PAR modulation of the UV-dependent levels of flavonoid metabolites in Arabidopsis thaliana (L.) Heynh. leaf rosettes: cumulative effects after a whole vegetative growth period.

Long-term effects of ultraviolet (UV) radiation on flavonoid biosynthesis were investigated in Arabidopsis thaliana using the sun simulators of the Helmholtz Zentrum München. The plants, which are widely used as a model system, were grown (1) at high photosynthetically active radiation (PAR; 1,310 micromol m(-2) s(-1)) and high biologically effective UV irradiation (UV-B(BE) 180 mW m(-2)) during a whole vegetative growth period. Under this irradiation regime, the levels of quercetin products were distinctively elevated with increasing UV-B irradiance. (2) Cultivation at high PAR (1,270 micromol m(-2) s(-1)) and low UV-B (UV-B(BE) 25 mW m(-2)) resulted in somewhat lower levels of quercetin products compared to the high-UV-B(BE) conditions, and only a slight increase with increasing UV-B irradiance was observed. On the other hand, when the plants were grown (3) at low PAR (540 micromol m(-2) s(-1)) and high UV-B (UV-B(BE) 180 mW m(-2)), the accumulation of quercetin products strongly increased from very low levels with increasing amounts of UV-B but the accumulation of kaempferol derivatives and sinapoyl glucose was less pronounced. We conclude (4) that the accumulation of quercetin products triggered by PAR leads to a basic UV protection that is further increased by UV-B radiation. Based on our data, (5) a combined effect of PAR and different spectral sections of UV radiation is satisfactorily described by a biological weighting function, which again emphasizes the additional role of UV-A (315-400 nm) in UV action on A. thaliana.

Intra- and interspecific differences of 10 barley and 10 tomato cultivars in response to short-time UV-B radiation: a study analysing thermoluminescence, fluorescence, gas-exchange and biochemical parameters.

The impact of UV-B radiation on 10 genotypically different barley and tomato cultivars was tested in a predictive study to screen for potentially UV-tolerant accessions and to analyze underlying mechanisms for UV-B sensitivity. Plant response was analyzed by measuring thermoluminescence, fluorescence, gas exchange and antioxidant status. Generally, barley cultivars proved to be much more sensitive against UV-B radiation than tomato cultivars. Statistical cluster analysis could resolve two barley groups with distinct differences in reaction patterns. The UV-B sensitive group showed a stronger loss in PSII photochemistry and a lower gas-exchange performance and regulation after UV-B radiation compared to the more tolerant group. The results indicate that photosynthetic light and dark reactions have to play optimally in concert to render plants more tolerant against UV-B radiation. Hence, measuring thermoluminescence/fluorescence and gas exchange in parallel will have much higher potential in identifying tolerant cultivars and will help to understand the underlying mechanisms.

Complex phenotypic profiles leading to ozone sensitivity in Arabidopsis thaliana mutants.

Genetically tractable model plants offer the possibility of defining the plant O(3) response at the molecular level. To this end, we have isolated a collection of ozone (O(3))-sensitive mutants of Arabidopsis thaliana. Mutant phenotypes and genetics were characterized. Additionally, parameters associated with O(3) sensitivity were analysed, including stomatal conductance, sensitivity to and accumulation of reactive oxygen species, antioxidants, stress gene-expression and the accumulation of stress hormones. Each mutant has a unique phenotypic profile, with O(3) sensitivity caused by a unique set of alterations in these systems. O(3) sensitivity in these mutants is not caused by gross deficiencies in the antioxidant pathways tested here. The rcd3 mutant exhibits misregulated stomata. All mutants exhibited changes in stress hormones consistent with the known hormonal roles in defence and cell death regulation. One mutant, dubbed re-8, is an allele of the classic leaf development mutant reticulata and exhibits phenotypes dependent on light conditions. This study shows that O(3) sensitivity can be determined by deficiencies in multiple interacting plant systems and provides genetic evidence linking these systems.

