Coat protein-mediated resistance to Turnip mosaic virus in oilseed rape (Brassica napus)

Oilseed rape (Brassica napus) lines transformedwith the coat protein (CP) gene of Turnip mosaic virus(TuMV) were used to determine the effectiveness of resistance to TuMV mediatedby CP RNA or coat protein. Lines with one, two, or more copies of transgeneswere produced. T₂ and T₃ lines containing the CP genewitha functional start codon synthesised coat protein and showed high, but variablelevels of resistance to TuMV (21–96% resistant plants per line). TheT₁ and T₂ progeny of all lines carrying the CP gene withamutated start codon so that RNA but not protein was expressed, were assusceptible to TuMV as controls. Thus, in these experiments we were able toinduce CP-mediated resistance, but not RNA-mediated resistance.     

cDNA cloning and expression analysis of a putative alternative oxidase HsAOX1 from wild barley (Hordeum spontaneum)

A novel alternative oxidase (AOX1) gene, designated HsAOX1 (GenBank accession number JF440341) was cloned by RT-PCR from wild barley (Hordeum spontaneum). The full length of HsAOX1 is 1115 bp with an open reading frame of 987 bp, encoding a protein of 328 amino acids with molecular weight of 36.89 kDa and a theoretical isoelectric point of 6.81. As found in other plant AOX1 proteins, sequence alignment showed that HsAOX1 had conserved metal binding and hydrophobic ??-helix regions and had high homology to other AOX1 in plants. The expression analysis by semi-quantitative RT-PCR revealed that HsAOX1 was induced in response to cold stress, H₂O₂ treatment, SA, antimycin A and KCN. These results showed that HsAOX1 functions not only during inhibition of cytochrome electron transport but also during oxidative stresses, thus suggesting a role of HsAOX1 in preventing the generation of free radicals by the mitochondrial electron transport chain. The cloning and characterization of the HsAOX1 gene will be useful for further studies of biological roles of HsAOX1 in plants.     

Evidence for H2O2 as the product of reduction of oxygen by alternative oxidase in mitochondria from potato tubers

Oxygen consumption by alternative oxidase (AOX), present in mitochondria of many angiosperms, is known to be cyanide-resistant in contrast to cytochrome oxidase. Its activity in potato tuber (Solanum tuberosum L.) was induced following chilling treatment at 4 °C. About half of the total O₂ consumption of succinate oxidation in such mitochondria was found to be sensitive to SHAM, a known inhibitor of AOX activity. Addition of catalase to the reaction mixture of AOX during the reaction decreased the rate of SHAM-sensitive oxygen consumption by nearly half, and addition at the end of the reaction released nearly half of the consumed oxygen by AOX, both typical of catalase action on H₂O₂. These findings with catalase suggest that the product of reduction of AOX is H₂O₂ and not H₂O, as previously surmised. In potatoes subjected to chill stress (4 °C) for periods of 3, 5 and ?8 days the activity of AOX in mitochondria increased progressively with a corresponding increase in the AOX protein detected by immunoblot of the protein.     

Molecular cloning,refined chromosomal mapping and structural analysis of the human gene encoding aldehyde oxidase (AOX1), a candidate for the ALS2 gene

Summary

Aldehyde oxidase (AOX) is a member of the xanthine oxidase (XO) family of molybdenum hydroxylase, iron-sulfur flavoproteins and is involved in the metabolism of a wide range of native and xenobiotic compounds. The potentially toxic reduced oxygen intermediates (ROI), hydrogen peroxide (H₂O₂) and superoxide anion (O₂^.-), are generated when reduced AOX becomes oxidized by molecular oxygen, raising the possibility for involvement of AOX in pathophysiology. Indeed, ROI generation by AOX has been directly implicated in hepatic ethanol toxicity. A cDNA encoding human AOX has been cloned, sequenced, and identified as AOX1. AOX1 was proposed as a candidate for an autosomal recessive form of amyotrophic lateral sclerosis (ALS2) because a YAC carrying AOX1 was mapped to the ALS2 locus and was expressed in microglial cells of the spinal cord. As a source of H₂O₂, AOX could mediate motor neuron degeneration. To provide a basis for further analysis of AOX1 in pathophysiology, and to examine the relationship of the human AOX1 gene to the gene for human xanthine dehydrogenase (XDH), we have studied the chromosomal locus encoding AOX1 in humans. In the present communication, we have analyzed P1 artificial chromosomes containing AOX1. Our refined chromosomal mapping by FISH locates AOX1 very centromere proximal in the 2q33 region at 2q32.3–2q33.1. We present the first complete structural map of an AOX gene and provide direct evidence that human XDH and AOX1 are related by a gene duplication event. In addition, 1500 bp of upstream DNA containing the putative AOX1 promoter were sequenced and expressed. In contrast to the amino acid coding regions, AOX1 and XDH promoter sequences exhibit marked divergence that reflects the differential activation of these closely related genes. Evidence is presented that AOX may be polygenic in humans as it is in plants, Dipterans, and mice.     

