The only African wild tobacco, Nicotiana africana: Alkaloid content and the effect of herbivory

Herbivory in some Nicotiana species is known to induce alkaloid production. This study examined herbivore-induced defenses in the nornicotine-rich African tobacco N. africana, the only Nicotiana species indigenous to Africa. We tested the predictions that: 1) N. africana will have high constitutive levels of leaf, flower and nectar alkaloids; 2) leaf herbivory by the African bollworm Helicoverpa armigera will induce increased alkaloid levels in leaves, flowers and nectar; and 3) increased alkaloid concentrations in herbivore-damaged plants will negatively affect larval growth. We grew N. africana in large pots in a greenhouse and exposed flowering plants to densities of one, three and six fourth-instar larvae of H. armigera, for four days. Leaves, flowers and nectar were analyzed for nicotine, nornicotine and anabasine. The principal leaf alkaloid was nornicotine (mean: 28 µg/g dry mass) followed by anabasine (4.9 µg/g) and nicotine (0.6 µg/g). Nornicotine was found in low quantities in the flowers, but no nicotine or anabasine were recorded. The nectar contained none of the alkaloids measured. Larval growth was reduced when leaves of flowering plants were exposed to six larvae. As predicted by the optimal defense theory, herbivory had a localized effect and caused an increase in nornicotine concentrations in both undamaged top leaves of herbivore damaged plants and herbivore damaged leaves exposed to one and three larvae. The nicotine concentration increased in damaged compared to undamaged middle leaves. The nornicotine concentration was lower in damaged leaves of plants exposed to six compared to three larvae, suggesting that N. africana rather invests in new growth as opposed to protecting older leaves under severe attack. The results indicate that the nornicotine-rich N. africana will be unattractive to herbivores and more so when damaged, but that potential pollinators will be unaffected because the nectar remains alkaloid-free even after herbivory

Olive fruits infested with olive fly larvae respond with an ethylene burst and the emission of specific volatiles

Olive fly (Bactrocera oleae R.) is the most harmful insect pest of olive (Olea europaea L.) which strongly affects fruits and oil production. Despite the expanding economic importance of olive cultivation, up to now, only limited information on plant responses to B. oleae is available. Here, we demonstrate that olive fruits respond to B. oleae attack by producing changes in an array of different defensive compounds including phytohormones, volatile organic compounds (VOCs), and defense proteins. Bactrocera oleae-infested fruits induced a strong ethylene burst and transcript levels of several putative ethylene-responsive transcription factors became significantly upregulated. Moreover, infested fruits induced significant changes in the levels of 12-oxo-phytodienoic acid and C12 derivatives of the hydroperoxide lyase. The emission of VOCs was also changed quantitatively and qualitatively in insect-damaged fruits, indicating that B. oleae larval feeding can specifically affect the volatile blend of fruits. Finally, we show that larval infestation maintained high levels of trypsin protease inhibitors in ripe fruits, probably by affecting post-transcriptional mechanisms. Our results provide novel and important information to understand the response of the olive fruit to B. oleae attack; information that can shed light onto potential new strategies to combat this pes

Methyl jasmonate-elicited herbivore resistance: does MeJA function as a signal without being hydrolyzed to JA?

Treatment with methyl jasmonate (MeJA) elicits herbivore resistance in many plant species and over-expression of JA carboxyl methyltransferase (JMT) constitutively increases JA-induced responses in Arabidopsis. When wild-type (WT) Nicotiana attenuata plants are treated with MeJA, a rapid transient endogenous JA burst is elicited, which in turn increases levels of nicotine and trypsin proteinase inhibitors (TPIs) and resistance to larvae of the specialist herbivore, Manduca sexta. All of these responses are impaired in plants silenced in lipoxygenase 3 expression (asLOX3) but are restored to WT levels by MeJA treatment. Whether these MeJA-induced responses are directly elicited by MeJA or by its cleavage product, JA, is unknown. Using virus-induced gene silencing (VIGS), we silenced MeJA-esterase (NaMJE) expression and found this gene responsible for most of the MeJA-cleaving activity in N. attenuata protein extracts. Silencing NaMJE in asLOX3, but not in WT plants, significantly reduced MeJA-induced nicotine levels and resistance to M. sexta, but not TPI levels. MeJA-induced transcript levels of threonine deaminase (NaTD) and phenylalanine ammonia lyase (NaPAL1) were also decreased in VIGS MJE (asLOX3) plants. Finally the performance of M. sexta larvae that fed on plants treated with JA or MeJA demonstrated that silencing NaMJE inhibited MeJA-induced but not JA-induced resistance in asLOX3 plants. From these results, we conclude that the resistance elicited by MeJA treatment is directly elicited not by MeJA but by its de-methylated product, JA