Induction of systemic resistance in tomato by N-acyl-L-homoserine lactone-producing rhizosphere bacteria.

N-acyl-L-homoserine lactone (AHL) signal molecules are utilized by Gram-negative bacteria to monitor their population density (quorum sensing) and to regulate gene expression in a density-dependent manner. We show that Serratia liquefaciens MG1 and Pseudomonas putida IsoF colonize tomato roots, produce AHL in the rhizosphere and increase systemic resistance of tomato plants against the fungal leaf pathogen, Alternaria alternata. The AHL-negative mutant S. liquefaciens MG44 was less effective in reducing symptoms and A. alternata growth as compared to the wild type. Salicylic acid (SA) levels were increased in leaves when AHL-producing bacteria colonized the rhizosphere. No effects were observed when isogenic AHL-negative mutant derivatives were used in these experiments. Furthermore, macroarray and Northern blot analysis revealed that AHL molecules systemically induce SA- and ethylene-dependent defence genes (i.e. PR1a, 26 kDa acidic and 30 kDa basic chitinase). Together, these data support the view that AHL molecules play a role in the biocontrol activity of rhizobacteria through the induction of systemic resistance to pathogens.

In situ quantitation of the spatial scale of calling distances and population density-independent N-acylhomoserine lactone-mediated communication by rhizobacteria colonized on plant roots.

We used computer-assisted microscopy at single cell resolution to quantify the in situ spatial scale of N-acylhomoserine lactone (AHL)-mediated cell-to-cell communication of Pseudomonas putida colonized on tomato and wheat root surfaces. The results of this in situ quantification study on close-to-natural surfaces challenge the conventional view of a quorum group requirement of high cell densities for this type of bacterial communication. In situ image analysis indicated that the effective 'calling distance' on root surfaces was most frequent at 4-5 microm, extended to 37 microm in the root tip/elongation zone and further out to 78 microm in the root hair zone. The spatial scale of these calling distances is very long-range in proportion to the size of individual bacteria. Geostatistical modeling analysis implicated the importance of AHL-gradients mediating effective communication between remote cells. We conclude that AHL-mediated cell-to-cell communication occurs not only within dense populations, but also in very small groups and over long ranges between individual bacteria, and therefore this cellular activity is more commonplace and effective than hitherto predicted. We propose that this cell-to-cell communication is governed more by the in situ spatial proximity of cells within AHL-gradients than the requirement for a quorum group of high population density.

Differences in the kinetics and scale of signalling molecule production modulate the ozone sensitivity of hybrid poplar clones: the roles of H2O2, ethylene and salicylic acid.

Hydrogen peroxide (H(2)O(2)), ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC) and salicylic acid (SA) concentrations and ACC synthase (ACS) gene expression were measured to establish whether the high sensitivity of the Populus deltoides x maximowiczii clone Eridano to ozone (O(3)) exposure, compared with the O(3)-resistant Populus deltoides x euramericana clone I-214, is attributable to differences in the modulation of signal transduction pathways. In a time-course experiment, Populus deltoides (poplar) clones were exposed to acute fumigation with 150 nl l(-1) O(3) for 5 h. The two poplar clones showed differences in ethylene evolution, I-214 displaying earlier and less pronounced ethylene emission than Eridano. In both clones, ethylene evolution was accompanied by increased ACS transcript levels and enhanced emission of free ACC. I-214 exhibited a greater basal concentration of free SA and a lower concentration of the conjugated pool. However, a slight accumulation of free SA at the end of the 5-h exposure was found only in Eridano, together with an earlier minimal increase in the concentration of conjugated SA. The results show that both clones react to O(3) by producing H(2)O(2), ethylene and SA, but the difference in sensitivity to the pollutant is probably attributable to differences in the kinetics and magnitude of this response.

Genome-wide analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a high light-induced transcriptional cluster involved in anthocyanin biosynthesis.

