Broad-spectrum acquired resistance in barley induced by the Pseudomonas pathosystem shares transcriptional components with Arabidopsis systemic acquired resistance.

Inducible resistance responses play a central role in the defense of plants against pathogen attack. Acquired resistance (AR) is induced alongside defense toward primary attack, providing broad-spectrum protection against subsequent pathogen challenge. The localization and molecular basis of AR in cereals is poorly understood, in contrast with the well-characterized systemic acquired resistance (SAR) response in Arabidopsis. Here, we use Pseudomonas syringae as a biological inducer of AR in barley, providing a clear frame of reference to the Arabidopsis-P. syringae pathosystem. Inoculation of barley leaf tissue with the nonadapted P. syringae pv. tomato avrRpm1 (PstavrRpm1) induced an active local defense response. Furthermore, inoculation of barley with PstavrRpm1 resulted in the induction of broad-spectrum AR at a distance from the local lesion, "adjacent" AR, effective against compatible isolates of P. syringae and Magnaporthe oryzae. Global transcriptional profiling of this adjacent AR revealed similarities with the transcriptional profile of SAR in Arabidopsis, as well as transcripts previously associated with chemically induced AR in cereals, suggesting that AR in barley and SAR in Arabidopsis may be mediated by analogous pathways.

Transient expression of Arabidopsis thaliana ascorbate peroxidase 3 in Nicotiana benthamiana plants infected with recombinant potato virus X.

We have explored the transient over-expression of Arabidopsis thaliana ascorbate peroxidase 3 (APX3) in Nicotiana benthamiana using a viral vector based on the potato virus X (PVX). Plants infected with a PVX:APX3 hybrid had a similar progression of viral particles compared to control plants infected with a PVX:GFP hybrid, indicating that infection was not affected by the over-expression of heterologous APX3. Our results also showed that in PVX:APX3-infected plants, the hybrid virus directed a high level of APX3 expression and the recombinant protein was functional, as inferred from the higher APX activity compared to mock and PVX:GFP hybrid-infected plants. The PVX recombinant expression system used is a simple and quick method for transient expression of heterologous APXs, which are expected to suffer specific processing in plant cells.

Are diverse signalling pathways integrated in the regulation of arabidopsis antioxidant defence gene expression in response to excess excitation energy?

When low-light-grown Arabidopsis rosettes are partially exposed to excess light (EL), the unexposed leaves become acclimated to excess excitation energy (EEE) and consequent photo-oxidative stress. This phenomenon, termed systemic acquired acclimation (SAA), is associated with redox changes in the proximity of photosystem II, changes in foliar H2O2 content and induction of antioxidant defences. The induction of extra-plastidial antioxidant systems is important in the protection of the chloroplast under EL conditions. A larger range of transcripts encoding different antioxidant defence enzymes may be induced in the systemically acclimated leaves and these include those encoded by the glutathione peroxidase (GPX2) and glutathione-S-transferase (GST) genes, which are also highly induced in the hypersensitive response and associated systemic acquired resistance (SAR) in incompatible plant-pathogen interactions. Furthermore, the expression of the SAR-inducible pathogenesis-related protein gene, PR2, is enhanced in SAA leaves. Wounded leaf tissue also shows enhanced systemic induction of a cytosolic ascorbate peroxidase gene (APX2) under EL conditions. These and other considerations, suggest H2O2 and other reactive oxygen species (ROS) could be the common factor in signalling pathways for diverse environmental stresses. These effects may be mediated by changes in the level and redox state of the cellular glutathione pool. Mutants with constitutive expression of a normally EL-inducible APX2 gene have much reduced levels of foliar glutathione. The expression of APX1 and APX3, encoding cytosolic and peroxisome-associated isoforms, respectively, are also under phytochrome-A-mediated control. The expression of these genes is tightly linked to the greening of plastids in etiolated seedlings. These data suggest that part of the developmental processes that bring about the acclimation of leaves to high light includes the configuration of antioxidant defences. Therefore, the linkage between immediate responses of leaves to EL, acclimation of chloroplasts to EEE and the subsequent changes to leaf form and function in high light could be mediated by the activity of foliar antioxidant defences and changes in the concentration of ROS.