Facial expressions depicting compassionate and critical emotions: the development and validation of a new emotional face stimulus set

Attachment with altruistic others requires the ability to appropriately process affiliative and kind facial cues. Yet there is no stimulus set available to investigate such processes. Here, we developed a stimulus set depicting compassionate and critical facial expressions, and validated its effectiveness using well-established visual-probe methodology. In Study 1, 62 participants rated photographs of actors displaying compassionate/kind and critical faces on strength of emotion type. This produced a new stimulus set based on N = 31 actors, whose facial expressions were reliably distinguished as compassionate, critical and neutral. In Study 2, 70 participants completed a visual-probe task measuring attentional orientation to critical and compassionate/kind faces. This revealed that participants lower in self-criticism demonstrated enhanced attention to compassionate/kind faces whereas those higher in self-criticism showed no bias. To sum, the new stimulus set produced interpretable findings using visual-probe methodology and is the first to include higher order, complex positive affect displays

Intraspecific Combinations of Flower and Leaf Volatiles Act Together in Attracting Hawkmoth Pollinators

Insects pinpoint mates, food and oviposition sites by olfactory cues. Recognizing and localizing a suitable target by olfaction is demanding. Odor sources emit characteristic blends of compounds that have to be identified against an environmentally derived olfactory background. This background, however, does not necessarily disturb the localization of a source. Rather, the contrary. Sex pheromones become more attractive to male moths when being presented against a relevant plant background. Here we asked whether such olfactory coaction also characterizes foraging cues. The tobacco hornworm Manduca sexta feeds on nectar from wild tobacco Nicotiana attenuata and sacred datura Datura wrightii flowers. We tested how leaf-derived volatile blends as a background affect the moths' approach to flower blends. We found coaction when a flower blend was presented against a conspecific leaf volatile background but not when the blend was presented against volatiles emitted by the other host plant or by a non-host plant. Hence, our results reveal a species-specific coaction between flower blend and leaf volatile background. The ability to integrate information from different odor sources on one plant might provide the moth with a fine-grained analysis of food site quality

Reverse Genetics in Ecological Research

By precisely manipulating the expression of individual genetic elements thought to be important for ecological performance, reverse genetics has the potential to revolutionize plant ecology. However, untested concerns about possible side-effects of the transformation technique, caused by Agrobacterium infection and tissue culture, on plant performance have stymied research by requiring onerous sample sizes. We compare 5 independently transformed Nicotiana attenuata lines harboring empty vector control (EVC) T-DNA lacking silencing information with isogenic wild types (WT), and measured a battery of ecologically relevant traits, known to be important in plant-herbivore interactions: phytohormones, secondary metabolites, growth and fitness parameters under stringent competitive conditions, and transcriptional regulation with microarrays. As a positive control, we included a line silenced in trypsin proteinase inhibitor gene (TPI) expression, a potent anti-herbivore defense known to exact fitness costs in its expression, in the analysis. The experiment was conducted twice, with 10 and 20 biological replicates per genotype. For all parameters, we detected no difference between any EVC and WT lines, but could readily detect a fitness benefit of silencing TPI production. A statistical power analyses revealed that the minimum sample sizes required for detecting significant fitness differences between EVC and WT was 2–3 orders of magnitude larger than the 10 replicates required to detect a fitness effect of TPI silencing. We conclude that possible side-effects of transformation are far too low to obfuscate the study of ecologically relevant phenotypes