In plants, reactive oxygen species and, more particularly, hydrogen peroxide (H(2)O(2)) play a dual role as toxic by-products of normal cell metabolism and as regulatory molecules in stress perception and signal transduction. Peroxisomal catalases are an important sink for photorespiratory H(2)O(2). Using ATH1 Affymetrix microarrays, expression profiles were compared between control and catalase-deficient Arabidopsis (Arabidopsis thaliana) plants. Reduced catalase levels already provoked differences in nuclear gene expression under ambient growth conditions, and these effects were amplified by high light exposure in a sun simulator for 3 and 8 h. This genome-wide expression analysis allowed us to reveal the expression characteristics of complete pathways and functional categories during H(2)O(2) stress. In total, 349 transcripts were significantly up-regulated by high light in catalase-deficient plants and 88 were down-regulated. From this data set, H(2)O(2) was inferred to play a key role in the transcriptional up-regulation of small heat shock proteins during high light stress. In addition, several transcription factors and candidate regulatory genes involved in H(2)O(2) transcriptional gene networks were identified. Comparisons with other publicly available transcriptome data sets of abiotically stressed Arabidopsis revealed an important intersection with H(2)O(2)-deregulated genes, positioning elevated H(2)O(2) levels as an important signal within abiotic stress-induced gene expression. Finally, analysis of transcriptional changes in a combination of a genetic (catalase deficiency) and an environmental (high light) perturbation identified a transcriptional cluster that was strongly and rapidly induced by high light in control plants, but impaired in catalase-deficient plants. This cluster comprises the complete known anthocyanin regulatory and biosynthetic pathway, together with genes encoding unknown proteins.

Response patterns in adult forest trees to chronic ozone stress: identification of variations and consistencies.

The responsiveness of adult beech and spruce trees to chronic O(3) stress was studied at a free-air O(3) exposure experiment in Freising/Germany. Over three growing seasons, gas exchange characteristics, biochemical parameters, macroscopic O(3) injury and the phenology of leaf organs were investigated, along with assessments of branch and stem growth as indications of tree performance. To assess response pattern to chronic O(3) stress in adult forest trees, we introduce a new evaluation approach, which provides a comprehensive, readily accomplishable overview across several tree-internal scaling levels, different canopy regions and growing seasons. This new approach, based on a three-grade colour coding, combines statistical analysis and the proficient ability of the "human eye" in pattern recognition.

Catalase deficiency drastically affects gene expression induced by high light in Arabidopsis thaliana.

In plants, hydrogen peroxide (H(2)O(2)) plays a major signaling role in triggering both a defense response and cell death. Increased cellular H(2)O(2) levels and subsequent redox imbalances are managed at the production and scavenging levels. Because catalases are the major H(2)O(2) scavengers that remove the bulk of cellular H(2)O(2), altering their levels allows in planta modulation of H(2)O(2) concentrations. Reduced peroxisomal catalase activity increased sensitivity toward both ozone and photorespiratory H(2)O(2)-induced cell death in transgenic catalase-deficient Arabidopsis thaliana. These plants were used as a model system to build a comprehensive inventory of transcriptomic variations, which were triggered by photorespiratory H(2)O(2) induced by high-light (HL) irradiance. In addition to an H(2)O(2)-dependent and -independent type of transcriptional response during light stress, microarray analysis on both control and transgenic catalase-deficient plants, exposed to 0, 3, 8, and 23 h of HL, revealed several specific regulatory patterns of gene expression. Thus, photorespiratory H(2)O(2) has a direct impact on transcriptional programs in plants.

Changes in hydrogen peroxide homeostasis trigger an active cell death process in tobacco.