Characterisation of pea cytosolic glutathione reductase expressed in transgenic tobacco.

Expression in transgenic tobacco (Nicotiana tabacum L.) of a pea (Pisum sativum L.) GOR2 cDNA, encoding an isoform of glutathione reductase (GOR2), resulted in a 3- to 7-fold elevation of total foliar glutathione reductase (GR) activity. The enzyme encoded by GOR2 was confirmed to be extraplastidial in organelle fractionation studies and was considered most likely to be localised in the cytosol. A partial purification of GOR2 was achieved but a standard affinity chromatography step, using adenosine-2',5'-diphosphate-Sepharose and often employed in the purification of GR from diverse sources, was unsuccessful with this isoform. Preparative isoelectric focussing was employed as part of the purification procedure of GOR2 and a complete separation from plastidial/mitochondrial glutathione reductase (GOR1) was achieved. The isoform GOR2 was shown to have a slower migration on non-denaturing polyacrylamide gels compared with GOR1 and properties typical of GR enzymes from plant sources.

Elevated glutathione biosynthetic capacity in the chloroplasts of transgenic tobacco plants paradoxically causes increased oxidative stress

Glutathione (GSH), a major antioxidant in most aerobic organisms, is perceived to be particularly important in plant chloroplasts because it helps to protect the photosynthetic apparatus from oxidative damage. In transgenic tobacco plants overexpressing a chloroplast-targeted gamma-glutamylcysteine synthetase (gamma-ECS), foliar levels of GSH were raised threefold. Paradoxically, increased GSH biosynthetic capacity in the chloroplast resulted in greatly enhanced oxidative stress, which was manifested as light intensity-dependent chlorosis or necrosis. This phenotype was associated with foliar pools of both GSH and gamma-glutamylcysteine (the immediate precursor to GSH) being in a more oxidized state. Further manipulations of both the content and redox state of the foliar thiol pools were achieved using hybrid transgenic plants with enhanced glutathione synthetase or glutathione reductase activity in addition to elevated levels of gamma-ECS. Given the results of these experiments, we suggest that gamma-ECS-transformed plants suffered continuous oxidative damage caused by a failure of the redox-sensing process in the chloroplast.

Systemic signaling and acclimation in response to excess excitation energy in Arabidopsis.

Land plants are sessile and have developed sophisticated mechanisms that allow for both immediate and acclimatory responses to changing environments. Partial exposure of low light-adapted Arabidopsis plants to excess light results in a systemic acclimation to excess excitation energy and consequent photooxidative stress in unexposed leaves. Thus, plants possess a mechanism to communicate excess excitation energy systemically, allowing them to mount a defense against further episodes of such stress. Systemic redox changes in the proximity of photosystem II, hydrogen peroxide, and the induction of antioxidant defenses are key determinants of this mechanism of systemic acquired acclimation.

Identification of cDNAS encoding plastid-targeted glutathione peroxidase.

A cDNA was isolated from pea leaf RNA which encodes a phospholipid hydroperoxide glutathione peroxidase (PHGPX; E.C. 1.1.1.1.9). The N-terminal section of this PHGPX encodes a recognisable chloroplast transit peptide. Efficient import in vitro of the pre-PHGPX protein into the stroma of isolated pea chloroplasts confirmed that the PHGPX is a chloroplast-located enzyme. The pea PHGPX has highly conserved homologues in Arabidopsis, citrus and Nicotiana sylvestris and the authors suggest that these proteins are also localised in the chloroplast and not in the cytosol as previously supposed.

Photosynthetic electron transport regulates the expression of cytosolic ascorbate peroxidase genes in Arabidopsis during excess light stress.

