Visual cues contribute to predator detection in anuran larvae

The ability of prey to detect predators directly affects their probability of survival. Chemical cues are known to be important for predator detection in aquatic environments, but the role of other potential cues is controversial. We tested for changes in behaviour of Rana temporaria tadpoles in response to chemical, visual, acoustic, and hydraulic cues originating from dragonfly larvae (Aeshna cyanea) and fish (Gasterosteus aculeatus). The greatest reduction in tadpole activity occurred when all cues were available, but activity was also significantly reduced by visual cues only. We did not find evidence for tadpoles lowering their activity in response to acoustic and hydraulic cues. There was no spatial avoidance of predators in our small experimental containers. The results show that anuran larvae indeed use vision for predator detection, while acoustic and hydraulic cues may be less important. Future studies of predator‐induced responses of tadpoles should not only concentrate on chemical cues but also consider visual stimuli. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ??, ??–??.     

Reproductive meristem fates in Gerbera

Flowering plants go through several phases between regular stem growth and the actual production of flower parts. The stepwise conversion of vegetative into inflorescence and floral meristems is usually unidirectional, but under certain environmental or genetic conditions, meristems can revert to an earlier developmental identity. Vegetative meristems are typically indeterminate, producing organs continuously, whereas flower meristems are determinate, shutting down their growth after reproductive organs are initiated. Inflorescence meristems can show either pattern. Flower and inflorescence development have been investigated in Gerbera hybrida, an ornamental plant in the sunflower family, Asteraceae. Unlike the common model species used to study flower development, Gerbera inflorescences bear a fixed number of flowers, and the architecture of the flowers differ in that Gerbera ovaries are inferior (borne below the perianth). This architectural difference has been exploited to show that floral meristem determinacy and identity are spatially and genetically distinct in Gerbera, and we have shown that a single SEPALLATA-like MADS domain factor controls both flower and inflorescence meristem fate in the plant. Although these phenomena have not been directly observed in Arabidopsis, the integrative role of the SEPALLATA function in reproductive meristem development may be general for all flowering plants.     

On the mechanism of apoplastic H<Subscript>2</Subscript>O<Subscript>2</Subscript> production during lignin formation and elicitation in cultured spruce cells—peroxidases after elicitation

A cell culture of Picea abies (L.) Karst. was used for studies of H₂O₂ generation during constitutive extracellular lignin formation and after elicitation by cell wall fragments of a pathogenic fungus, Heterobasidium parviporum. Stable, micromolar levels of H₂O₂ were present in the culture medium during lignin formation. Elicitation induced a burst of H₂O₂, peaking at ca. 90 min after elicitation. Of exogenous reducing substrates that may be responsible for the synthesis of H₂O₂ from O₂, NADH stimulated H₂O₂ production irrespective of elicitation. Cysteine (Cys) and glutathione (GSH) partially scavenged the constitutive H₂O₂, but usually increased or prolonged elicitor-induced H₂O₂ formation. Culture medium peroxidases were not able to generate H₂O₂ in vitro with Cys or GSH as reductants. These thiols, however, generated H₂O₂ non-enzymically at pH 4.5. [³⁵S]Sulphate feeding to spruce cells showed that endogenous sulphur-containing compounds (including GSH, GSSG and cysteic acid) existed in the culture medium. The apoplastic levels of these were, however, undetectable by the monobromobimane method suggesting that their contribution to apoplastic H₂O₂ formation is probably minor. Azide, an inhibitor of haem-containing enzymes, slightly inhibited constitutive H₂O₂ generation but strongly delayed the elicitor-induced H₂O₂ accumulation. Diphenylene iodonium, an inhibitor of flavin-containing enzymes, efficiently inhibited H₂O₂ production irrespective of elicitation. Elicitation led to downregulation of the expression of several peroxidase genes, and peroxidase activity in the culture medium was slightly reduced. Expression of three other peroxidase genes and a respiratory burst oxidase homologue (rboh) gene were upregulated. These data suggest that both peroxidases and rboh may contribute to H₂O₂ generation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D

The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations.The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K(M) and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3)+Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.     

Antifungal activity of stilbenes in in vitro bioassays and in transgenic<Emphasis Type="Italic"> Populus</Emphasis> expressing a gene encoding pinosylvin synthase

The effect of two stilbene compounds, pinosylvin and resveratrol, on the growth of several fungi was evaluated in plate tests. Wood decay tests were carried out with birch and aspen samples impregnated with the two stilbenes. In plate experiments, resveratrol had an enhancing effect on growth at concentrations where pinosylvin was already enough to prevent the growth of most fungi studied. Pinosylvin impregnated at 0.2% (w/w) concentration significantly reduced the decay caused by all fungi except Phellinus tremulae. In contrast, a resveratrol content of 0.8%, did not protect the wood from decay. A pinosylvin-synthase-encoding gene from Pinus sylvestris was transferred into aspen (Populus tremula) and two hybrid aspen clones (Populus tremula×tremuloides) by Agrobacterium tumefaciens-mediated transformation. Transgenic plants accumulated pinosylvin synthase-specific mRNA and showed stilbene synthase enzyme activity in vitro. Transgenic aspen line H4 showed increased resistance to Phellinus tremulae, while two hybrid aspen transformants decayed faster than the control trees. However, we were unable to detect the accumulation of stilbenes in the transgenic plantlets.Communicated by P. Debergh     

Probing pH-dependent functional elements in proteins: modification of carboxylic acid pairs in Trichoderma reesei cellobiohydrolase Cel6A

Two carboxylic acid side chains can, depending on their geometry and environment, share a proton in a hydrogen bond and form a carboxyl-carboxylate pair. In the Trichoderma reesei cellobiohydrolase Cel6A structure, five carboxyl-carboxylate pairs are observed. One of these pairs (D175-D221) is involved in catalysis, and three other pairs are found in, or close to the two surface loops covering the active site tunnel of the catalytic domain. To stabilize Cel6A at alkaline pH values, where deprotonation of the carboxylic acids leads to repulsion of their side chains, we designed two mutant enzymes. In the first mutant, one carboxyl-carboxylate pair (E107-E399) was replaced by a corresponding amide-carboxylate pair (Q107-E399), and in the second mutant, all three carboxyl-carboxylate pairs (E107-E399, D170-E184, and D366-D419) were mutated in a similar manner. The unfolding studies using both intrinsic tryptophan fluorescence and far-ultraviolet circular dichroism spectroscopy at different pH values demonstrate that the unfolding temperature (T(m)) of both mutants has changed, resulting in destabilization of the mutant enzymes at acidic pH and stabilization at alkaline pH. The effect of stabilization seems additive, as a Cel6A triple mutant is the most stable enzyme variant. This increased stability is also reflected in the 2- or 4-fold increased half-life of the two mutants at alkaline pH, while the catalytic rate on cellotetraose (at t = 0) has not changed. Increased operational stability at alkaline pH was also observed on insoluble cellulosic substrates. Local conformational changes are suggested to take place in the active site loops of Cel6A wild-type enzyme at elevated pHs (pH 7), affecting to the end-product spectrum on insoluble cellulose. The triple mutant does not show such pH-dependent behavior. Overall, our results demonstrate that carboxyl-carboxylate pair engineering is a useful tool to alter pH-dependent protein behavior.     

Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus

In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites.