Molecular and ecological plant defense responses along an elevational gradient in a boreal ecosystem.

Plants have the capacity to alter their phenotype in response to environmental factors, such as herbivory, a phenomenon called phenotypic plasticity. However, little is known on how plant responses to herbivory are modulated by environmental variation along ecological gradients. To investigate this question, we used bilberry (Vaccinium myrtillus L.) plants and an experimental treatment to induce plant defenses (i.e., application of methyl jasmonate; MeJA), to observe ecological responses and gene expression changes along an elevational gradient in a boreal system in western Norway. The gradient included optimal growing conditions for bilberry in this region (ca. 500 m a.s.l.), and the plant's range limits at high (ca. 900 m a.s.l.) and low (100 m a.s.l.) elevations. Across all altitudinal sites, MeJA-treated plants allocated more resources to herbivory resistance while reducing growth and reproduction than control plants, but this response was more pronounced at the lowest elevation. High-elevation plants growing under less herbivory pressure but more resource-limiting conditions exhibited consistently high expression levels of defense genes in both MeJA-treated and untreated plants at all times, suggesting a constant state of "alert." These results suggest that plant defense responses at both the molecular and ecological levels are modulated by the combination of climate and herbivory pressure, such that plants under different environmental conditions differentially direct the resources available to specific antiherbivore strategies. Our findings are important for understanding the complex impact of future climate changes on plant-herbivore interactions, as this is a major driver of ecosystem functioning and biodiversity.

Transcriptional profiling of methyl jasmonate-induced defense responses in bilberry (Vaccinium myrtillus L.).

BACKGROUND: Bilberry (Vaccinium myrtillus L.) is one of the most abundant wild berries in the Northern European ecosystems. This species plays an important ecological role as a food source for many vertebrate and invertebrate herbivores. It is also well-recognized for its bioactive compounds, particularly substances involved in natural defenses against herbivory. These defenses are known to be initiated by leaf damage (e.g. chewing by insects) and mediated by activation of the jasmonic acid (JA) signaling pathway. This pathway can be activated by exogenous application of methyl jasmonate (MeJA), the volatile derivative of JA, which is often used to stimulate plant defense responses in studies of plant-herbivore interactions at ecological, biochemical, and molecular organismal levels. As a proxy for herbivore damage, wild V. myrtillus plants were treated in the field with MeJA and changes in gene expression were compared to untreated plants. RESULTS: The de novo transcriptome assembly consisted of 231,887 unigenes. Nearly 71% of the unigenes were annotated in at least one of the databases interrogated. Differentially expressed genes (DEGs), between MeJA-treated and untreated control bilberry plants were identified using DESeq. A total of 3590 DEGs were identified between the treated and control plants, with 2013 DEGs upregulated and 1577 downregulated. The majority of the DEGs identified were associated with primary and secondary metabolism pathways in plants. DEGs associated with growth (e.g. those encoding photosynthesis-related components) and reproduction (e.g. flowering control genes) were frequently down-regulated while those associated with defense (e.g. encoding enzymes involved in biosynthesis of flavonoids, lignin compounds, and deterrent/repellent volatile organic compounds) were up-regulated in the MeJA treated plants. CONCLUSIONS: Ecological studies are often limited by controlled conditions to reduce the impact of environmental effects. The results from this study support the hypothesis that bilberry plants, growing in natural conditions, shift resources from growth and reproduction to defenses while in a MeJA-induced state, as when under insect attack. This study highlights the occurrence of this trade-off at the transcriptional level in a realistic field scenario and supports published field observations wherein plant growth is retarded and defenses are upregulated.

Multiannual effects of induced plant defenses: Are defended plants good or bad neighbors?

Defenses induced by herbivore feeding or phytohormones such as methyl jasmonate (MeJA) can affect growth, reproduction, and herbivory, not only on the affected individual but also in its neighboring plants. Here, we report multiannual defense, growth, and reproductive responses of MeJA-treated bilberry (Vaccinium myrtillus) and neighboring ramets. In a boreal forest in western Norway, we treated bilberry ramets with MeJA and water (control) and measured responses over three consecutive years. We observed the treatment effects on variables associated with herbivory, growth, and reproduction in the MeJA-treated and untreated ramet and neighboring ramets distanced from 10 to 500 cm. MeJA-treated ramets had fewer grazed leaves and browsed shoots compared to control, with higher effects in 2014 and 2015, respectively. In 2013, growth of control ramets was greater than MeJA-treated ramets. However, MeJA-treated ramets had more flowers and berries than control ramets 2 years after the treatment. The level of insect and mammalian herbivory was also lower in untreated neighboring ramets distanced 10-150 cm and, consistent with responses of MeJA-treated ramets, the stronger effect was also one and 2 years delayed, respectively. The same neighboring ramets had fewer flowers and berries than untreated ramets, indicating a trade-off between defense and reproduction. Although plant-plant effects were observed across all years, the strength varied by the distance between the MeJA-treated ramets and its untreated neighbors. We document that induced defense in bilberry reduces both insect and mammalian herbivory, as well as growth, over multiple seasons. The defense responses occurred in a delayed manner with strongest effects one and 2 years after the induction. Additionally, our results indicate defense signaling between MeJA-treated ramets and untreated neighbors. In summary, this study shows that induced defenses are important ecological strategies not only for the induced individual plant but also for neighboring plants across multiple years in boreal forests.

How to induce defense responses in wild plant populations? Using bilberry (Vaccinium myrtillus) as example.

Inducible plant defense is a beneficial strategy for plants, which imply that plants should allocate resources from growth and reproduction to defense when herbivores attack. Plant ecologist has often studied defense responses in wild populations by biomass clipping experiments, whereas laboratory and greenhouse experiments in addition apply chemical elicitors to induce defense responses. To investigate whether field ecologists could benefit from methods used in laboratory and greenhouse studies, we established a randomized block-design in a pine-bilberry forest in Western Norway. We tested whether we could activate defense responses in bilberry (Vaccinium myrtillus) by nine different treatments using clipping (leaf tissue or branch removal) with or without chemical treatment by methyljasmonate (MeJA). We subsequently measured consequences of induced defenses through vegetative growth and insect herbivory during one growing season. Our results showed that only MeJA-treated plants showed consistent defense responses through suppressed vegetative growth and reduced herbivory by leaf-chewing insects, suggesting an allocation of resources from growth to defense. Leaf tissue removal reduced insect herbivory equal to the effect of the MeJa treatments, but had no negative impact on growth. Branch removal did not reduce insect herbivory or vegetative growth. MeJa treatment and clipping combined did not give an additional defense response. In this study, we investigated how to induce defense responses in wild plant populations under natural field conditions. Our results show that using the chemical elicitor MeJA, with or without biomass clipping, may be a better method to induce defense response in field experiments than clipping of leaves or branches that often has been used in ecological field studies.