The two α-dox genes of Nicotiana attenuata: overlapping but distinct functions in development and stress responses

<p>Abstract</p> <p>Background</p> <p>Plant fatty acid α-dioxygenases (α-DOX) are oxylipin-forming enzymes induced by biotic and abiotic stresses, which also participate in developmental processes. In <it>Nicotiana attenuata</it>, herbivory strongly induces the expression of an <it>α-dox1 </it>gene. To determine its role, we silenced its expression using <it>Agrobacterium</it>-mediated plant transformation with an inverted repeat construct. More than half of the transformed lines showed a severe dwarf growth phenotype that was very similar to the phenotype of tomato plants mutated at a second <it>α-dox </it>isoform. This led us to identify the corresponding <it>α-dox2 </it>gene in <it>N. attenuata </it>and examine the regulation of both <it>α-dox </it>genes as well as the consequences of their silencing in plant development and anti-herbivore defense.</p> <p>Results</p> <p>The transformed lines exhibiting a dwarf growth phenotype are co-silenced for both <it>α-dox </it>genes resulting in a nearly complete suppression of α-DOX activity, which is associated with increases in ABA, JA and anthocyanin levels, all metabolic signatures of oxidative stress. The other lines, only silenced for <it>α-dox1</it>, developed similarly to wild-type plants, exhibited a 40% reduction of α-DOX activity resulting in a 50% reduction of its main product <it>in planta </it>(2-HOT) and showed no signs of oxidative stress. In contrast to <it>α-dox1</it>, the expression of <it>α-dox2 </it>gene is not induced by wounding or elicitors in the oral secretions of <it>Manduca sexta</it>. Instead, <it>α-dox2 </it>is expressed in roots and flowers which lack <it>α-dox1 </it>expression, but both genes are equally regulated during leaf maturation. We transiently silenced <it>α-dox </it>gene copies with gene-specific constructs using virus induced gene silencing and determined the consequences for plant development and phytohormone and 2-HOT levels. While individual silencing of <it>α-dox1 </it>or <it>α-dox2 </it>had no effects on plant growth, the co-suppression of both <it>α-dox </it>genes decreased plant growth. Plants transiently silenced for both <it>α-dox </it>genes had increased constitutive levels of JA and ABA but silencing <it>α-dox1 </it>alone resulted in lower <it>M. sexta</it>-induced levels of JA, 2-HOT and ABA.</p> <p>Conclusions</p> <p>Thus, both <it>α-dox </it>isoforms function in the development of <it>N. attenuata</it>. In leaf maturation, the two <it>α-dox </it>genes have overlapping functions, but only <it>α-dox2 </it>is involved in root and flower development and only <it>α-dox1 </it>functions in anti-herbivore defense.</p

Diverting the Flux of the JA Pathway in Nicotiana attenuata Compromises the Plant's Defense Metabolism and Fitness in Nature and Glasshouse

A plant's inducible defenses against herbivores as well as certain developmental processes are known to be controlled by the jasmonic acid (JA) pathway. We have previously shown that ectopically expressing Arabidopsis thaliana JA O-methyltransferase in Nicotiana attenuata (35S-jmt) strongly reduces the herbivory-elicited jasmonate bursts by acting as metabolic sink that redirects free JA towards methylation; here we examine the consequences of this metabolic sink on N. attenuata's secondary metabolism and performance in nature. In the glasshouse, 35S-jmt plants produced fewer seed capsules due to shorter floral styles, which could be restored to wild type (WT) levels after hand-pollination, and were more susceptible to Manduca sexta larvae attack. When transplanted into the Great Basin Desert in Utah, 35S-jmt plants grew as well as WT empty vector, but were highly attacked by native herbivores of different feeding guilds: leaf chewers, miners, and single cell feeders. This greater susceptibility was strongly associated with reduced emissions of volatile organic compounds (hexenylesters, monoterpenes and sesquiterpenes) and profound alterations in the production of direct defenses (trypsin proteinase inhibitors [TPI], nicotine, diterpene glycosides [DTGs] and phenylpropanoid-polyamine conjugates) as revealed by a combination of targeted and metabolomics analyses of field collected samples. Complementation experiments with JA-Ile, whose formation is outcompeted in 35S-jmt plants by the methylation reaction, restored the local TPI activation to WT levels and partially complemented nicotine and DTG levels in elicited but not systemic leaves. These findings demonstrate that MeJA, the major JA metabolite in 35S-jmt plants, is not an active signal in defense activation and highlights the value of creating JA sinks to disrupt JA signaling, without interrupting the complete octadecanoid pathway, in order to investigate the regulation of plants' defense metabolism in nature

