Effect of ascorbate oxidase over-expression on ascorbate recycling gene expression in response to agents imposing oxidative stress

Ascorbate oxidase (AO) is a cell wall-localized enzyme that uses oxygen to catalyse the oxidation of ascorbate (AA) to the unstable radical monodehydroascorbate (MDHA) which rapidly disproportionates to yield dehydroascorbate (DHA) and AA, and thus contributes to the regulation of the AA redox state. Here, it is reported that in vivo lowering of the apoplast AA redox state, through increased AO expression in transgenic tobacco (Nicotiana tabacum L. cv. Xanthi), exerts no effects on the expression levels of genes involved in AA recycling under normal growth conditions, but plants display enhanced sensitivity to various oxidative stress-promoting agents. RNA blot analyses suggest that this response correlates with a general suppression of the plant's antioxidative metabolism as demonstrated by lower expression levels of AA recycling genes. Furthermore, studies using Botrytis cinerea reveal that transgenic plants exhibit increased sensitivity to fungal infection, although the response is not accompanied by a similar suppression of AA recycling gene expression. Our current findings, combined with previous studies which showed the contribution of AO in the regulation of AA redox state, suggest that the reduction in the AA redox state in the leaf apoplast of these transgenic plants results in shifts in their capacity to withstand oxidative stress imposed by agents imposing oxidative stress.     

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.     

Ascorbic Acid-Dependent Regulation of Redox Levels of Chlorogenic Acid and its Isomers in the Apoplast of Leaves of Nicotiana tabacum L.

There is a question whether ascorbic acid (AA) can control redoxlevels of phenolics in the apoplast. The present study was designedto answer this question. AA, dehydroascorbic acid (DHA), chlorogenicacid (CGA) and its two structural isomers were present in theapoplast of leaves of tobacco (Nicotiana tabacum L. cv. BelW3).The levels of AA plus DHA (AA + DHA) and the ratios of AA to(AA + DHA) decreased while the levels of CGA plus its isomersincreased during leaf aging. o-Quinones of CGA plus its isomerswere found in the apoplast only in aged leaves of which apoplasticlevel of AA was nearly zero. In addition, activity of apoplasticperoxidase that could oxidize CGA and its isomers increasedduring leaf aging. From the observations, it is concluded thatAA can regulate the accumulation of the o-quinones of CGA andits isomers in the apoplast. Based on the conclusion, it isproposed that soluble peroxidase in the apoplast has two functions,namely, (i) scavenging of H₂O₂ and/or regulation of the levelof apoplastic H₂O₂ in the presence of AA, and (ii) accumulationof oxidation products of the phenolics in the absence of AA. (Received January 30, 1998; Accepted April 7, 1998)     

Combined Effects of Auxin Transport Inhibitors and Cytokinin: Alterations of Organ Development in Tobacco

We have examined the effects of the auxin transport inhibitors1-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid(TIBA) on leaf morphogenesis of transgenic Nicotiana tabacum(cv. Xanthi) plants expressing the Agrobacterium tumefacienscytokinin biosynthetic gene, ipt. We have observed the formationof saucer-shaped leaf-like organs at the shoot apex and at lateralbuds. The formation of apical saucer-shaped leaf-like organscan be duplicated by the application of exogenous NPA and cytokininto wild-type tobacco seedlings. We have also observed adventitiousleaf-like organs with altered petiole and blade morphology inthe transgenic plants treated with auxin transport inhibitors.These results suggest that the combination of diminished auxintransport and elevated cytokinin can lead to alterations inleaf development in tobacco. ⁴Present address: Genesis Research and Development Corporation,P.O. Box 50, Auckland, New Zealand     

Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase gene

To analyze the physiological role of dehydroascorbate reductase (DHAR, EC 1.8.5.1) catalyzing the reduction of DHA to ascorbate in environmental stress adaptation, T1 transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants expressing a human DHAR gene in chloroplasts were biochemically characterized and tested for responses to various stresses. Fully expanded leaves of transgenic plants had about 2.29 times higher DHAR activity (units/g fresh wt) than non-transgenic (NT) plants. Interestingly, transgenic plants also showed a 1.43 times higher glutathione reductase activity than NT plants. As a result, the ratio of AsA/DHA was changed from 0.21 to 0.48, even though total ascorbate content was not significantly changed. When tobacco leaf discs were subjected to methyl viologen (MV) at 5 mumol/L and hydrogen peroxide (H2O2) at 200 mmol/L, transgenic plants showed about a 40% and 25% reduction in membrane damage relative to NT plants, respectively. Furthermore, transgenic seedlings showed enhanced tolerance to low temperature (15 degrees C) and NaCl (100 mmol/L) compared to NT plants. These results suggest that a human derived DHAR properly works for the protection against oxidative stress in plants.     

