Summary 1.Plant defence theory predicts trade-offs among defence traits as a result of resource limitation or pleiotropic effects. Although theoretically widely accepted, empirical demonstrations of such trade-offs are surprisingly scarce and mechanistic explanations are usually lacking. 2. We quantified cyanogenesis (the release of hydrogen cyanide (HCN)) as a direct defence and the emission of volatile organic compounds (VOCs) as an indirect defence against herbivores. To elucidate whether the trade-offs occur at the genetic or phenotypic level we investigated cultivated and wild-type accessions of lima bean (Fabaceae: Phaseolus lunatus L.) and compared different leaf developmental stages. Genetic relationships among the accessions were studied using amplified fragment length polymorphism (AFLP) analysis. 3. Cyanogenesis and the release of VOCs differed significantly among the accessions and were negatively correlated: high cyanogenic accessions released low amounts of VOCs and vice versa. The same remained true for the ontogenetic stages, since primary leaves of all accessions hardly ever produced HCN at all, yet regularly showed high release rates of VOCs. 4. Low and high cyanogenic accessions of lima bean formed distinct clades in an AFLP-based dendrogram, while wild-types and cultivars did not separate. The first pattern indicates that the underlying defensive syndromes are genetically conserved, while the latter is likely to be caused by a multiple origin of cultivated lima beans or an extensive gene flow among cultivated and wild plants. 5. Synthesis . Trade-offs between cynogenesis and VOC release were obvious both between accessions and at the ontogenetic level, and thus cannot be explained by pleiotropy. We contend that allocation restrictions and/or adaptations to different enemy pressures are most likely to explain why lima bean can invest into cyanogenesis or VOCs, but not both.

We investigated the wound response of the commercially important red alga, Gracilaria chilensis, in order to obtain insight into its interaction with epiphytic pests. After wounding, the host releases free fatty acids as well as the hydroxylated eicosanoids, 8R-hydroxy eicosatetraenoic acid (8-HETE) and 7S,8R-dihydroxy eicosatetraenoic acid (7,8-di-HETE). While the release of free arachidonic acid and subsequent formation of 8-HETE is controlled by phospholipase A, 7,8-di-HETE production is independent of this lipase. This dihydroxylated fatty acid might be directly released from galactolipids. Physiologically relevant concentrations of oxylipins reduced spore settlement of Acrochaetium sp. (Rhodophyta, Acrochaetiaceae) and suppressed the development of hapteria in Ceramium rubrum (Rhodophyta, Ceramiaceae) when these model epiphytes were exposed to artificial surfaces that contained 8-HETE or 7,8-di-HETE. Thus, the immediate release of oxylipins can be seen as G. chilensis defence against epiphytes.

From Penicillium janczewskii, obtained from a marine sample, two new diastereomeric quinolinones, 3S,4R-dihydroxy-4-(4'-methoxyphenyl)-3,4-dihydro-2(1H)-quinolinone (1) and 3R,4R-dihydroxy-4-(4'-methoxyphenyl)-3,4-dihydro-2(1H)-quinolinone (2), were identified, along with two known alkaloids, peniprequinolone (3) and 3-methoxy-4-hydroxy-4-(4'-methoxyphenyl)-3,4-dihydro-2(1H)-quinolinone (4). Cytotoxicity testing on eight tumor cell lines revealed a moderate specificity of 2 on SKOV-3 cells.

The facultative red algal epiphyte Acrochaetium sp. liberated spores preferentially and recruited more successfully in laboratory cultures when its host Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira was present. The same effect was also induced by cell-free medium from G. chilensis, suggesting it contained a molecular signal. Antibiotics prevented spore release in Acrochaetium sp., even when G. chilensis was present, suggesting a prokaryotic origin of the signal. Simultaneous application of N-butyl-homoserine-lactone (BHL) restored the spore-release capacity, which demonstrated that spore release was not directly inhibited by the antibiotics and indicated that bacterially generated Nacyl-homoserine-lactones (AHLs) regulate spore release. An involvement of AHL was further indicated by the fact that two different halofuranone inhibitors of AHL receptors also inhibited spore release when they were applied at relatively low concentrations. Of seven different AHLs tested, only BHL induced the effect. However, BHL was only active at relatively high concentrations (100 lM), and it was not detected in spore-release-inducing medium of G. chilensis. Another water-soluble AHL or an AHL structure analog is therefore probably the active compound in G. chilensis cultures. The data presented demonstrate that life cycle completion in Acrochaetium sp. strongly depends on bacteria, which are not always present in sufficient numbers on the alga itself. Exogenous bacteria that are associated with G. chilensis or with other potential substrates may therefore trigger timely spore liberation in Acrochaetium sp., provided that the necessary concentration of AHL is reached. This first finding of AHL perception in a red alga confirms that AHL signalling is more widespread among eukaryotes than was thought until recently. However, spore release of a second red alga, Sahlingia subintegra (Rosenv.) Kornmann, was unaffected by AHL, and the reaction observed is therefore not universal.Algal spore release is known to be controlled by numerous factors, including light and temperature