Competitive Performance of Transgenic Wheat Resistant to Powdery Mildew

Genetically modified (GM) plants offer an ideal model system to study the influence of single genes that confer constitutive resistance to pathogens on the ecological behaviour of plants. We used phytometers to study competitive interactions between GM lines of spring wheat Triticum aestivum carrying such genes and control lines. We hypothesized that competitive performance of GM lines would be reduced due to enhanced transgene expression under pathogen levels typically encountered in the field. The transgenes pm3b from wheat (resistance against powdery mildew Blumeria graminis) or chitinase and glucanase genes from barley (resistance against fungi in general) were introduced with the ubiquitin promoter from maize (pm3b and chitinase genes) or the actin promoter from rice (glucanase gene). Phytometers of 15 transgenic and non-transgenic wheat lines were transplanted as seedlings into plots sown with the same 15 lines as competitive environments and subject to two soil nutrient levels. Pm3b lines had reduced mildew incidence compared with control lines. Chitinase and chitinase/glucanase lines showed the same high resistance to mildew as their control in low-nutrient treatment and slightly lower mildew rates than the control in high-nutrient environment. Pm3b lines were weaker competitors than control lines. This resulted in reduced yield and seed number. The Pm3b line with the highest transgene expression had 53.2% lower yield than the control whereas the Pm3b line which segregated in resistance and had higher mildew rates showed only minor costs under competition. The line expressing both chitinase and glucanase genes also showed reduced yield and seed number under competition compared with its control. Our results suggest that single transgenes conferring constitutive resistance to pathogens can have ecological costs and can weaken plant competitiveness even in the presence of the pathogen. The magnitude of these costs appears related to the degree of expression of the transgenes

Pyrethrins Protect Pyrethrum Leaves Against Attack by Western Flower Thrips, Frankliniella occidentalis

Pyrethrins are active ingredients extracted from pyrethrum flowers (Tanacetum cinerariifolium), and are the most widely used botanical insecticide. However, several thrips species are commonly found on pyrethrum flowers in the field, and are the dominant insects found inside the flowers. Up to 80 % of western flower thrips (WFT, Frankliniella occidentalis) adults died within 3 days of initiating feeding on leaves of pyrethrum, leading us to evaluate the role of pyrethrins in the defense of pyrethrum leaves against WFT. The effects of pyrethrins on WFT survival, feeding behavior, and reproduction were measured both in vitro and in planta (infiltrated leaves). The lethal concentration value (LC50) for pyrethrins against WFT adults was 12.9 mg/ml, and pyrethrins at 0.1 % (w/v) and 1 % (w/v) had significantly negative effects on feeding, embryo development, and oviposition. About 20-70 % of WFT were killed within 2 days when they were fed chrysanthemum leaves containing 0.01-1 % pyrethrins. Chrysanthemum leaves containing 0.1 % or 1 % pyrethrins were significantly deterrent to WFT. In a no-choice assay, the reproduction of WFT was reduced significantly when the insects were fed leaves containing 0.1 % pyrethrins, and no eggs were found in leaves containing 1 % pyrethrins. Our results suggest that the natural concentrations of pyrethrins in the leaves may be responsible for the observed high mortality of WFT on pyrethrum

Transcriptional Analysis of Arabidopsis thaliana Response to Lima Bean Volatiles

Exposure of plants to herbivore-induced plant volatiles (HIPVs) alters their resistance to herbivores. However, the whole-genome transcriptional responses of treated plants remain unknown, and the signal pathways that produce HIPVs are also unclear.Time course patterns of the gene expression of Arabidopsis thaliana exposed to Lima bean volatiles were examined using Affymetrix ATH1 genome arrays. Results showed that A. thaliana received and responded to leafminer-induced volatiles from Lima beans through up-regulation of genes related to the ethylene (ET) and jasmonic acid pathways. Time course analysis revealed strong and partly qualitative differences in the responses between exposure at 24 and that at 48 h. Further experiments using either A. thaliana ET mutant ein2-1 or A. thaliana jasmonic acid mutant coi1-2 indicated that both pathways are involved in the volatile response process but that the ET pathway is indispensable for detecting volatiles. Moreover, transcriptional comparisons showed that plant responses to larval feeding do not merely magnify the volatile response process. Finally, (Z)-3-hexen-ol, ocimene, (3E)-4,8-dimethyl-1,3,7-nonatriene, and (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene triggered responses in A. thaliana similar to those induced by the entire suite of Lima bean volatiles after 24 and 48 h.This study shows that the transcriptional responses of plants to HIPVs become stronger as treatment time increases and that ET signals are critical during this process