Fraxinus mandshurica 4-coumarate-CoA ligase 2 enhances drought and osmotic stress tolerance of tobacco by increasing coniferyl alcohol content.

4-Coumarate-CoA ligase (4CL) is an important branch point in the phenylpropane pathway and plays important roles in plant growth and development. In this study, the 4CL2 gene from Fraxinus mandshurica (designated Fm4CL2) was identified and isolated. Sequence analysis revealed that Fm4CL2 is a type I 4CL gene involved in lignin biosynthesis. Analysis of cell wall components revealed that Fm4CL2-overexpressing (OE-Fm4CL2) tobacco showed increased lignin content (by 58.9%) and decreased hemicellulose content (by 41.2%). Detection of small-molecule metabolites in the lignin pathway revealed that coumaric acid content decreased by 48% and coniferyl alcohol content increased by 250% compared with the control values. Compared with wild type, OE-Fm4CL2 tobacco showed increased xylem cell layer number (by 120%) and cell wall thickness (by 54.5%). Under osmotic stress, transgenic tobacco showed higher growth than wild-type tobacco. The germination rate of transgenic tobacco was higher than that of wild type. Reactive oxygen species accumulation and malondialdehyde content were significantly lower in transgenic tobacco than in wild type. Under drought, the expression of stress-related genes was higher in 35S-Fm4CL2-infected Fraxinus mandshurica plants than in control plants. These results indicate that Fm4CL2 overexpression can enhance drought and osmotic stress tolerance of plants.

Higher Activities of Defense-Associated Enzymes may Contribute to Greater Resistance of Manchurian Ash to Emerald Ash Borer Than A closely Related and Susceptible Congener.

Emerald ash borer (EAB) is an invasive beetle native to Asia that infests and kills ash (Fraxinus spp.) in North America. Previous experiments indicated that larvae feeding on co-evolved, resistant Manchurian ash (F. mandshurica) have increased antioxidant and quinone-protective enzyme activities compared to larvae feeding on susceptible North American species. Here, we examined mechanisms of host-generated oxidative and quinone-based stress and other putative defenses in Manchurian ash and the closely related and chemically similar, but susceptible, black ash (F. nigra), with and without exogenous application of methyl jasmonate (MeJA) to induce resistance mechanisms. Peroxidase activities were 4.6-13.3 times higher in Manchurian than black ash, although both species appeared to express the same three peroxidase isozymes. Additionally, peroxidase-mediated protein cross-linking activity was stronger in Manchurian ash. Polyphenol oxidase, ß-glucosidase, chitinase, and lipoxygenase activities also were greater in Manchurian ash, but only lipoxygenase activity increased with MeJA application. Phloem H2O2 levels were similar and were increased by MeJA application in both species. Lastly, trypsin inhibitor activity was detected in methanol and water extracts that were not allowed to oxidize, indicating the presence of phenolic-based trypsin inhibitors. However, no proteinaceous trypsin inhibitor activity was detected in either species. In response to MeJA application, Manchurian ash had higher trypsin inhibitor activity than black ash using the unoxidized water extracts, but no treatment effects were detected using methanol extracts. Based on these results we hypothesize that peroxidases, lignin polymerization, and quinone generation contribute to the greater resistance to EAB displayed by Manchurian ash.

The C-terminal domains of two homologous Oleaceae ß-1,3-glucanases recognise carbohydrates differently: Laminarin binding by NMR.

Ole e 9 and Fra e 9 are two allergenic ß-1,3-glucanases from olive and ash tree pollens, respectively. Both proteins present a modular structure with a catalytic N-terminal domain and a carbohydrate-binding module (CBM) at the C-terminus. Despite their significant sequence resemblance, they differ in some functional properties, such as their catalytic activity and the carbohydrate-binding ability. Here, we have studied the different capability of the recombinant C-terminal domain of both allergens to bind laminarin by NMR titrations, binding assays and ultracentrifugation. We show that rCtD-Ole e 9 has a higher affinity for laminarin than rCtD-Fra e 9. The complexes have different exchange regimes on the NMR time scale in agreement with the different affinity for laminarin observed in the biochemical experiments. Utilising NMR chemical shift perturbation data, we show that only one side of the protein surface is affected by the interaction and that the binding site is located in the inter-helical region between α1 and α2, which is buttressed by aromatic side chains. The binding surface is larger in rCtD-Ole e 9 which may account for its higher affinity for laminarin relative to rCtD-Fra e 9.

Drought stress has contrasting effects on antioxidant enzymes activity and phenylpropanoid biosynthesis in Fraxinus ornus leaves: an excess light stress affair?

The experiment was conducted using Fraxinus ornus plants grown outside under full sunlight irradiance, and supplied with 100% (well-watered, WW), 40% (mild drought, MD), or 20% (severe drought, SD) of the daily evapotranspiration demand, with the main objective of exploring the effect of excess light stress on the activity of antioxidant enzymes and phenylpropanoid biosynthesis. Net CO2 assimilation rate at saturating light and daily assimilated CO2 were significantly smaller in SD than in WW and MD plants. Xanthophyll-cycle pigments supported nonphotochemical quenching to a significantly greater extent in SD than in MD and WW leaves. As a consequence, the actual efficiency of PSII (Φ(PSII)) was smaller, while the excess excitation-energy in the photosynthetic apparatus was greater in SD than in WW or MD plants. The concentrations of violaxanthin-cycle pigments relative to total chlorophyll (Chl(tot)) exceeded 200 mmol mol⁻¹ Chl(tot) in SD leaves at the end of the experiment. This leads to hypothesize for zeaxanthin a role not only as nonphotochemical quencher, but also as chloroplast antioxidant. Reductions in ascorbate peroxidase and catalase activities, as drought-stress progressed, were paralleled by greater accumulations of esculetin and quercetin 3-O-glycosides, both phenylpropanoids having effective capacity to scavenge H2O2. The drought-induced accumulation of esculetin and quercetin 3-O-glycosides in the vacuoles of mesophyll cells is consistent with their putative functions as reducing agents for H2O2 in excess light-stressed leaves. Nonetheless, the concentration of H2O2 and the lipid peroxidation were significantly greater in SD than in MD and WW leaves. It is speculated that vacuolar phenylpropanoids may constitute a secondary antioxidant system, even on a temporal basis, activated upon the depletion of primary antioxidant defences, and aimed at keeping whole-cell H2O2 within a sub-lethal concentration range.