Evolutionary footprints of a cold relic in a rapidly warming world

With accelerating global warming, understanding the evolutionary dynamics of plant adaptation to environmental change is increasingly urgent. Here we reveal the enigmatic history of the genus Cochlearia (Brassicaceae), a Pleistocene relic that originated from a drought-adapted Mediterranean sister genus during the Miocene. Cochlearia rapidly diversified and adapted to circum-Arctic regions and other cold-characterized habitat types during the Pleistocene. This sudden change in ecological preferences was accompanied by a highly complex, reticulate polyploid evolution, which was apparently triggered by the impact of repeated Pleistocene glaciation cycles. Our results illustrate that two early diversified arctic-alpine diploid gene pools contributed differently to the evolution of this young polyploid genus now captured in a cold-adapted niche. Metabolomics revealed central carbon metabolism responses to cold in diverse species and ecotypes, likely due to continuous connections to cold habitats that may have facilitated widespread adaptation to alpine and subalpine habitats, and which we speculate were coopted from existing drought adaptations. Given the growing scientific interest in adaptive evolution of temperature-related traits, our results provide much-needed taxonomic and phylogenomic resolution of a model system as well as first insights into the origins of its adaptation to cold

Nicotiana attenuata Data Hub (NaDH): an integrative platform for exploring genomic, transcriptomic and metabolomic data in wild tobacco

Background: Nicotiana attenuata (coyote tobacco) is an ecological model for studying plant-environment interactions and plant gene function under real-world conditions. During the last decade, large amounts of genomic, transcriptomic and metabolomic data have been generated with this plant which has provided new insights into how native plants interact with herbivores, pollinators and microbes. However, an integrative and open access platform that allows for the efficient mining of these -omics data remained unavailable until now. Description: We present the Nicotiana attenuata Data Hub (NaDH) as a centralized platform for integrating and visualizing genomic, phylogenomic, transcriptomic and metabolomic data in N. attenuata. The NaDH currently hosts collections of predicted protein coding sequences of 11 plant species, including two recently sequenced Nicotiana species, and their functional annotations, 222 microarray datasets from 10 different experiments, a transcriptomic atlas based on 20 RNA-seq expression profiles and a metabolomic atlas based on 895 metabolite spectra analyzed by mass spectrometry. We implemented several visualization tools, including a modified version of the Electronic Fluorescent Pictograph (eFP) browser, co-expression networks and the Interactive Tree Of Life (iTOL) for studying gene expression divergence among duplicated homologous. In addition, the NaDH allows researchers to query phylogenetic trees of 16,305 gene families and provides tools for analyzing their evolutionary history. Furthermore, we also implemented tools to identify co-expressed genes and metabolites, which can be used for predicting the functions of genes. Using the transcription factor NaMYB8 as an example, we illustrate that the tools and data in NaDH can facilitate identification of candidate genes involved in the biosynthesis of specialized metabolites. Conclusion: The NaDH provides interactive visualization and data analysis tools that integrate the expression and evolutionary history of genes in Nicotiana, which can facilitate rapid gene discovery and comparative genomic analysis. Because N. attenuata shares many genome-wide features with other Nicotiana species including cultivated tobacco, and hence NaDH can be a resource for exploring the function and evolution of genes in Nicotiana species in general. The NaDH can be accessed at: http://nadh.ice.mpg.de

Current European Labyrinthula zosterae Are Not Virulent and Modulate Seagrass (Zostera marina) Defense Gene Expression

Pro- and eukaryotic microbes associated with multi-cellular organisms are receiving increasing attention as a driving factor in ecosystems. Endophytes in plants can change host performance by altering nutrient uptake, secondary metabolite production or defense mechanisms. Recent studies detected widespread prevalence of Labyrinthula zosterae in European Zostera marina meadows, a protist that allegedly caused a massive amphi-Atlantic seagrass die-off event in the 1930's, while showing only limited virulence today. As a limiting factor for pathogenicity, we investigated genotype×genotype interactions of host and pathogen from different regions (10–100 km-scale) through reciprocal infection. Although the endophyte rapidly infected Z. marina, we found little evidence that Z. marina was negatively impacted by L. zosterae. Instead Z. marina showed enhanced leaf growth and kept endophyte abundance low. Moreover, we found almost no interaction of protist×eelgrass-origin on different parameters of L. zosterae virulence/Z. marina performance, and also no increase in mortality after experimental infection. In a target gene approach, we identified a significant down-regulation in the expression of 6/11 genes from the defense cascade of Z. marina after real-time quantitative PCR, revealing strong immune modulation of the host's defense by a potential parasite for the first time in a marine plant. Nevertheless, one gene involved in phenol synthesis was strongly up-regulated, indicating that Z. marina plants were probably able to control the level of infection. There was no change in expression in a general stress indicator gene (HSP70). Mean L. zosterae abundances decreased below 10% after 16 days of experimental runtime. We conclude that under non-stress conditions L. zosterae infection in the study region is not associated with substantial virulence