Effect of lead (Pb) on the systemic movement of RNA viruses in tobacco (Nicotiana tabacum var. Turkish)

Effect of various lead (Pb) concentrations on the systemic movement of RNA viruses was examined in tobacco plants. Prior to inoculation, plants were grown hydroponically for 6 days in Hoagland’s solution supplemented with five concentrations of lead nitrate [Pb(NO₃)₂]: 0.0 (control), 10, 15, 50, and 100 μM. Four different RNA viruses with different cell-to-cell movement mechanisms were used. Two weeks after inoculation lower and upper leaves of each treatment were harvested and examined for the presence of viral coat protein. In plants inoculated with Tobacco mosaic virus, Potato virus X, and Tobacco etch virus, TEM images and western blot assays confirmed the presence of viral coat proteins in the upper leaves of all lead treatments. However, in plants inoculated with Turnip vein-clearing virus (TVCV), no signs of viral particles were detected in the upper leaves of plants treated with 10 μM or 15 μM lead nitrate. In contrast, plants treated with high concentrations of lead nitrate (50 μM or 100 μM) showed viral particles in their upper leaves. In plants treated with 10 μM or 15 μM lead nitrate, callose accumulation was the same as in control plants. This suggests that non-toxic concentrations of lead nitrate may trigger the production of putative cellular factors in addition to callose that interfere with the TVCV systemic movement. In contrast, plants treated with 100 μM lead nitrate showed less callose as compared to control plants, facilitating the systemic movement of TVCV.     

Jasmonic Acid-Mediated-Induced Resistance in Groundnut (Arachis hypogaea L.) Against Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae)

Jasmonic acid (JA) acts as a signal molecule to induce resistance in plants against herbivores and its levels are elevated in plants after wounding or insect damage. Groundnut is an important crop in many tropical and subtropical regions worldwide, but there is surprisingly little knowledge on its induced defenses against herbivores. The effect of JA as a spray on induced resistance in three groundnut genotypes, namely, ICGV 86699 (resistant), NCAc 343 (resistant), and TMV 2 (susceptible), against Helicoverpa armigera was studied. The activity of oxidative enzymes [peroxidase (POD) and polyphenol oxidase (PPO)] and the amounts of other host plant defense components [total phenols, hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and protein content] were recorded at 24, 48, 72, and 96 h after pretreatment (1 day) with JA followed by infestation with H. armigera (PJA + HIN) and H. armigera infestation with simultaneous JA application (HIN + JA) to understand the consequences of induced resistance in groundnut. The plant damage, larval survival, and larval weights were also recorded. There was a significant increase in POD and PPO activities and in the amounts of total phenols, H₂O₂, MDA, and proteins in PJA + HIN- and JA + HIN-treated plants as compared to the plants treated with JA and infested with H. armigera individually and to untreated control plants. Among all the genotypes, the strongest induction of defense was observed in the ICGV 86699 genotype. It is concluded that pretreatment with JA and its application during low levels of insect infestation can increase the levels of host plant resistance against herbivorous insects and reduce the pest-associated losses in groundnut.     

A novel role for cyanide in the control of cucumber (Cucumis sativus L.) seedlings response to environmental stress

The effects of potassium cyanide (KCN) pretreatment on the response of cucumber (Cucumis sativus L.) plants to salt, polyethylene glycol (PEG) and cold stress were investigated in the present study. Here, we found that KCN pretreatment improved cucumber seedlings tolerance to stress conditions with maximum efficiency at a concentration of 20 µM. The results showed that pretreatment with 20 µM KCN alleviated stress‐induced oxidative damage in plant cells and clearly induced the activity of alternative oxidase (AOX) and the ethylene production. Furthermore, the structures of thylakoids and mitochondria in the KCN‐pretreated seedlings were less damaged by the stress conditions, which maintained higher total chlorophyll content, photosynthetic rate and photosystem II (PSII) proteins levels than the control. Importantly, the addition of the AOX inhibitor salicylhydroxamic acid (1 mm ; SHAM) decreased plant resistance to environmental stress and even compromised the cyanide (CN)‐enhanced stress tolerance. Therefore, our findings provide a novel role of CN in plant against environmental stress and indicate that the CN‐enhanced AOX might contribute to the reactive oxygen species (ROS) scavenging and the protection of photosystem by maintaining energy charge homoeostasis from chloroplast to mitochondria.     

