Promoters of the barley germin-like GER4 gene cluster enable strong transgene expression in response to pathogen attack.

Immunity of plants triggered by pathogen-associated molecular patterns (PAMPs) is based on the execution of an evolutionarily conserved defense response that includes the accumulation of pathogenesis-related (PR) proteins as well as multiple other defenses. The most abundant PR transcript of barley (Hordeum vulgare) leaf epidermis attacked by the powdery mildew fungus Blumeria graminis f. sp hordei encodes the germin-like protein GER4, which has superoxide dismutase activity and functions in PAMP-triggered immunity. Here, we show that barley GER4 is encoded by a dense cluster of tandemly duplicated genes (GER4a-h) that underwent several cycles of duplication. The genomic organization of the GER4 locus also provides evidence for repeated gene birth and death cycles. The GER4 promoters contain multiple WRKY factor binding sites (W-boxes) preferentially located in promoter fragments that were exchanged between subfamily members by gene conversion. Mutational analysis of TATA-box proximal W-boxes used GER4c promoter-beta-glucuronidase fusions to reveal their enhancing effects and functional redundancy on pathogen-induced promoter activity. The data suggest enhanced transcript dosage as an evolutionary driving force for the local expansion and functional redundancy of the GER4 locus. In addition, the GER4c promoter provides a tool to study signal transduction of PAMP-triggered immunity and to engineer strictly localized and pathogen-regulated disease resistance in transgenic cereal crops.

Screening for rapidly evolving genes in the ectomycorrhizal fungus Paxillus involutus using cDNA microarrays.

We have examined the variations in gene content and sequence divergence that could be associated with symbiotic adaptations in the ectomycorrhizal fungus Paxillus involutus and the closely related species Paxillus filamentosus. Strains with various abilities to form mycorrhizae were analysed by comparative genomic hybridizations using a cDNA microarray containing 1076 putative unique genes of P. involutus. To screen for genes diverging at an enhanced and presumably non-neutral rate, we implemented a simple rate test using information from both the variations in hybridizations signal and data on sequence divergence of the arrayed genes relative to the genome of Coprinus cinereus. C. cinereus is a free-living saprophyte and is the closest evolutionary relative to P. involutus that has been fully sequenced. Approximately 17% of the genes investigated were detected as rapidly diverging within Paxillus. Furthermore, 6% of the genes varied in copy numbers between the analysed strains. Genome rearrangements associated with this variation including duplications and deletions may also play a role in adaptive evolution. The cohort of divergent and duplicated genes showed an over-representation of either orphans, genes whose products are located at membranes, or genes encoding for components of stress/defence reactions. Some of the identified genomic changes may be associated with the variation in host specificity of ectomycorrhizal fungi. The proposed procedure could be generally applicable to screen for rapidly evolving genes in closely related strains or species where at least one has been sequenced or characterized by expressed sequence tag analysis.

Divergence in gene expression related to variation in host specificity of an ectomycorrhizal fungus.

Ectomycorrhizae are formed by mutualistic interactions between fungi and the roots of woody plants. During symbiosis the two organisms exchange carbon and nutrients in a specific tissue that is formed at the contact between a compatible fungus and plant. There is considerable variation in the degree of host specificity among species and strains of ectomycorrhizal fungi. In this study, we have for the first time shown that this variation is associated with quantitative differences in gene expression, and with divergence in nucleotide sequences of symbiosis-regulated genes. Gene expression and sequence evolution were compared in different strains of the ectomycorrhizal fungus Paxillus involutus; the strains included Nau, which is not compatible with birch and poplar, and the two compatible strains Maj and ATCC200175. On a genomic level, Nau and Maj were very similar. The sequence identity was 98.9% in the 16 loci analysed, and only three out of 1075 genes analysed by microarray-based hybridizations had signals indicating differences in gene copy numbers. In contrast, 66 out of the 1075 genes were differentially expressed in Maj compared to Nau after contact with birch roots. Thirty-seven of these symbiosis-regulated genes were also differentially expressed in the ATCC strain. Comparative analysis of DNA sequences of the symbiosis-regulated genes in different strains showed that two of them have evolved at an enhanced rate in Nau. The sequence divergence can be explained by a decreased selection pressure, which in turn is determined by lower functional constraints on these proteins in Nau as compared to the compatible strains.

Differential effects of elevated ozone on two hybrid aspen genotypes predisposed to chronic ozone fumigation. Role of ethylene and salicylic acid.

