Christian Langebartels

Catalase is a sink for H2O2 and is indispensable for stress defence in C3 plants

Hydrogen peroxide (H2O2) has been implicated in many stress conditions. Control of H2O2 levels is complex and dissection of mechanisms generating and relieving H2O2 stress is difficult, particularly in intact plants. We have used transgenic tobacco with ∼10% wild‐type catalase activity to study the role of catalase and effects of H2O2 stress in plants. Catalase‐deficient plants showed no visible disorders at low light, but in elevated light rapidly developed white necrotic lesions on the leaves. Lesion formation required photorespiratory activity since damage was prevented under elevated CO2. Accumulation of H2O2 was not detected during leaf necrosis. Alternative H2O2‐scavenging mechanisms may have compensated for reduced catalase activity, as shown by increased ascorbate peroxidase and glutathione peroxidase levels. Leaf necrosis correlated with accumulation of oxidized glutathione and a 4‐fold decrease in ascorbate, indicating that catalase is critical for maintaining the redox balance during oxidative stress. Such control may not be limited to peroxisomal H2O2 production. Catalase functions as a cellular sink for H2O2, as evidenced by complementation of catalase deficiency by exogenous catalase, and comparison of catalase‐deficient and control leaf discs in removing external H2O2. Stress analysis revealed increased susceptibility of catalase‐deficient plants to paraquat, salt and ozone, but not to chilling.

Ozone-Sensitive Arabidopsis rcd1 Mutant Reveals Opposite Roles for Ethylene and Jasmonate Signaling Pathways in Regulating Superoxide-Dependent Cell Death

We have isolated a codominant Arabidopsis mutant, radical-induced cell death1 (rcd1), in which ozone (O3) and extracellular superoxide (O2•–), but not hydrogen peroxide, induce cellular O2•– accumulation and transient spreading lesions. The cellular O2•– accumulation is ethylene dependent, occurs ahead of the expanding lesions before visible symptoms appear, and is required for lesion propagation. Exogenous ethylene increased O2•–-dependent cell death, whereas impairment of ethylene perception by norbornadiene in rcd1 or ethylene insensitivity in the ethylene-insensitive mutant ein2 and in the rcd1 ein2 double mutant blocked O2•– accumulation and lesion propagation. Exogenous methyl jasmonate inhibited propagation of cell death in rcd1. Accordingly, the O3-exposed jasmonate-insensitive mutant jar1 displayed spreading cell death and a prolonged O2•– accumulation pattern. These results suggest that ethylene acts as a promoting factor during the propagation phase of developing oxyradical-dependent lesions, whereas jasmonates have a role in lesion containment. Interaction and balance between these pathways may serve to fine-tune propagation and containment processes, resulting in alternate lesion size and formation kinetics.

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 (H2O2) 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 H2O2. 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 H2O2 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, H2O2 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 H2O2 transcriptional gene networks were identified. Comparisons with other publicly available transcriptome data sets of abiotically stressed Arabidopsis revealed an important intersection with H2O2-deregulated genes, positioning elevated H2O2 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.

Ozone: An abiotic elicitor of plant defence reactions

The air pollutant ozone has recently been found to resemble fungal elicitors — it can induce plant signal molecules such as ethylene and salicylic acid, as well as certain genes and biosynthetic pathways associated with pathogen and oxidative defence. The action of ambient ozone on the plant defence system may predispose the plant to enhanced attack by pathogens, but may also lead to induced resistance. The results mean that ozone can be regarded as a new experimental tool for analyzing stress responses.

Polyamines as radical scavengers and protectants against ozone damage

Abstract Leaf injury of the ozone-sensitive tobacco cultivar Bel W 3 caused by ozone treatments was prevented to a large extent by root application of putrescine, spermidine or spermine. The titres of soluble free and conjugated putrescine and spermidine were concomitantly increased two- to three-fold after putrescine or spermidine application. The amounts of putrescine and spermidine associated with cell wall or membrane pellet fractions were elevated four to six times above levels of control plants. In order to establish whether the protective effect of polyamines against ozone damage may be caused by their proposed radical scavenging properties, the reactivities of polyamines and putrescine conjugates towards hydroxyl, tert -butoxyl, sulphite radicals and superoxide anions were determined. Free polyamines showed relatively low rate constants with all types of radicals. Only putrescine conjugates with the effective radical scavengers caffeic, ferulic and p -coumaric acid had consistently high rate constants. It is concluded that scavenging of radicals by free polyamines cannot explain the protection against ozone damage observed after exogenous application.