Effect of abiotic stresses on the activity of antioxidative enzymes and contents of phytohormones in wild type and AtCKX2 transgenic tobacco plants

The responses of antioxidant enzymes (AOE) ascorbate peroxidase (APX), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) in soluble protein extracts from leaves and roots of tobacco (Nicotiana tabacum L. cv. Samsun NN) plants to the drought stress, salinity and enhanced zinc concentration were investigated. The studied tobacco included wild-type (WT) and transgenic plants (AtCKX2) harbouring the cytokinin oxidase/dehydrogenase gene under control of 35S promoter from Arabidopsis thaliana (AtCKX2). The transgenic plants exhibited highly enhanced CKX activity and decreased contents of cytokinins and abscisic acid in both leaves and roots, altered phenotype, retarded growth, and postponed senescence onset. Under control conditions, the AtCKX2 plants exhibited noticeably higher activity of GR in leaves and APX and SOD in roots. CAT activity in leaves always decreased upon stresses in WT while increased in AtCKX2 plants. On the contrary, the SOD activity was enhanced in WT but declined in AtCKX2 leaves. In roots, the APX activity prevailingly increased in WT while mainly decreased in AtCKX2 in response to the stresses. Both WT and AtCKX2 leaves as well as roots exhibited elevated abscisic acid content and increased CKX activity under all stresses while endogenous CKs and IAA contents were not much affected by stress treatments in either WT or transgenic plants.     

Expansion of transgenic tobacco protoplasts expressing pumpkin ascorbate oxidase is more rapid than that of wild-type protoplasts

 When pumpkin (Cucurbita spp., cv. Ebisu Nankin) ascorbate oxidase cDNA was introduced into cultured cells of tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow No. 2) by Agrobacterium-mediated transformation, the transgenic cells expressed and secreted the recombinant pumpkin ascorbate oxidase into the culture medium. These transgenic cells showed no morphological difference from wild-type cells. However, in the presence of applied hormones protoplasts prepared from the transgenic cells elongated more rapidly than those of wild-type cells. We propose that ascorbate oxidase may play a key role in the regulation of cell expansion perhaps by controlling transport processes through the plasma membrane, but not by affecting the cell wall. Received: 28 October 1999 / Accepted: 18 January 2000     

Overexpression of Pp14-3-3 from Pyrus pyrifolia fruit increases drought and salt tolerance in transgenic tobacco plant

Drought and salinity are the major abiotic stresses, which reduce agricultural productivity. In plants, 14-3-3s function as regulators of many target proteins through direct protein-protein interactions and play an important role during response to abiotic stresses. Here we report that CaMV 35S promoter driven overexpression of a Pyrus pyrifolia 14-3-3 gene, Pp14-3-3, improves drought and NaCl tolerance in T1 generation plants of transgenic tobacco (Nicotiana tabacum L. cv Xanthi). Under drought and NaCl stresses, the Pp14-3-3 was largely expressed in T1 transgenic tobacco lines, and compared with the wild-type (WT), transgenic tobacco plants showed relatively normal growth condition. In addition, the levels of membrane lipid peroxidation in T1 transgenic lines were definitely lower than that in WT according to the significantly decreased content of malondialdehyde. Meanwhile, the T1 transgenic tobacco lines showed significantly slower superoxide anion production rate than the WT under abiotic stress. Moreover, both the glutathione S-transferase (GST) and ascorbate peroxidase (APX) activities in T1 transgenic lines were markedly higher than those in WT. GSTs and APXs are important components of plant antioxidant system, and the results of present study suggested that Pp14-3-3 should play a crucial role in reducing oxidative damage caused by drought and salt stresses.     

