Dieter Ernst

Crosstalk and differential response to abiotic and biotic stressors reflected at the transcriptional level of effector genes from secondary metabolism

Plant secondary metabolism significantly contributes to defensive measures against adverse abiotic and biotic cues. To investigate stress-induced, transcriptional alterations of underlying effector gene families, which encode enzymes acting consecutively in secondary metabolism and defense reactions, a DNA array (MetArray) harboring gene-specific probes was established. It comprised complete sets of genes encoding 109 secondary product glycosyltransferases and 63 glutathione-utilizing enzymes along with 62 cytochrome P450 monooxygenases and 26 ABC transporters. Their transcriptome was monitored in different organs of unstressed plants and in shoots in response to herbicides, UV-B radiation, endogenous stress hormones, and pathogen infection. A principal component analysis based on the transcription of these effector gene families defined distinct responses and crosstalk. Methyl jasmonate and ethylene treatments were separated from a group combining reactions towards two sulfonylurea herbicides, salicylate and an avirulent strain of Pseudomonas syringae pv. tomato. The responses to the herbicide bromoxynil and UV-B radiation were distinct from both groups. In addition, these analyses pinpointed individual effector genes indicating their role in these stress responses. A small group of genes was diagnostic in differentiating the response to two herbicide classes used. Interestingly, a subset of genes induced by P. syringae was not responsive to the applied stress hormones. Small groups of comprehensively induced effector genes indicate common defense strategies. Furthermore, homologous members within branches of these effector gene families displayed differential expression patterns either in both organs or during stress responses arguing for their non-redundant functions.

Biochemical Plant Responses to Ozone (IV. Cross-Induction of Defensive Pathways in Parsley (Petroselinum crispum L.) Plants).

Parsley (Petroselinum crispum L.) is known to respond to ultraviolet irradiation by the synthesis of flavone glycosides, whereas fungal or elicitor stress leads to the synthesis of furanocoumarin phytoalexins. We tested how these defensive pathways are affected by a single ozone treatment (200 nL L-1; 10 h). Assays were performed at the levels of transcripts, for enzyme activities, and for secondary products. The most rapid transcript accumulation was maximal at 3 h, whereas flavone glycosides and furanocoumarins were maximally induced at 12 and 24 h, respectively, after the start of ozone treatment. Ozone acted as a cross-inducer because the two distinct pathways were simultaneously induced. These results are consistent with the previously observed ozone induction of fungal and viral defense reactions in tobacco, spruce, and pine.

An ozone-responsive region of the grapevine resveratrol synthase promoter differs from the basal pathogen-responsive sequence.

Stilbene synthase (STS) is an enzyme involved in the biosynthesis of stilbenes, which are synthesized in various plants in response to pathogen attack, UV irradiation or exposure to ozone. We describe analysis of an ozone inducible STS transcript and its corresponding promoter (Vst1), combined with the β-glucuronidase (GUS) reporter gene. A single ozone pulse (0.1 µl/l, 10 h) resulted in 11-fold GUS expression. Histochemical localization of GUS activity revealed small spots distributed over the whole leaf. Cross-sections of leaf tissue showed that the Vst1 promoter was induced in palisade and spongy parenchyma cells and to a lesser extent in epidermal cells. Deletions at the 5′ end showed that a partial promoter sequence between position − 430 and− 280 constituted the ozone-responsive region, whereas for effective pathogen-inducibility sequences from− 280 to −140 have been shown to be necessary.

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.

The challenge of making ozone risk assessment for forest trees more mechanistic.

Upcoming decades will experience increasing atmospheric CO2 and likely enhanced O3 exposure which represents a risk for the carbon sink strength of forests, so that the need for cause-effect related O3 risk assessment increases. Although assessment will gain in reliability on an O3 uptake basis, risk is co-determined by the effective dose, i.e. the plants sensitivity per O3 uptake. Recent progress in research on the molecular and metabolic control of the effective O3 dose is reported along with advances in empirically assessing O3 uptake at the whole-tree and stand level. Knowledge on both O3 uptake and effective dose (measures of stress avoidance and tolerance, respectively) needs to be understood mechanistically and linked as a pre-requisite before practical use of process-based O3 risk assessment can be implemented. To this end, perspectives are derived for validating and promoting new O3 flux-based modelling tools.