Exposure of Arabidopsis plants that were maintained under low light (200 mumol of photons m-2 sec-1) to excess light (2000 mumol of photons m-2 sec-1) for 1 hr caused reversible photoinhibition of photosynthesis. Measurements of photosynthetic parameters and the use of electron transport inhibitors indicated that a novel signal transduction pathway was initiated at plastoquinone and regulated, at least in part, by the redox status of the plastoquinone pool. This signal, which preceded the photooxidative burst of hydrogen peroxide (H2O2) associated with photoinhibition of photosynthesis, resulted in a rapid increase (within 15 min) in mRNA levels of two cytosolic ascorbate peroxidase genes (APX1 and APX2). Treatment of leaves with exogenous reduced glutathione abolished this signal, suggesting that glutathione or the redox status of the glutathione pool has a regulatory impact on this signaling pathway. During recovery from photooxidative stress, transcripts for cytosolic glutathione reductase (GOR2) increased, emphasizing the role of glutathione in this stress.

Cloning and characterisation of a cytosolic glutathione reductase cDNA from pea (Pisum sativum L.) and its expression in response to stress.

A second glutathione reductase (GR) cDNA has been cloned and sequenced from pea (Pisum sativum L. cv. Birte). This new GR cDNA (GOR2) does not encode a pre-protein with a transit peptide and therefore is most likely to represent a cytosolic GR. It is significantly different at the DNA level from the previously cloned chloroplastidial/mitochondrial pea GR (GOR1), but retains the features characteristic of GRs from all sources and has GR activity when expressed in Escherichia coli. GOR2 maps to linkage group 6 on the pea genome map and it seems likely that this is the only locus for this gene. In contrast to GOR1, transcript levels of GOR2 increase in the recovery (post-stress) phases of both drought and chilling by about ten- and three-fold respectively. GOR2 therefore may play a role in the restoration of the post-stress redox state of the cytosolic glutathione pool.

Manipulation of glutathione metabolism in transgenic plants.

There is clear potential for the genetic manipulation of key enzymes involved in stress metabolism in transgenic plants. However, the data emerging so far from such experiments are equivocal. The detailed analysis of stress responses in progeny of primary transgenics, coupled with comparisons with control transgenic plants that do not contain the GR transgene, allows us to take into account the possible variation in response to stress associated with regeneration of plants from tissue culture. The picture that is now beginning to emerge with respect to the role of GR in stress protection is that, although there are clearly benefits to be had from overexpression of the enzymes, there is no direct correlation between enzyme levels and stress tolerance. It may be that overexpression of the cytosolic isoform (gor2) will prove to be of greater benefit. Furthermore, the types of stresses to which transgenic plants have been exposed in order to assess the consequences of oxidative stress tolerance cannot reproduce those that will experienced in field conditions. Only when plants with higher GR levels and increased glutathione synthesis capacity are grown in field trials will it be possible to make a full assessment of the benefits of engineering plants with altered glutathione metabolism.

Characterisation of a glutathione reductase gene and its genetic locus from pea (Pisum sativum L.).

A cDNA encoding the chloroplast/mitochondrial form of glutathione reductase (GR; EC 1.6.4.2) from pea (Pisum sativum L.) was used to map a single GR locus, named GOR1. In two domesticated genotypes of pea (cv. Birte and JI 399) it is likely that the GOR1 locus contains a single gene. However, in a semi-domesticated land race of pea (JI 281) two distinct but closely related sets of GR gene sequences were detected at the GOR1 locus. The extra GR sequences in JI 281 represent either a second intact gene or a partial or pseudogene copy. A GR gene was cloned from cv. Birte, sequenced and its structure analysed. No feature of the transcription or structure of the gene suggested a mechanism for generating any more than one form of GR. From these data plus previously published biochemical evidence we suggest that a second, distinct gene encoding for the cytosolic form of GR should be present in peas. The GOR1-encoded GR mRNA can be detected in all main organs of the plant and no alternative spliced species was present which could perhaps account for the generation of multiple isoforms of GR. The mismatch between the number of charge-separable isoforms in pea and the proposed number of genes suggests that different GR isoforms arise by some form of post-translational modification.

Cytosolic ascorbate peroxidase from Arabidopsis thaliana L. is encoded by a small multigene family.