The two agar-producing red algae, Gracilaria chilensis C. J. Bird, McLachlan & E. C. Oliveira and Gracilaria conferta (Schousboe ex Montagne) Montagne, responded with hydrogen peroxide (H 2 O 2 ) release when agar oligosaccharides were added to the medium. In G. conferta, a transient release was observed, followed by a refractory state of 6 h. This response was sensitive to chemical inhibitors of NADPH oxidase, protein kinases, protein phosphatases, and calcium translocation in the cell, whereas it was insensitive to inhibitors of metalloenzymes. Transmission electron microscopic observations of the H 2 O 2 -dependent formation of cerium peroxide from cerium chloride indicated oxygen activation at the plasma membrane of G. conferta. A putative system, consisting of a receptor specific to agar oligosaccharides and a plasma membranelocated NADPH oxidase, appears to be responsible for the release of H 2 O 2 in G. conferta. Subcellular examination of G. chilensis showed that the H 2 O 2 release was located in the cell wall. It was sensitive to inhibitors of metalloenzymes and flavoenzymes, and no refractory state was observed. The release was correlated with accumulation of an aldehyde in the algal medium, suggesting that an agar oligosaccharide oxidase is present in the apoplast of G. chilensis. The presence of this enzyme could also be demonstrated by polyacrylamide electrophoresis under nondenaturating conditions and proven to be variable. Cultivation of G. chilensis at 16 to 171 C resulted in significantly stronger expression of agar oligosaccharide oxidase than cultivation at 121 C, which indicates that the enzyme is used under conditions that generally favor microbial agar macerating activity.Specific cell wall-derived oligosaccharides are known to regulate growth, development, and defense responses in higher plants (Darvill et al. 1992, Aldington and Fry 1993, John et al. 1997, Braam 1999, Vorwerk et al. 2004. Examples of recognition of oligosaccharides have also been reported for marine algae. Agar oligosaccharides released from the cell wall matrix of Gracilaria conferta (Schousboe ex Montagne) Montagne control the abundance of agar degrading bacteria at the surface of this alga (Weinberger and Friedlander 2000). Similarly, alginate oligosaccharides from the cell wall matrix of kelp sporophytes play a key role during the induction of resistance against algal endophytes and bacteria in the brown algae Laminaria digitata and Macrocystis pyrifera, respectively (Küpper et al. 2002). 1

Herbivore-induced volatile organic compounds (VOCs) are widely appreciated as an indirect defense mechanism since carnivorous arthropods use VOCs as cues for host localization and then attack herbivores. Another function of VOCs is plant-plant signaling. That VOCs elicit defensive responses in neighboring plants has been reported from various species, and different compounds have been found to be active. In order to search for a structural motif that characterizes active VOCs, we used lima bean (Phaseolus lunatus), which responds to VOCs released from damaged plants with an increased secretion of extrafloral nectar (EFN). We exposed lima bean to (Z)-3-hexenyl acetate, a substance naturally released from damaged lima bean and known to induce EFN secretion, and to several structurally related compounds. (E)-3-hexenyl acetate, (E)-2-hexenyl acetate, 5-hexenyl acetate, (Z)-3-hexenylisovalerate, and (Z)-3-hexenylbutyrate all elicited significant increases in EFN secretion, demonstrating that neither the (Z)-configuration nor the position of the double-bond nor the size of the acid moiety are critical for the EFN-inducing effect. Our result is not consistent with previous concepts that postulate reactive electrophile species (Michael-acceptor-systems) for defense-induction in Arabidopsis. Instead, we postulate that physicochemical processes, including interactions with odorant binding proteins and resulting in changes in transmembrane potentials, can underlie VOCs-mediated signaling processes.

The red alga Gracilaria chilensis is commercially farmed for the production of agar hydrocolloids, but some susceptible algae in farms suffer from intense epiphyte growth. We investigated the induced chemical defense response of G. chilensis against epiphytes and demonstrated that an extract of an epiphyte-challenged alga can trigger a defense response. The hormonally active metabolites were purified by RP-HPLC. Treatment with the extract or the purified fraction changed the chemical profile of the alga and increased resistance against epiphyte spores. Semi-quantitative RT-PCR and enzyme assays demonstrated that this metabolic response occurs after an increase in lipoxygenase and phospholipase A2 activity. Although this suggests the involvement of regulatory oxylipins, neither jasmonic acid nor the algal metabolite prostaglandin E2 triggers comparable defense responses.

Studies on direct and indirect defenses of lima bean (Phaseolus lunatus L.) revealed a quantitative trade-off between cyanogenesis and the total quantitative release of herbivore-induced volatile organic compounds (VOCs). In this addendum we focus on the qualitative variability in the VOC bouquets. We found intraspecific and ontogenetic variation. Five out of eleven lima bean accessions lacked particular VOCs in the bouquets released from secondary and/or primary leaves. These compounds (cis-3-hexenyl acetate, methyl salicylate and methyl jasmonate) can induce and prime indirect defenses in neighboring plants. Thus, the variability in VOC quality as described here might have substantial effects on plant-plant communication.