-sensitive phenotype in rice. In addition, both the knockdown and overexpression of OsTRXh1 decrease abscisic acid sensitivity during seed germination and seedling growth. We also analyzed the levels of hydrogen peroxide produced in transgenic plants, and the results show that more hydrogen peroxide is produced in the extracellular space of OsTRXh1 knockdown plants than in wild-type plants, whereas the OsTRXh1 overexpression plants produce less hydrogen peroxide under salt stress. These results show that OsTRXh1 regulates the redox state of the apoplast and influences plant development and stress responses.     

OsTRXh1 regulates the redox state of the apoplast and influences stress responses in rice

The plant cell apoplast is the compartment beyond the cell plasmalemma, including the cell wall and intercellular space. Many environmental elements can trigger reactive oxygen species (ROS) burst at the plasma membrane which then alters the redox state of the apoplast. Recently, h-type thioredoxin (Trx), OsTRXh1, was identified to be involved in apoplastic redox state regulation in rice. OsTRXh1 is conserved redox-active Trx and can be secreted into the extracellular regions. Through transgenic rice plant, we found that OsTRXh1 regulated ROS accumulation in apoplast and influenced plant development and stress responses. This provides new insights into apoplastic redox state regulation pathway and expands our understanding of h-type Trxs function.     

Transport and action of ascorbate at the plant plasma membrane

The plasmalemma is both a bridge and a barrier between the cytoplasm and the outside world. It is a dynamic interface that perceives and transmits information concerning changes in the environment to the nucleus to modify gene expression. In plants, ascorbate is an essential part of this dialogue. The concentration and ratio of reduced to oxidized ascorbate in the apoplast, for example, possibly modulates cell division and growth. The leaf apoplast contains millimolar amounts of ascorbate that protect the plasmalemma against oxidative damage. The apoplastic ascorbate-dehydroascorbate redox couple is linked to the cytoplasmic ascorbate-dehydroascorbate redox couple by specific transporters for either or both metabolites. Although evidence about the mechanisms driving ascorbate or dehydroascorbate transport remains inconclusive, these carrier proteins potentially regulate the level and redox status of ascorbate in the apoplast. The redox coupling between compartments facilitated by these transport systems allows coordinated control of key physiological responses to environmental cues.     

Recent advances in ascorbate biosynthesis and the physiological significance of ascorbate peroxidase in photosynthesizing organisms

Ascorbate (AsA), the most abundant water-soluble redox compound in plants and eukaryotic algae, has multiple functions. There is compelling genetic evidence that the biosynthesis of AsA proceeds via a D-mannose/L-galactose pathway and is the most significant source of AsA in plants. AsA plays important roles in antioxidative defense, particularly via the AsA/glutathione cycle. AsA peroxidase (APX) plays a central role in the cycle and is emerging as a key enzyme in cellular H(2)O(2) metabolism. Plants possess diverse APX isoenzymes in cellular compartments, including the chloroplast, cytosol, and microbody. In algae, however, the number and distribution of APX proteins are quite limited. Recent progress in molecular biological analysis of APX isoenzymes has revealed elaborate mechanisms for the tissue-dependent regulation of two chloroplastic APX isoenzymes by alternative splicing, and for redox regulation of cytosolic APX gene expression in response to light stress. Furthermore, transgenic plants overexpressing a chloroplastic APX isoenzyme enable us to evaluate the behavior of the enzyme under conditions of photo-oxidative stress. Molecular physiological analysis has revealed that cytosolic APX is part of the system modulating the cellular H(2)O(2) level in redox signaling.     

Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress

To evaluate the physiological importance of thylakoid membrane-bound ascorbate peroxidase (tAPX) in the active oxygen species-scavenging system of chloroplasts, the level of tAPX in tobacco plants was altered by expression of the tAPX cDNA in both sense and antisense orientation. The tobacco plants transformed with constructs of antisense tAPXs from spinach and tobacco could not be obtained, suggesting that the suppression of tAPX in higher plants had a severe effect on the growth even under normal conditions. In contrast, the transgenic tobacco plants (TpTAP-12) overexpressing tAPX, which had approximately 37-fold higher activity than that of the wild-type plants, were generated. The TpTAP-12 plants showed increased tolerance to oxidative stress caused by application of methylviologen (MV, 50 microm) under light intensity (300 and 1600 microE m(-2) sec(-1)) and by chilling stress with high light intensity (4 degrees C, 1000 microE m(-2) sec(-1)). At 24 h after the MV treatment under illumination at 300 microE m-2 sec-1, destruction of chlorophyll was observed in the wild-type plants, but not in the TpTAP-12 plants. The activities of thiol-modulated enzymes in the Calvin cycle, the level and redox status of ascorbate (AsA), and the activity of tAPX in the wild-type plants significantly decreased, while those in the TpTAP-12 plants were hardly changed. These observations suggest that tAPX is a limiting factor of antioxidative systems under photo-oxidative stress in chloroplasts, and that the enhanced activity of tAPX functions to maintain the AsA content and the redox status of AsA under stress conditions.     