Enhanced tolerances of transgenic tobacco plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against methyl viologen-mediated oxidative stress

In order to better understand the role of antioxidant enzymes in plant stress protection mechanisms, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants were developed that overexpress both superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts. These plants were evaluated for protection against methyl viologen (MV, paraquat)‐mediated oxidative damage both in leaf discs and whole plants. Transgenic plants that express either chloroplast‐targeted CuZnSOD (C) or MnSOD (M) and APX (A) were developed (referred to as CA plants and AM plants, respectively). These plant lines were crossed to produce plants that express all three transgenes (CMA plants and AMC plants). These plants had higher total APX and SOD activities than non‐transgenic (NT) plants and exhibit novel APX and SOD isoenzymes not detected in NT plants. As expected, transgenic plants that expressed single SODs showed levels of protection from MV that were only slightly improved compared to NT plants. The expression of either SOD isoform along with APX led to increased protection while expression of both SODs and APX provided the highest levels of protection against membrane damage in leaf discs and visual symptoms in whole plants.     

Modulation of flowering responses in different Nicotiana varieties

We have identified and characterized a FLOWERING PROMOTING FACTOR 1(FPF1) gene from tobacco (NtFPF1). Over-expression of NtFPF1 leads to early flowering in the day-neutral tobacco Nicotiana tabacum cv. Hicks, and under inductive photoperiods also in the short-day Nicotiana tabacum cv. Hicks Maryland Mammoth (MM) tobacco and the long-day plant Nicotiana sylvestris. N. sylvestris wild-type plants remained in the rosette stage and never flowered under non-inductive short-days, whereas 35S::NtFPF1 transgenic plants bolted but did not flower. However, if treated with gibberellins, transgenic N. sylvestrisplants flowered much faster under non-inductive short days than corresponding wild type plants, indicating an additive effect of gibberellins and the NtFPF1 protein in flowering time control. The day-neutral wild type cv. Hicks and the short-day cv. Hicks MM plants exhibit an initial rosette stage, both under short- and long-days. In the transgenic lines, this rosette stage was completely abolished. Wild-type plants of cv. Hicks MM never flowered under long days; however, all transgenic lines over-expressing NtFPF1 flowered under this otherwise non-inductive photoperiod.     

Mise en Évidence d'un Effet Régulateur de la Proline Exogène sur le Cycle du Glycolate Chez Nicotiana tabacum cv. Xanthi

Demonstration of a regulatory effect of exogenous proline on the glycolate cycle in Nicotiana tabacum cv. Xanthi n.c. An exogenous proline supply in the light provokes an increase in free glycine concentration in apical tissues or leaf disks of vegetative Nicotiana tabacum cv. Xanthi n.c. The same phenomenon does not occur in the equivalent tissues of tobacco plants after floral induction, these being naturally rich in proline. Under different environmental conditions (light, dark, varying concentrations of CO₂ and O₂), the exogenous proline appears to modify one or more reactions of the glycolate pathway.     

Antioxidant protection during ageing and senescence in transgenic tobacco with enhanced activity of cytokinin oxidase/dehydrogenase

We studied changes in physiological parameters of whole leaves and in antioxidant protection of chloroplasts during ageing and senescence of tobacco (Nicotiana tabacum L. cv. Samsun NN) leaves with enhanced cytokinin oxidase/dehydrogenase activity (CKX) or without it (WT). Old leaves of CKX plants maintained higher pigment content and photosystem 2 activity compared to WT leaves of the same age. Chloroplasts of old CKX plants showed better antioxidant capacity represented by higher superoxide dismutase, dehydroascorbate reductase and glutathione reductase activities.     

Hydrogen peroxide generated by copper amine oxidase is involved in abscisic acid-induced stomatal closure in Vicia faba

H₂O₂ is an essential signal in absicic acid (ABA)-induced stomatalclosure. It can be synthesized by several enzymes in plants.In this study, the roles of copper amine oxidase (CuAO) in H₂O₂production and stomatal closure were investigated. ExogenousABA stimulated apoplast CuAO activity, increased H₂O₂ productionand [Ca²⁺]_cyt levels in Vicia faba guard cells, and inducedstomatal closure. These processes were impaired by CuAO inhibitor(s).In the metabolized products of CuAO, only H₂O₂ could inducestomatal closure. By the analysis of enzyme kinetics and polyaminecontents in leaves, putrescine was regarded as a substrate ofCuAO. Putrescine showed similar effects with ABA on the regulationof H₂O₂ production, [Ca²⁺]_cyt levels, as well as stomatal closure.The results suggest that CuAO in V. faba guard cells is an essentialenzymatic source for H₂O₂ production in ABA-induced stomatalclosure via the degradation of putrescine. Calcium messengeris an important intermediate in this process. Key words: Abscisic acid, calcium, copper amine oxidase, hydrogen peroxide, putrescine, stomatal closure, Vicia faba Received 13 October 2007; Revised 16 December 2007 Accepted 20 December 2007     

Action de la proline exogène sur l'activité de la voie du glycolate chez Nicotiana tabacum cv. Xanthi n.c.

The effect of exogenous proline on the activity of the glycolate pathway in Nicotiana tabacum cv. Xanthi n.c. An exogenous proline supply in the light provokes an increase in free glycine concentration in apical tissues or in leaf disks of vegetative Nicotiana tabacum L. cv. Xanthi n.c. This does not occur in the equivalent tissues of tobacco plants after floral induction, these being naturally rich in proline. In vegetative tobacco, we have tried to determine this specific action of exogenous proline. With ¹⁴C glycine, ¹⁴CO₂ experiments (Pulse-chase) and glycine decarboxylase activity determinations, we observed that glycine-serine transformation was inhibited by proline supply. Presently it is important to determine if endogenous proline acts on the same reaction.     

Developmental Pattern of Root and Shoot Organogenesis in Cultured Leaf Explants of Nicotiana tabacum cv. Xanthi nc.

Caulogenesis and rhizogenesis were studied in cultured leafexplants of Nicotiana tabacum cv. Xanthi nc. using both lightand scanning electron microscopy. The timing of organ appearancewas also recorded. The patterns of development seen were comparedto each other and to that in explants grown on growth regulator-freemedium. Shoots first appeared after 12 d in culture and rootsafter 7 d. In caulogenesis nodules appear at the explant edgeand from these the shoots arise. The nodules are mainly derivedfrom palisade mesophyll cells, along with some spongy mesophylland bundle-sheath cells. The nodules form a continuous row alongthe edge of the explant and their initiation appears to be centredon veins. Shoots are produced indirectly. Roots are produceddirectly from bundle-sheath and vein parenchyma cells. Withoutplant growth regulators bundle-sheath cells still divide, althoughonly a few divisions were seen. Key words: Nicotiana tabacum, in vitro, caulogenesis, rhizogenesis     

The Association of Ascorbate and Ascorbate Oxidase in the Apoplast with IAA-Enhanced Elongation of Epicotyls from Vigna angularis

The level of (ascorbic acid (AA) plus dehydroascorbic acid (DHA))and the ratio of the level of AA to that of AA plus DHA in intercellularwashing fluid (IWF) of epicotyl segments from Vigna angularisdecreased from 2.8±0.3 to 1.2±0.5nmol (g fr wt)^–1and from 0.23±0.03 to 0.13±0.01, respectively,after incubation of the segments without IAA for 20 h at 27°C.However, these values changed to 5.3±1.7 nmol (g fr wt)^–1and 0.07±0.05 after incubation with 0.1 mM IAA. The activityof cell wall-bound ascorbate oxidase increased by about 20%and 70% after incubation without IAA and with IAA, respectively.However, the activity of cell wall-bound peroxidase was notaffected by incubation with or without IAA. The activities ofascorbate oxidase and peroxidase in IWF decreased by about 85and 75% after incubation without IAA. IAA did not affect thesedecreases to any great extent. A lignin-like compound was formedduring the incubation of epicotyl segments in the absence ofIAA. Formation of this compound was inhibited by IAA. The resultssuggest that one of the causes of the enhancement of elongationgrowth by IAA is the inhibition of peroxidase-dependent lignificationas a result of increases in levels of AA and DHA and in ascorbateoxidase activity. (Received August 16, 1993; Accepted December 6, 1993)     

Expression of HPV-11 L1 protein in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

Background  

We have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv. Xanthi as potential sources for an inexpensive subunit vaccine.