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 anLE-ACO1 promoter-β-glucuronidase fusion construct, β-glucuronidase activity increased rapidly at the beginning of the O3 exposure and had a spatial distribution resembling the pattern of extracellular H2O2 production at 7 h, which coincided with the cell death pattern after 24 h. Ethylene synthesis and perception were required for active H2O2 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 H2O2production, in regulating the spread of cell death.

Oxidative burst and cell death in ozone-exposed plants

The phytotoxic air pollutant ozone spontaneously generates reactive oxygen species (ROS) in the leaf apoplast, provokes hypersensitive response-like lesions and induces defence reactions that significantly overlap with pathogen and other oxidative stress responses. Consequently, ozone has been used as a tool to unravel in planta ROS-induced plant defence and cell death mechanisms. Ozone exposure stimulates an oxidative burst in leaves of sensitive plants, resulting in the generation and accumulation of hydrogen peroxide or superoxide anions in distinct species. Accumulation of these ROS precedes the induction of cell death, and both responses co-occur spatially in the periveinal regions of the leaves. The review summarizes some of the recent results that have been obtained concerning the molecular basis of apoplastic ROS production in monocot and dicot species. Signal molecules, in particular ethylene and salicylic acid, control and potentiate the oxidative burst and subsequent cell death in its initiation and propagation phases while jasmonate leads to lesion containment. Amplification mechanisms that result in the production of excess ROS and hypersensitive cell death are discussed as major factors in ozone sensitivity of plant species and cultivars.

Ozone-induced changes of mRNA levels of β-1,3-glucanase, chitinase and 'pathogenesis-related' protein 1b in tobacco plants

Treatment of the ozone-sensitive tobacco cultivar Bel W3 with an ozone pulse (0.15 μl/l, 5 h) markedly increased the mRNA level of basic β-1,3-glucanase and to a lower degree that of basic chitinase. The increase of β-1,3-glucanase mRNA level occurred within 1 h and showed a transient maximum. Seventeen hours after ozone treatment, the β-1,3-glucanase mRNA level decreased to lower values. The increase of basic chitinase mRNA level was delayed and was less pronounced than that of β-1,3-glucanase mRNA. Cultivar Bel B showed only a small increase of β-1,3-glucanase mRNA level after the same ozone treatment, whereas its basic chitinase mRNA was more strongly induced. Prolonged ozone treatment for 2 days of tobacco Bel W3 led to a persistent level of β-1,3-glucanase and basic chitinase mRNAs, as well as to an increase of acidic chitinase and ‘pathogenesis-related’ (PR) 1b mRNA levels. The results indicate that genes so far considered to code for PR proteins may also be involved in the plant response to oxidative stress.

Anthocyanin accumulation and PAL activity in a suspension culture of Daucus carota L.

Cells of Daucus carota grown in a liquid medium produced large amounts of cyanidin as the only flavonoid aglycon. After inoculation in fresh medium a maximum activity of phenylalanine ammonia lyase (PAL; EC 4.3.1.5) was observed within 24 h. L-α-aminooxy-β-phenylpropionic acid (L-AOPP), thought to be a competitive inhibitor of PAL, inhibited cyanidin accumulation up to 80%. In order to study the regulatory role of PAL, the effects of L-AOPP and t-cinnamic acid, the product of the deamination of phenylalanine, were investigated. Cinnamic acid, applied in vivo (10-4 M), was not able to compensate for the inhibition of cyanidin production caused by L-AOPP (10-4 M) in the same sample. Carrot cells treated with L-AOPP exhibited a “super-induction” of PAL already described for gherkin hypocotyls (Amrhein and Gerhardt 1979). This effect was not influenced by t-cinnamic acid. L-AOPP seems to be a very specific inhibitor since it affected neither growth nor soluble protein content, whereas t-cinnamic acid inhibited both. Investigations on the content of soluble amino acids in L-AOPP-treated cells revealed a specific accumulation of soluble phenylalanine, whereas treatment with t-cinnamic acid led to an increase of amino acids in general, thus indicating that the latter compound has a rather unspecific effect on cellular metabolism. In vitro studies with PAL isolated from Daucus carota revealed that L-AOPP inhibited the enzyme at very low doses (KI=2.4·10-9), whereas t-cinnamic acid, by comparison, affected the enzyme at high concentrations (KI=1.8·10-4).

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.