In transgenic tobacco plants with reduced catalase activity, high levels of hydrogen peroxide (H2O2) can accumulate under photorespiratory conditions. Such a perturbation in H2O2 homeostasis induced cell death in clusters of palisade parenchyma cells, primarily along the veins. Ultrastructural alterations, such as chromatin condensation and disruption of mitochondrial integrity, took place before cell death. Furthermore, enhanced transcript levels of mitochondrial defense genes accompanied these mitochondrial changes. Pharmacological data indicated that the initiation and execution of cell death require de novo protein synthesis and that the signal transduction pathway leading to cell death involved changes in ion homeostasis, (de)phosphorylation events and an oxidative burst, as observed during hypersensitive responses. This oxidase-dependent oxidative burst is essential for cell death, but it is not required for the accumulation of defense proteins, suggesting a more prominent role for the oxidative burst in abiotic stress-induced cell death.

Ozone-induced gene expression occurs via ethylene-dependent and -independent signalling.

Recent studies suggest that ethylene is involved in signalling ozone-induced gene expression. We show here that application of ozone increased glucuronidase (GUS) expression of chimeric reporter genes regulated by the promoters of the tobacco class I beta-1,3-glucanases (GLB and Gln2) and the grapevine resveratrol synthase (Vst1) genes in transgenic tobacco leaves. 5'-deletion analysis of the class I beta-1,3-glucanase promoter revealed that ozone-induced gene regulation is mainly mediated by the distal enhancer region containing the positively acting ethylene-responsive element (ERE). In addition, application of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, blocked ozone-induced class I beta-1,3-glucanase promoter activity. Enhancer activity and ethylene-responsiveness depended on the integrity of the GCC boxes, cis-acting elements present in the ERE of the class I beta-1,3-glucanase and the basic-type pathogenesis-related PR-1 protein (PRB-1b) gene promoters. The minimal PRB-1b promoter containing only the ERE with intact GCC boxes, was sufficient to confer 10-fold ozone inducibility to a GUS-reporter gene, while a substitution mutation in the GCC box abolished ozone responsiveness. The ERE region of the class I beta-1,3-glucanase promoter containing two intact GCC boxes confered strong ozone inducibility to a minimal cauliflower mosaic virus (CaMV) 35S RNA promoter, whereas two single-base substitution in the GCC boxes resulted in a complete loss of ozone inducibility. Taken together, these datastrongly suggest that ethylene is signalling ozone-induced expression of class I beta-l,3-glucanase and PRB-1b genes. Promoter analysis of the stilbene synthase Vst1 gene unravelled different regions for ozone and ethylene-responsiveness. Application of 1-MCP blocked ethylene-induced Vst1 induction, but ozone induction was not affected. This shows that ozone-induced gene expression occurs via at least two different signalling mechanisms and suggests an additional ethylene independent signalling pathway for ozone-induced expression of genes involved in phytoalexin biosynthesis.

Ethylene synthesis regulated by biphasic induction of 1-aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase genes is required for hydrogen peroxide accumulation and cell death in ozone-exposed tomato.

We show that above a certain threshold concentration, ozone leads to leaf injury in tomato (Lycopersicon esculentum). Ozone-induced leaf damage was preceded by a rapid increase in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activity, ACC content, and ethylene emission. Changes in mRNA levels of specific ACC synthase, ACC oxidase, and ethylene receptor genes occurred within 1 to 5 h. Expression of the genes encoding components of ethylene biosynthesis and perception, and biochemistry of ethylene synthesis suggested that ozone-induced ethylene synthesis in tomato is under biphasic control. In transgenic plants containing an LE-ACO1 promoter-beta-glucuronidase fusion construct, beta-glucuronidase activity increased rapidly at the beginning of the O(3) exposure and had a spatial distribution resembling the pattern of extracellular H(2)O(2) production at 7 h, which coincided with the cell death pattern after 24 h. Ethylene synthesis and perception were required for active H(2)O(2) production and cell death resulting in visible tissue damage. The results demonstrate a selective ozone response of ethylene biosynthetic genes and suggest a role for ethylene, in combination with the burst of H(2)O(2) production, in regulating the spread of cell death.