The alternative pathway in cucumber seedlings under low temperature stress was enhanced by salicylic acid

The alternative pathway is a cyanide-resistant and non-phosphorylatory electron transport pathway in mitochondria of higher plants. Alternative oxidase (AOX) is the terminal oxidase of this pathway. Our present study investigated the effect of exogenous salicylic acid (SA) on alternative pathway in cucumber (Cucumis sativus L.) seedlings under low temperature stress. Results showed that during the process of low temperature stress, the alternative pathway capacity was enhanced as AOX expression increased in SA pretreated seedlings. Compared with seedlings without SA pretreatment, slower decrease of relative water content and lower levels of electrolyte leakage, H₂O₂ and malonyldialdehyde content were detected in SA pretreated seedlings. These results indicated that SA could alleviate the injury caused by low temperature on cucumber seedlings. Since the special protective functions of alternative pathway and AOX in plants, we suggested that the alternative pathway was related to SA-mediated plant resistance to environmental stresses such as low temperature.     

Identification of a region of the Arabidopsis <Emphasis Type="Italic">AtAOX1a</Emphasis> promoter necessary for mitochondrial retrograde regulation of expression

Chemical inhibition of the mitochondrial electron transport chain (mtETC) by antimycin A (AA) or the TCA cycle by monofluoroacetate (MFA) causes increased expression of nucleus-encoded alternative oxidase (AOX) genes in plants. In order to better understand the mechanisms of this mitochondrial retrograde regulation (MRR) of gene expression, constructs containing deleted and mutated versions of a promoter region of the Arabidopsis thaliana AOX1a gene (AtAOX1a) controlling expression of the coding region of the enhanced firefly luciferase gene were employed to identify regions of the AtAOX1a promoter important for induction in response to mtETC or TCA cycle inhibition. Transient transformation coupled with in vitro and in vivo assays as well as plants containing transgenes with truncated promoter regions were used to identify a 93 base pair portion of the promoter, termed the MRR region, that was necessary for induction. Further mutational analyses showed that most of the 93 bp MRR region is important for both AA and MFA induction. Sub-regions within the MRR region that are especially important for strong induction by both AA or MFA were identified. Specific mutations in a W-box and Dof motifs in the MRR region indicate that these specific motifs are not important for induction. Recent evidence suggests that MRR of AOX genes following inhibition of the mtETC is via a separate signaling pathway from MRR resulting from metabolic shifts, such as those that result from MFA treatment. Our data suggest that these signaling pathways share regulatory regions in the AtAOX1a promoter. Arabidopsis proteins interacted specifically with a probe containing the MRR region, as shown by electrophoretic mobility shift assays and Southwestern blotting. These interactions were eliminated under reducing conditions.     

Demonstration of the Specific Involvement of Coat Protein in Tobacco Mosaic Virus (TMV) Cross Protection using a TMV Coat Protein Mutant

The DT-1G mutant of tobacco mosaic virus (TMV) which has no coat protein was used to study the specific involvement of coat protein in TMV cross protection in N. sylvestris. Leaves of N. sylvestris previously inoculated with the mutantor the common strain of TMV were challenged with either turnip mosaic virus (TuMV) or a strain of TMV (TMV-N). Both TuMV and TMV-N produce necrotic lesions on N. sylvestris. About one-half as many lesions were produced by TuMV and TMV-N on leaves, inoculated with the DT-1G mutant compared with lesions produced by the same inoculum on control leaves. When leaves of N. sylvestris previously inoculated with the common strain of TMV were challenged with either TuMV or TMV-N, TuMV produced about one-half as many lesions as on control leaves whereas TMV-N produced about one-tenth as many lesions as on control leaves. A high level of non-specific resistance was induced by the mutant without coat protein, but it did not specifically protect against TMV.     

