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1.  Current European Labyrinthula zosterae Are Not Virulent and Modulate Seagrass (Zostera marina) Defense Gene Expression 
PLoS ONE  2014;9(4):e92448.
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.
PMCID: PMC3972160  PMID: 24691450
2.  Silencing an N-Acyltransferase-Like Involved in Lignin Biosynthesis in Nicotiana attenuata Dramatically Alters Herbivory-Induced Phenolamide Metabolism 
PLoS ONE  2013;8(5):e62336.
In a transcriptomic screen of Manduca sexta-induced N-acyltransferases in leaves of Nicotiana attenuata, we identified an N-acyltransferase gene sharing a high similarity with the tobacco lignin-biosynthetic hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) gene whose expression is controlled by MYB8, a transcription factor that regulates the production of phenylpropanoid polyamine conjugates (phenolamides, PAs). To evaluate the involvement of this HCT-like gene in lignin production as well as the resulting crosstalk with PA metabolism during insect herbivory, we transiently silenced (by VIGs) the expression of this gene and performed non-targeted (UHPLC-ESI/TOF-MS) metabolomics analyses. In agreement with a conserved function of N. attenuata HCT-like in lignin biogenesis, HCT-silenced plants developed weak, soft stems with greatly reduced lignin contents. Metabolic profiling demonstrated large shifts (up to 12% deregulation in total extracted ions in insect-attacked leaves) due to a large diversion of activated coumaric acid units into the production of developmentally and herbivory-induced coumaroyl-containing PAs (N′,N′′-dicoumaroylspermidine, N′,N′′-coumaroylputrescine, etc) and to minor increases in the most abundant free phenolics (chlorogenic and cryptochlorogenic acids), all without altering the production of well characterized herbivory-responsive caffeoyl- and feruloyl-based putrescine and spermidine PAs. These data are consistent with a strong metabolic tension, exacerbated during herbivory, over the allocation of coumaroyl-CoA units among lignin and unusual coumaroyl-containing PAs, and rule out a role for HCT-LIKE in tuning the herbivory-induced accumulation of other PAs. Additionally, these results are consistent with a role for lignification as an induced anti-herbivore defense.
PMCID: PMC3660383  PMID: 23704878
3.  NaMYC2 transcription factor regulates a subset of plant defense responses in Nicotiana attenuata 
BMC Plant Biology  2013;13:73.
To survive herbivore attack, plants have evolved potent mechanisms of mechanical or chemical defense that are either constitutively present or inducible after herbivore attack. Due to the costs of defense deployment, plants often regulate their biosynthesis using various transcription factors (TFs). MYC2 regulators belong to the bHLH family of transcription factors that are involved in many aspects of plant defense and development. In this study, we identified a novel MYC2 TF from N. attenuata and characterized its regulatory function using a combination of molecular, analytic and ecological methods.
The transcript and targeted metabolite analyses demonstrated that NaMYC2 is mainly involved in the regulation of the biosynthesis of nicotine and phenolamides in N. attenuata. In addition, using broadly-targeted metabolite analysis, we identified a number of other metabolite features that were regulated by NaMYC2, which, after full annotation, are expected to broaden our understanding of plant defense regulation. Unlike previous reports, the biosynthesis of jasmonates and some JA-/NaCOI1-dependent metabolites (e.g. HGL-DTGs) were not strongly regulated by NaMYC2, suggesting the involvement of other independent regulators. No significant differences were observed in the performance of M. sexta on MYC2-silenced plants, consistent with the well-known ability of this specialist insect to tolerate nicotine.
By regulating the biosynthesis of nicotine, NaMYC2 is likely to enhance plant resistance against non-adapted herbivores and contribute to plant fitness; however, multiple JA/NaCOI1-dependent mechanisms (perhaps involving other MYCs) that regulate separate defense responses are likely to exist in N. attenuata. The considerable variation observed amongst different plant families in the responses regulated by jasmonate signaling highlights the sophistication with which plants craft highly specific and fine-tuned responses against the herbivores that attack them.
PMCID: PMC3655906  PMID: 23634896
bHLH; Manduca sexta; MYC2; Transcription factors; Nicotiana attenuata; Nicotine; Phenolamides; Plant-insect interactions; Transcriptional regulation
4.  Honing in on phenotypes: comprehensive two-dimensional gas chromatography of herbivory-induced volatile emissions and novel opportunities for system-level analyses 
AoB Plants  2013;5:plt002.
Here we discuss opportunities for system-wide analysis of plant volatiles provided by the implementation of non-supervised data processing. We illustrate the value of such approaches by presenting recent findings on wild tobacco volatile emissions using two-dimensional gas chromatography.