Molecular cloning, sequence analysis and elicitor-/ozone-induced accumulation of cinnamyl alcohol dehydrogenase from Norway spruce (Picea abies L.)

Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. We have previously isolated pure CAD enzyme from Norway spruce (Picea abies L.) cell culture. Here we report on partial protein sequences of the 42 kDa CAD polypeptide. A cDNA encoding CAD was isolated from the spruce cell culture. The open reading frame of a full-length cDNA coded for a 357 amino acid polypeptide with a calculated Mr of 38 777 Da. The identity of the deduced polypeptide was verified by comparison with amino acid sequences of tryptic peptides from the purified enzyme. Southern blot analysis showed the presence of only one gene for CAD. Sequence comparison with CAD from tobacco and with a N-terminal protein sequence from loblolly pine CAD showed an identity of 69.7% and 91.5%, respectively. Treatment of spruce cell cultures with elicitor, as well as of seedlings with ozone both markedly increased the CAD mRNA level.

Isolation of functional RNA from plant tissues rich in phenolic compounds

A method for the isolation of RNA from different tissues of trees (seedlings, saplings, and adult trees) is described. Using this procedure it is possible to remove large amounts of disturbing polyphenolic compounds from nucleic acids. The method involves an acetone treatment of the freeze-dried and powdered plant material, the use of high salt concentrations in the extraction buffer and an aqueous two-phase system. These steps were combined with the conventional phenol/chloroform extraction and CsCl centrifugation. The method has been successfully applied to the isolation and purification of RNA from pine (Pinus sylvestris L. and Pinus mugo Turr.), Norway spruce (Picea abies L.), and beech (Fagus sylvatica L.). The functional quality of RNA extracted by this procedure has been characterized by its uv spectrum, by agarose gel electrophoresis with ethidium bromide staining, Northern blot hybridization, and in vitro translation.

Forests under climate change and air pollution: Gaps in understanding and future directions for research

Forests in Europe face significant changes in climate, which in interaction with air quality changes, may significantly affect forest productivity, stand composition and carbon sequestration in both vegetation and soils. Identified knowledge gaps and research needs include: (i) interaction between changes in air quality (trace gas concentrations), climate and other site factors on forest ecosystem response, (ii) significance of biotic processes in system response, (iii) tools for mechanistic and diagnostic understanding and upscaling, and (iv) the need for unifying modelling and empirical research for synthesis. This position paper highlights the above focuses, including the global dimension of air pollution as part of climate change and the need for knowledge transfer to enable reliable risk assessment. A new type of research site in forest ecosystems (supersites) will be conducive to addressing these gaps by enabling integration of experimentation and modelling within the soil-plant-atmosphere interface, as well as further model development.

Ethylenediurea (EDU): A research tool for assessment and verification of the effects of ground level ozone on plants under natural conditions

Ethylenediurea (EDU) has been widely used to prevent ozone (O(3)) injury and crop losses in crop plants and growth reductions in forest trees. Successful use requires establishing a dose/response curve for EDU and the proposed plant in the absence of O(3) and in the presence of O(3) before initiating multiple applications to prevent O(3) injury. EDU can be used to verify foliar O(3) symptoms in the field, and to screen plants for sensitivity to O(3) under ambient conditions. Despite considerable research, the mode of action of EDU remains elusive. Additional research on the mode of action of EDU in suppressing O(3) injury in plants may also be helpful in understanding the mode of action of O(3) in causing injury in plants.

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; 1,310xa0µmolxa0m−2u2009s−1) and high biologically effective UV irradiation (UV-BBE 180xa0mWxa0m−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 (1,270xa0µmolxa0m−2u2009s−1) and low UV-B (UV-BBE 25xa0mWxa0m−2) resulted in somewhat lower levels of quercetin products compared to the high-UV-BBE 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 (540xa0µmolxa0m−2u2009s−1) and high UV-B (UV-BBE 180xa0mWxa0m−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 (315–400xa0nm) in UV action on A. thaliana.