The role of ethylene (ET) signaling in the responses of two hybrid aspen (Populus tremula L. x P. tremuloides Michx.) clones to chronic ozone (O(3); 75 nL L(-1)) was investigated. The hormonal responses differed between the clones; the O(3)-sensitive clone 51 had higher ET evolution than the tolerant clone 200 during the exposure, whereas the free salicylic acid concentration in clone 200 was higher than in clone 51. The cellular redox status, measured as glutathione redox balance, did not differ between the clones suggesting that the O(3) lesions were not a result of deficient antioxidative capacity. The buildup of salicylic acid during chronic O(3) exposure might have prevented the up-regulation of ET biosynthesis in clone 200. Blocking of ET perception with 1-methylcyclopropene protected both clones from the decrease in net photosynthesis during chronic exposure to O(3). After a pretreatment with low O(3) for 9 d, an acute 1.5-fold O(3) elevation caused necrosis in the O(3)-sensitive clone 51, which increased substantially when ET perception was blocked. The results suggest that in hybrid aspen, ET signaling had a dual role depending on the severity of the stress. ET accelerated leaf senescence under low O(3), but under acute O(3) elevation, ET signaling seemed to be required for protection from necrotic cell death.

Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization.

The aim of this review is to assess the mode of action and role of antioxidants as protection from heavy metal stress in roots, mycorrhizal fungi and mycorrhizae. Based on their chemical and physical properties three different molecular mechanisms of heavy metal toxicity can be distinguished: (a) production of reactive oxygen species by autoxidation and Fenton reaction; this reaction is typical for transition metals such as iron or copper, (b) blocking of essential functional groups in biomolecules, this reaction has mainly been reported for non-redox-reactive heavy metals such as cadmium and mercury, (c) displacement of essential metal ions from biomolecules; the latter reaction occurs with different kinds of heavy metals. Transition metals cause oxidative injury in plant tissue, but a literature survey did not provide evidence that this stress could be alleviated by increased levels of antioxidative systems. The reason may be that transition metals initiate hydroxyl radical production, which can not be controlled by antioxidants. Exposure of plants to non-redox reactive metals also resulted in oxidative stress as indicated by lipid peroxidation, H(2)O(2) accumulation, and an oxidative burst. Cadmium and some other metals caused a transient depletion of GSH and an inhibition of antioxidative enzymes, especially of glutathione reductase. Assessment of antioxidative capacities by metabolic modelling suggested that the reported diminution of antioxidants was sufficient to cause H(2)O(2) accumulation. The depletion of GSH is apparently a critical step in cadmium sensitivity since plants with improved capacities for GSH synthesis displayed higher Cd tolerance. Available data suggest that cadmium, when not detoxified rapidly enough, may trigger, via the disturbance of the redox control of the cell, a sequence of reactions leading to growth inhibition, stimulation of secondary metabolism, lignification, and finally cell death. This view is in contrast to the idea that cadmium results in unspecific necrosis. Plants in certain mycorrhizal associations are less sensitive to cadmium stress than non-mycorrhizal plants. Data about antioxidative systems in mycorrhizal fungi in pure culture and in symbiosis are scarce. The present results indicate that mycorrhization stimulated the phenolic defence system in the Paxillus-Pinus mycorrhizal symbiosis. Cadmium-induced changes in mycorrhizal roots were absent or smaller than those in non-mycorrhizal roots. These observations suggest that although changes in rhizospheric conditions were perceived by the root part of the symbiosis, the typical Cd-induced stress responses of phenolics were buffered. It is not known whether mycorrhization protected roots from Cd-induced injury by preventing access of cadmium to sensitive extra- or intracellular sites, or by excreted or intrinsic metal-chelators, or by other defence systems. It is possible that mycorrhizal fungi provide protection via GSH since higher concentrations of this thiol were found in pure cultures of the fungi than in bare roots. The development of stress-tolerant plant-mycorrhizal associations may be a promising new strategy for phytoremediation and soil amelioration measures.

Characterisation of antioxidative systems in the ectomycorrhiza-building basidiomycete Paxillus involutus (Bartsch) Fr. and its reaction to cadmium.

The effect of different cadmium (Cd) concentrations (5, 50 and 500 microM) on growth, Cd accumulation and antioxidative systems was studied in Paxillus involutus, grown in liquid medium. Cd was rapidly accumulated by P. involutus and resulted in growth inhibition within 24 h. Antioxidative enzymes (superoxide dismutase (SOD), EC 1.15.1.1; catalase (CAT), EC 1.11.1.6; monodehydroascorbate radical reductase (MDAR), EC 1.6.5.4; dehydroascorbate reductase (DAR) glutathione reductase (GR), EC 1.8.1.7 and glutathione-dependent peroxidase (GPx), EC 1.11.1.9) were active in the investigated fungus. Furthermore, high concentrations of glutathione but no ascorbate were detected. Cd exposure resulted in a significant induction of SOD activity. However, activities of enzymes responsible for the detoxification of H2O2 showed no Cd-dependent increase or were only transiently induced (CAT, GPx) and no accumulation of H2O2 was detected. Exposure to low Cd concentrations (5 and 50 microM) caused an increase in GR, while 500 microM Cd led to an inhibition of GR and CAT. Increased glutathione concentrations were observed as a consequence of all Cd treatments. These results suggest that the antioxidative protection of the investigated strain of P. involutus was sufficient to avoid Cd-mediated oxidative stress. It is likely that this strain was able to detoxify high concentrations of Cd by transport of Cd into the vacuole because a high correlation between Cd and sulphur in the vacuole was detected by EDX.