A second cytosolic ascorbate peroxidase (cAPX; EC 1.11.1.11) gene from Arabidopsis thaliana has been characterised. This second gene (designated APX1b) maps to linkage group 3 and potentially encodes a cAPX as closely related to that from other dicotyledonous species as to the other member of this gene family (Kubo et al., 1993, FEBS Lett 315: 313 317; here designated APX1a), which maps to linkage group 1. In contrast, the lack of sequence similarity in non-coding regions of the genes implies that they are differentially regulated. Under non-stressed conditions only APX1a is expressed. APX1b was identified during low-stringency probing using a cDNA coding for pea cAPX which, in turn, was recovered from a cDNA library by immunoscreening with an antiserum raised against tea plastidial APX (pAPX). No pAPX cDNAs were recovered, despite the antiserum displaying specificity for pAPX in Western blots.

Simultaneous targeting of pea glutathione reductase and of a bacterial fusion protein to chloroplasts and mitochondria in transgenic tobacco.

N-terminal presequences from cDNAs encoding mitochondrion- or chloroplast-specific proteins are able, with variable efficiencies, to target preproteins to their respective organelles. In the few cases studied in which a nuclear-encoded protein is found in both these organelles, each compartment-specific isoform is encoded by a separate gene. Glutathione reductase (GR) from peas is encoded by a single nuclear gene and yet GR is distributed between chloroplasts, mitochondria and the cytosol. Previous sequence analysis of a full-length GR cDNA revealed the presence of a putative plastid transit peptide. However, expression of this cDNA in transgenic tobacco resulted in substantially elevated GR activities in both chloroplasts and mitochondria in four independent lines examined. There was no effect on expression of the endogenous tobacco GR genes. Replacement of the GR presequence with presequences from pea rbcS (chloroplast) and Nicotiana plumbaginifolia Mn-SOD (mitochondrion) resulted in targeting of GR only into the appropriate organelle. Expression of a fusion protein between the amino terminal region of GR and phosphinothricin acetyl transferase resulted in targeting of the foreign protein to chloroplasts and mitochondria. Thus, the pea GR presequence is capable of co-targeting this enzyme or a foreign protein to chloroplasts and mitochondria in vivo. This is the first example of co-targeting by a higher plant preprotein.

Cloning and characterisation of glutathione reductase cDNAs and identification of two genes encoding the tobacco enzyme.

We have isolated 4 cDNA clones (GRT1-4) encoding glutathione reductase (GR) from a tobacco (Nicotiana tabacum L.) leaf cDNA library. The cDNAs were almost identical: GRT1, GRT3 and GRT4 represented the same gene, differing only in that GRT4 contained an intron within the C-terminal part of the coding sequence. Failure to splice out this intron resulted in a substitution of the final 13 amino acids of the deduced amino acid sequence. A second gene was represented by GRT2. Southern blots indicated that there were two related GR genes in tobacco. The presence of multiple isoforms of GR in tobacco may be explained in part by the expression of a small gene family. In addition, alternative isoforms may result from translation of different mRNAs derived from the same gene by intron skipping during the splicing of nascent GR mRNAs.

Molecular characterization of glutathione reductase cDNAs from pea (Pisum sativum L.).

A cDNA for pea glutathione reductase has been cloned and sequenced. The derived amino acid sequence of 562 residues shows a high degree of homology to the previously published GR sequences from human erythrocytes and from two prokaryotes: Escherichia coli and Pseudomonas aeruginosa. The pea enzyme differs from other GRs in having an N-terminal leader sequence of about 60-70 residues which may be a chloroplast transit peptide and a 20 amino acid C-terminal extension of unknown function.

Agrobacterium - and microprojectile - mediated viral DNA delivery into barley microspore-derived cultures.

Anther cultures of barley (Hordeum vulgare L. var. "Igri") were used as targets for Agrobacterium-mediated DNA transfer and direct DNA uptake by particle bombardment. A wheat dwarf virus construct which can replicate to a high copy number in cereal cells provided a sensitive marker for successful DNA delivery. Although DNA delivery was achieved using both procedures, particle bombardment gave more reproducible and higher levels of infection. The ability to deliver DNA into cereal cells which have a high regeneration capacity may provide a route for stable transformation.