The cis-expression of the coat protein of turnip mosaic virus is essential for viral intercellular movement in plants

To establish infection, plant viruses are evolutionarily empowered with the ability to spread intercellularly. Potyviruses represent the largest group of known plant-infecting RNA viruses, including many agriculturally important viruses. To better understand intercellular movement of potyviruses, we used turnip mosaic virus (TuMV) as a model and constructed a double-fluorescent (green and mCherry) protein-tagged TuMV infectious clone, which allows distinct observation of primary and secondary infected cells. We conducted a series of deletion and mutation analyses to characterize the role of TuMV coat protein (CP) in viral intercellular movement. TuMV CP has 288 amino acids and is composed of three domains: the N-terminus (amino acids 1–97), the core (amino acids 98–245), and the C-terminus (amino acids 246–288). We found that deletion of CP or its segments amino acids 51–199, amino acids 200–283, or amino acids 265–274 abolished the ability of TuMV to spread intercellularly but did not affect virus replication. Interestingly, deletion of amino acids 6–50 in the N-terminus domain resulted in the formation of aberrant virions but did not significantly compromise TuMV cell-to-cell and systemic movement. We identified the charged residues R178 and D222 within the core domain that are essential for virion formation and TuMV local and systemic transport in plants. Moreover, we found that trans-expression of the wild-type CP either by TuMV or through genetic transformation-based stable expression could not rescue the movement defect of CP mutants. Taken together these results suggest that TuMV CP is not essential for viral genome replication but is indispensable for viral intercellular transport where only the cis-expressed CP is functional.     

Effects of light on cyanide‐resistant respiration and alternative oxidase function in Arabidopsis seedlings

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy wasteful cyanide (CN)‐resistant respiration and plays a role in optimizing photosynthesis. Although it has been demonstrated that leaf AOX is upregulated after illumination, the in vivo mechanism of AOX upregulation by light and its physiological significance are still unknown. In this report, red light and blue light‐induced AOX (especially AOX1a) expressions were characterized. Phytochromes, phototropins and cryptochromes, all these photoreceptors mediate the light‐response of AOX1a gene. When aox1a mutant seedlings were grown under a high‐light (HL) condition, photobleaching was more evident in the mutant than the wild‐type plants. More reactive oxygen species (ROS) accumulation and inefficient dissipation of chloroplast reducing‐equivalents in aox1a mutant may account for its worse adaptation to HL stress. When etiolated seedlings were exposed to illumination for 4 h, chlorophyll accumulation was largely delayed in aox1a plants. We first suggest that more reduction of the photosynthetic electron transport chain and more accumulation of reducing‐equivalents in the mutant during de‐etiolation might be the main reasons.     

The apparent non‐host resistance of Ethiopian mustard to a radish‐infecting strain of Turnip mosaic virus is largely determined by the C‐terminal region of the P3 viral protein

Two different isolates of Turnip mosaic virus (TuMV: UK 1 and JPN 1) belonging to different virus strains were tested on three different Brassica species, namely turnip (Brassica rapa L.), Indian mustard (Brassica juncea L.) and Ethiopian mustard (Brassica carinata A. Braun). Although all three hosts were readily infected by isolate UK 1, isolate JPN 1 was able to establish a visible systemic infection only in the first two. Ethiopian mustard plants showed no local or systemic symptoms, and no virus antigens could be detected by enzyme‐linked immunosorbent assay (ELISA). Thus, this species looks like a non‐host for JPN 1, an apparent situation of non‐host resistance (NHR). Through an experimental approach involving chimeric viruses made by gene interchange between two infectious clones of both virus isolates, the genomic region encoding the C‐terminal domain of viral protein P3 was found to bear the resistance determinant, excluding any involvement of the viral fusion proteins P3N‐PIPO and P3N‐ALT in the resistance. A further determinant refinement identified two adjacent positions (1099 and 1100 of the viral polyprotein) as the main determinants of resistance. Green fluorescent protein (GFP)‐tagged viruses showed that the resistance of Ethiopian mustard to isolate JPN 1 is only apparent, as virus‐induced fluorescence could be found in discrete areas of both inoculated and non‐inoculated leaves. In comparison with other plant–virus combinations of extreme resistance, we propose that Ethiopian mustard shows an apparent NHR to TuMV JPN 1, but not complete immunity or extreme resistance.