Plant volatile organic compound (VOC) production requires a complex network of biochemical pathways, which, although well mapped from a biochemical point of view, remains only partly understood with regard to its physiological and genetic regulation. Additionally, although analytical procedures for plant VOC measurement have become increasingly faster and more sensitive in recent years, pinpointing relevant shifts in VOC production from the thousands of molecular fragments that are generated by modern mass spectrometer instruments remains challenging. Here we discuss novel opportunities for system-wide analysis provided by the implementation of non-targeted data processing and multivariate statistics in VOC analysis. We illustrate the value of implementing non-targeted data processing with examples of recent findings from our group on the interactive control exerted by salivary components of a lepidopteran herbivore, Manduca sexta, on herbivory-induced VOC emissions in the wild tobacco Nicotiana attenuata. Finally, we briefly discuss the use of multi-platform data integration for probing the nature of metabolic and regulatory systems underlying VOC emissions.
PMCID: PMC3581813  PMID: 23444146
Herbivory; metabolomics; Nicotiana attenuata; plant volatiles; two-dimensional gas chromatography; untargeted analysis.
5.  Tissue Specific Diurnal Rhythms of Metabolites and Their Regulation during Herbivore Attack in a Native Tobacco, Nicotiana attenuata 
PLoS ONE  2011;6(10):e26214.
Ecological performance is all about timing and the endogenous clock that allows the entrainment of rhythms and anticipation of fitness-determining events is being rapidly characterized. How plants anticipate daily abiotic stresses, such as cold in early mornings and drought at noon, as well as biotic stresses, such as the timing of pathogen infections, is being explored, but little is known about the clock's role in regulating responses to insect herbivores and mutualists, whose behaviors are known to be strongly diurnally regulated and whose attack is known to reconfigure plant metabolomes. We developed a liquid chromatography-mass spectrometry procedure and analyzed its output with model-based peak picking algorithms to identify metabolites with diurnal accumulation patterns in sink/source leaves and roots in an unbiased manner. The response of metabolites with strong diurnal patterns to simulated attack from the specialist herbivore, Manduca sexta larvae was analyzed and annotated with in-house and public databases. Roots and leaves had largely different rhythms and only 10 ions of 182 oscillating ions in leaves and 179 oscillating ions in roots were rhythmic in both tissues: root metabolites mainly peaked at dusk or night, while leaf metabolites peaked during the day. Many oscillating metabolites showed tissue-specific regulation by simulated herbivory of which systemic responses in unattacked tissues were particularly pronounced. Diurnal and herbivory-elicited accumulation patterns of disaccharide, phenylalanine, tyrosine, lyciumoside I, coumaroyl tyramine, 12-oxophytodienoic acid and jasmonic acid and those of their related biosynthetic transcripts were examined in detail. We conclude that oscillating metabolites of N. attenuata accumulate in a highly tissue-specific manner and the patterns reveal pronounced diurnal rhythms in the generalized and specialized metabolism that mediates the plant's responses to herbivores and mutualists. We propose that diurnal regulation will prove to an important element in orchestrating a plant's responses to herbivore attack.
PMCID: PMC3196511  PMID: 22028833
6.  Diverting the Flux of the JA Pathway in Nicotiana attenuata Compromises the Plant's Defense Metabolism and Fitness in Nature and Glasshouse 
PLoS ONE  2011;6(10):e25925.
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.
PMCID: PMC3189938  PMID: 22022469
7.  The two α-dox genes of Nicotiana attenuata: overlapping but distinct functions in development and stress responses 
BMC Plant Biology  2010;10:171.
Plant fatty acid α-dioxygenases (α-DOX) are oxylipin-forming enzymes induced by biotic and abiotic stresses, which also participate in developmental processes. In Nicotiana attenuata, herbivory strongly induces the expression of an α-dox1 gene. To determine its role, we silenced its expression using Agrobacterium-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 α-dox isoform. This led us to identify the corresponding α-dox2 gene in N. attenuata and examine the regulation of both α-dox genes as well as the consequences of their silencing in plant development and anti-herbivore defense.
The transformed lines exhibiting a dwarf growth phenotype are co-silenced for both α-dox 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 α-dox1, developed similarly to wild-type plants, exhibited a 40% reduction of α-DOX activity resulting in a 50% reduction of its main product in planta (2-HOT) and showed no signs of oxidative stress. In contrast to α-dox1, the expression of α-dox2 gene is not induced by wounding or elicitors in the oral secretions of Manduca sexta. Instead, α-dox2 is expressed in roots and flowers which lack α-dox1 expression, but both genes are equally regulated during leaf maturation. We transiently silenced α-dox 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 α-dox1 or α-dox2 had no effects on plant growth, the co-suppression of both α-dox genes decreased plant growth. Plants transiently silenced for both α-dox genes had increased constitutive levels of JA and ABA but silencing α-dox1 alone resulted in lower M. sexta-induced levels of JA, 2-HOT and ABA.
Thus, both α-dox isoforms function in the development of N. attenuata. In leaf maturation, the two α-dox genes have overlapping functions, but only α-dox2 is involved in root and flower development and only α-dox1 functions in anti-herbivore defense.
PMCID: PMC3017789  PMID: 20701756

Results 1-7 (7)