转铜/锌超氧化物歧化酶和抗坏血酸过氧化物酶基因马铃薯的耐氧化和耐盐性研究

高盐等逆境可以加剧植物体内活性氧的产生,进而引起植物细胞死亡。为开发抗逆境作物,以置于氧化诱导型启动子下定位于叶绿体的转铜/锌超氧化物歧化酶（Cu/ZnSOD）和抗坏血酸过氧化物酶基因(APX)马铃薯为材料,研究了其对MV和 NaCl所引起的氧化胁迫的耐受性。结果表明, MV胁迫下,转基因马铃薯叶片膜的相对电导率明显低于对照; NaCl胁迫下,其叶绿素含量高于对照。 在含NaCl 的培养基上,转基因幼苗生根率明显大于对照。另外,NaCl胁迫下转基因马铃薯叶片的SOD和APX酶活性显著高于对照,与其耐盐性的提高相一致。这些研究表明,转入Cu/ZnSOD和APX基因的马铃薯清除活性氧的能力增强,抗逆性得到提高。本实验采用氧化诱导型启动子调控下的SOD和APX两个基因协同作用,使外源基因只有在逆境胁迫时才特异性表达,增强转基因植株的抗逆效果,为培育抗逆经济作物开阔了思路。     

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.     

Increasing tolerance to ozone by elevating foliar ascorbic acid confers greater protection against ozone than increasing avoidance

Ascorbic acid (Asc) is the most abundant antioxidant in plants and serves as a major contributor to the cell redox state. Exposure to environmental ozone can cause significant damage to plants by imposing conditions of oxidative stress. We examined whether increasing the level of Asc through enhanced Asc recycling would limit the deleterious effects of environmental oxidative stress. Plants overexpressing dehydroascorbate reductase (DHAR), which results in an increase in the endogenous level of Asc, were exposed to acute or chronic levels of ozone. DHAR-overexpressing plants had a lower oxidative load, a lower level of oxidative-related enzyme activities, a higher level of chlorophyll, and a higher level of photosynthetic activity 24 h following an acute exposure (2 h) to 200 ppb ozone than control plants, despite exhibiting a larger stomatal area. Reducing the size of the Asc pool size through suppression of DHAR expression had the opposite effect. Following a chronic exposure (30 d) to 100 ppb ozone, plants with a larger Asc pool size maintained a larger stomatal area and a higher oxidative load, but retained a higher level of photosynthetic activity than control plants, whereas plants suppressed for DHAR had a substantially reduced stomatal area, but also a substantially lower level of photosynthetic activity. Together, these data indicate that, despite a reduced ability to respond to ozone through stomatal closure, increasing the level of Asc through enhanced Asc recycling provided greater protection against oxidative damage than reducing stomatal area.     

Increased Vitamin C Content Accompanied by an Enhanced Recycling Pathway Confers Oxidative Stress Tolerance in Arabidopsis

Vitamin C (L-ascorbic acid, AsA) has important antioxidant and metabolic functions in both plants and animals. Once used, ascorbic acid can be regenerated from its oxidized form in a reaction catalyzed by dehydroascorbate reductase (DHAR, EC 1.8.5.1). To analyze the physiological role of DHAR catalyzing the reduction of DHA to ascorbate in environmental stress adaptation, we examined whether increasing the level of AsA through enhanced AsA recycling would limit the deleterious effects of oxidative stress. A chimeric construct consisting of the double CaMV35S promoter fused to the Myc-dhar gene was introduced into Arabidopsis thaliana. Transgenic plants were biochemically characterized and tested for responses to oxidative stress. Western blot indicated that the dhar-transgene was successfully expressed. In homozygous T_4 transgenic seedlings, DHAR overexpression was increased up to 1.5 to 5.4 fold, which enhanced foliar ascorbic acid levels 2- to 4.25-fold and ratio of AsA/DHA about 3- to 16-fold relative to wild type. In addition, the level of glutathione, the reductant used by DHAR, also increased as did its redox state. When whole plants were treated with high light and high temperature stress or in vitro leaf discs were subjected to 10 μM paraquat, transgenic plants showed a larger AsA pool size, lower membrane damage, and a higher level of chlorophyll compared with controls. These data suggested that increasing the plant vitamin C content through enhanced ascorbate recycling could limit the deleterious effects of environmental oxidative stress.     

Overexpression of Rice Sphingosine-1-Phoshpate Lyase Gene OsSPL1 in Transgenic Tobacco Reduces Salt and Oxidative Stress Tolerance

Sphingolipids, including sphingosine-1-phosphate (S1P), have been shown to function as signaling mediators to regulate diverse aspects of plant growth, development, and stress response. In this study, we performed functional analysis of a rice (Oryza sativa) S1P lyase gene OsSPL1 in transgenic tobacco plants and explored its possible involvement in abiotic stress response. Overexpression of OsSPL1 in transgenic tobacco resulted in enhanced sensitivity to exogenous abscisic acid (ABA), and decreased tolerance to salt and oxidative stress, when compared with the wild type. Furthermore, the expression levels of some selected stress-related genes in OsSPL1-overexpressing plants were reduced after application of salt or oxidative stress, indicating that the altered responsiveness of stress-related genes may be responsible for the reduced tolerance in OsSPL1-overexpressing tobacco plants under salt and oxidative stress. Our results suggest that rice OsSPL1 plays an important role in abiotic stress responses.