A rapid and sensitive method for the simultaneous analysis of aliphatic and polar molecules containing free carboxyl groups in plant extracts by LC-MS/MS

<p>Abstract</p> <p>Background</p> <p>Aliphatic molecules containing free carboxyl groups are important intermediates in many metabolic and signalling reactions, however, they accumulate to low levels in tissues and are not efficiently ionized by electrospray ionization (ESI) compared to more polar substances. Quantification of aliphatic molecules becomes therefore difficult when small amounts of tissue are available for analysis. Traditional methods for analysis of these molecules require purification or enrichment steps, which are onerous when multiple samples need to be analyzed. In contrast to aliphatic molecules, more polar substances containing free carboxyl groups such as some phytohormones are efficiently ionized by ESI and suitable for analysis by LC-MS/MS. Thus, the development of a method with which aliphatic and polar molecules -which their unmodified forms differ dramatically in their efficiencies of ionization by ESI- can be simultaneously detected with similar sensitivities would substantially simplify the analysis of complex biological matrices.</p> <p>Results</p> <p>A simple, rapid, specific and sensitive method for the simultaneous detection and quantification of free aliphatic molecules (e.g., free fatty acids (FFA)) and small polar molecules (e.g., jasmonic acid (JA), salicylic acid (SA)) containing free carboxyl groups by direct derivatization of leaf extracts with Picolinyl reagent followed by LC-MS/MS analysis is presented. The presence of the N atom in the esterified pyridine moiety allowed the efficient ionization of 25 compounds tested irrespective of their chemical structure. The method was validated by comparing the results obtained after analysis of <it>Nicotiana attenuata </it>leaf material with previously described analytical methods.</p> <p>Conclusion</p> <p>The method presented was used to detect 16 compounds in leaf extracts of <it>N. attenuata </it>plants. Importantly, the method can be adapted based on the specific analytes of interest with the only consideration that the molecules must contain at least one free carboxyl group.</p

Rapid modification of the insect elicitor N-linolenoyl-glutamate via a lipoxygenase-mediated mechanism on Nicotiana attenuata leaves

<p>Abstract</p> <p>Background</p> <p>Some plants distinguish mechanical wounding from herbivore attack by recognizing specific constituents of larval oral secretions (OS) which are introduced into plant wounds during feeding. Fatty acid-amino acid conjugates (FACs) are major constituents of <it>Manduca sexta </it>OS and strong elicitors of herbivore-induced defense responses in <it>Nicotiana attenuata </it>plants.</p> <p>Results</p> <p>The metabolism of one of the major FACs in <it>M. sexta </it>OS, <it>N</it>-linolenoyl-glutamic acid (18:3-Glu), was analyzed on <it>N. attenuata </it>wounded leaf surfaces. Between 50 to 70% of the 18:3-Glu in the OS or of synthetic 18:3-Glu were metabolized within 30 seconds of application to leaf wounds. This heat-labile process did not result in free α-linolenic acid (18:3) and glutamate but in the biogenesis of metabolites both more and less polar than 18:3-Glu. Identification of the major modified forms of this FAC showed that they corresponded to 13-hydroxy-18:3-Glu, 13-hydroperoxy-18:3-Glu and 13-oxo-13:2-Glu. The formation of these metabolites occurred on the wounded leaf surface and it was dependent on lipoxygenase (LOX) activity; plants silenced in the expression of <it>NaLOX2 </it>and <it>NaLOX3 </it>genes showed more than 50% reduced rates of 18:3-Glu conversion and accumulated smaller amounts of the oxygenated derivatives compared to wild-type plants. Similar to 18:3-Glu, 13-oxo-13:2-Glu activated the enhanced accumulation of jasmonic acid (JA) in <it>N. attenuata </it>leaves whereas 13-hydroxy-18:3-Glu did not. Moreover, compared to 18:3-Glu elicitation, 13-oxo-13:2-Glu induced the differential emission of two monoterpene volatiles (β-pinene and an unidentified monoterpene) in ir<it>lox2 </it>plants.</p> <p>Conclusions</p> <p>The metabolism of one of the major elicitors of herbivore-specific responses in <it>N. attenuata </it>plants, 18:3-Glu, results in the formation of oxidized forms of this FAC by a LOX-dependent mechanism. One of these derivatives, 13-oxo-13:2-Glu, is an active elicitor of JA biosynthesis and differential monoterpene emission.</p

Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants

BACKGROUND: Genetically modified plants are widely used in agriculture and increasingly in ecological research to enable the selective manipulation of plant traits in the field. Despite their broad usage, many aspects of unwanted transgene silencing throughout plant development are still poorly understood. A transgene can be epigenetically silenced by a process called RNA directed DNA methylation (RdDM), which can be seen as a heritable loss of gene expression. The spontaneous nature of transgene silencing has been widely reported, but patterns of acquirement remain still unclear. RESULTS: Transgenic wild tobacco plants (Nicotiana attenuata) expressing heterologous genes coding for antimicrobial peptides displayed an erratic and variable occurrence of transgene silencing. We focused on three independently transformed lines (PNA 1.2, PNA 10.1 and ICE 4.4) as they rapidly lost the expression of the resistance marker and down-regulated transgene expression by more than 200 fold after only one plant generation. Bisulfite sequencing indicated hypermethylation within the 35S and NOS promoters of these lines. To shed light on the progress of methylation establishment, we successively sampled leaf tissues from different stages during plant development and found a rapid increase in 35S promoter methylation during vegetative growth (up to 77% absolute increase within 45 days of growth). The levels of de novo methylation were inherited by the offspring without any visible discontinuation. A secondary callus regeneration step could interfere with the establishment of gene silencing and we found successfully restored transgene expression in the offspring of several regenerants. CONCLUSIONS: The unpredictability of the gene silencing process requires a thorough selection and early detection of unstable plant lines. De novo methylation of the transgenes was acquired solely during vegetative development and did not require a generational change for its establishment or enhancement. A secondary callus regeneration step provides a convenient way to rescue transgene expression without causing undesirable morphological effects, which is essential for experiments that use transformed plants in the analysis of ecologically important traits

Application of silicone tubing for robust, simple, high - throughput , and time - resolved analysis of plant volatiles in field experiments

Kallenbach M, Veit D, Eilers E, Schuman MC. Application of silicone tubing for robust, simple, high - throughput , and time - resolved analysis of plant volatiles in field experiments. Bio-Protocol. 2015;5(3): e1391

How scent and nectar influence floral antagonists and mutualists

Abstract Many plants attract and reward pollinators with floral scents and nectar, respectively, but these traits can also incur fitness costs as they also attract herbivores. This dilemma, common to most flowering plants, could be solved by not producing nectar and/or scent, thereby cheating pollinators. Both nectar and scent are highly variable in native populations of coyote tobacco, Nicotiana attenuata, with some producing no nectar at all, uncorrelated with the tobacco&apos;s main floral attractant, benzylacetone. By silencing benzylacetone biosynthesis and nectar production in all combinations by RNAi, we experimentally uncouple these floral rewards/attractrants and measure their costs/benefits in the plant&apos;s native habitat and experimental tents. Both scent and nectar increase outcrossing rates for three, separately tested, pollinators and both traits increase oviposition by a hawkmoth herbivore, with nectar being more influential than scent. These results underscore that it makes little sense to study floral traits as if they only mediated pollination services

An Evaluation of the Causes, Consequences, and Potential Solutions to Increased Red River Flooding in the Caddo Parish Regions of Northwest Louisiana

Urban flooding is a challenge for many parts of the world, and Caddo Parish, Louisiana, is no exception. Caddo Parish, located in Northwestern Louisiana on the banks of the Red River, has been the subject of intense flooding for decades, issuing widespread devastation to many areas of the parish. As waters from rain events and upstream reservoirs deluged the Red River, countless individuals and communities were affected. In addition to damage and destruction of homes and personal belongings, sectors of the economy were also impacted, notably agriculture and industry. Rising waters jeopardized public infrastructure, affecting commerce throughout the parish, particularly waterway systems. This report, prepared by graduate students of the Bush School of Government and Public Service at Texas A&M University, per request of the Caddo Parish Sheriff’s Office, outlines policy solutions to protect the individuals and communities of Caddo Parish from future flooding

The ecological role of the activation of oxylipin biosynthesis in plants as a response to insect herbivory

In this work, I describe the activation and regulation of oxylipin signaling pathways in Nicotiana attenuata in response to herbivory. When the tobacco hornworm, Manduca sexta, feeds on N. attenuata, the plant perceives fatty acid-amino acid conjugates like 18:3-Glu present in the insect’s oral secretions. After contact with wounded leaf tissue, 18:3-Glu is rapidly modified into active and inactive metabolites catalyzed by LOX2. This metabolism of 18:3-Glu may play a role in the fine tuning of plant responses to insect herbivory. In response to herbivory, the plant induces the jasmonic acid (JA) biosynthesis. Here, 18:3-Glu together with the regulatory factors salicylate induced protein kinase (SIPK) and nonexpressor of PR-1 (NPR1) affect JA biosynthesis by enhancing the supply of α-linolenic acid through glycerolipase A1 (GLA1) whereas wound induced protein kinase (WIPK) affect basal allene oxide synthase (AOS) activity. After stimulus, the produced JA is conjugated to isoleucine by JASMONATE RESISTANT 4 and 6 (JAR4/6) to form JA-Ile. JA-Ile binds to SCFCOI which participates in the activation of defense response against insect herbivores. The plants’ capacity to mediate this JA signaling is essential to survive in nature. Besides the activation of JA biosynthesis, leaf wounding and insect herbivory does also results in the generation of green leaf volatiles such as (3Z)-hexenal and C12 molecules such as (9Z)-traumatin through the cleavage of e.g. 13-OOH-18:3 by hydroperoxy lyase (HPL). 98% of the (9Z)-traumatin formed is converted into 9-OH-traumatin: two-thirds by product recycling through LOX2 and one-third by non-enzymatic oxidation. In summary I showed that metabolism of insect elicitors at the wound site can tune the induction of oxylipin signaling pathways, how these pathways are activated upon insect herbivore attack and which function these signaling pathways fulfill in nature

Nicotiana attenuata SIPK, WIPK, NPR1, and Fatty Acid-Amino Acid Conjugates Participate in the Induction of Jasmonic Acid Biosynthesis by Affecting Early Enzymatic Steps in the Pathway1[W][OA]

Wounding and herbivore attack elicit the rapid (within minutes) accumulation of jasmonic acid (JA) that results from the activation of previously synthesized biosynthetic enzymes. Recently, several regulatory factors that affect JA production have been identified; however, how these regulators affect JA biosynthesis remains at present unknown. Here we demonstrate that Nicotiana attenuata salicylate-induced protein kinase (SIPK), wound-induced protein kinase (WIPK), nonexpressor of PR-1 (NPR1), and the insect elicitor N-linolenoyl-glucose (18:3-Glu) participate in mechanisms affecting early enzymatic steps of the JA biosynthesis pathway. Plants silenced in the expression of SIPK and NPR1 were affected in the initial accumulation of 13-hydroperoxy-linolenic acid (13-OOH-18:3) after wounding and 18:3-Glu elicitation by mechanisms independent of changes in 13-lipoxygenase activity. Moreover, 18:3-Glu elicited an enhanced and rapid accumulation of 13-OOH-18:3 that depended partially on SIPK and NPR1 but was independent of increased 13-lipoxygenase activity. Together, the results suggested that substrate supply for JA production was altered by 18:3-Glu elicitation and SIPK- and NPR1-mediated mechanisms. Consistent with a regulation at the level of substrate supply, we demonstrated by virus-induced gene silencing that a wound-repressed plastidial glycerolipase (NaGLA1) plays an essential role in the induction of de novo JA biosynthesis. In contrast to SIPK and NPR1, mechanisms mediated by WIPK did not affect the production of 13-OOH-18:3 but were critical to control the conversion of this precursor into 12-oxo-phytodienoic acid. These differences could be partially accounted for by reduced allene oxide synthase activity in WIPK-silenced plants