Unpredictability of nectar nicotine promotes outcrossing by hummingbirds in Nicotiana attenuata

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118

119

Significance When hornworm ( Manduca sexta ) larvae feed on Nicotiana attenuata plants in native habitats, more disappear at night when they feed on transgenic, nicotine-free N. attenuata plants because wolf spiders ( Camptocosa parallela ) selectively prey on nicotine-free larvae. When larvae consume nicotine-replete plants, their midgut-expressed cytochrome P450 6B46 ( CYP6B46 ) is upregulated. The larvae in which CYP6B46 is silenced by plant-mediated RNAi excrete more of their ingested nicotine. Silencing CYP6B46 impairs the mechanisms of passing ingested nicotine from the midgut to the hemolymph to be exhaled from the spiracles during spider attack. Spiders are deterred by this nicotine-rich halitosis. Thus, the CYP6B46 -silenced larvae exhale less nicotine and become ready spider prey, demonstrating that CYP6B46 functions to repurpose the normally excreted nicotine for defense.

“…Clear supernatant was collected and analyzed using a HPLC/ESI-Q3-MS (Varian 1200) with 35V capillary voltage as described previously (46). Analysis of Cotinine, CNO and NNO.…”

Section: Methodsmentioning

confidence: 99%

Natural history-driven, plant-mediated RNAi-based study reveals CYP6B46 ’s role in a nicotine-mediated antipredator herbivore defense

Kumar

Pandit

Steppuhn

et al. 2013

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118

119

“…However, in flowers their levels are relatively low, and in the case of nicotine, their concentrations change between day and night (68), and in nectar, nicotine occurs at highly variable and unpredictable concentrations that function to increase the outcrossing behavior of hummingbird pollinators (69). Given that high nicotine levels in flowers repel pollinators in N. attenuata (70), we speculate that high levels of HGL-DTGs or phenolamides in flowers might have similar effects.…”

Section: Discussionmentioning

confidence: 94%

Flower-specific jasmonate signaling regulates constitutive floral defenses in wild tobacco

Wang

et al. 2017

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Optimal defense (OD) theory predicts that within a plant, tissues are defended in proportion to their fitness value and risk of predation. The fitness value of leaves varies greatly and leaves are protected by jasmonate (JA)-inducible defenses. Flowers are vehicles of Darwinian fitness in flowering plants and are attacked by herbivores and pathogens, but how they are defended is rarely investigated. We used Nicotiana attenuata, an ecological model plant with well-characterized herbivore interactions to characterize defense responses in flowers. Early floral stages constitutively accumulate greater amounts of two well-characterized defensive compounds, the volatile (E)-α-bergamotene and trypsin proteinase inhibitors (TPIs), which are also found in herbivore-induced leaves. Plants rendered deficient in JA biosynthesis or perception by RNA interference had significantly attenuated floral accumulations of defensive compounds known to be regulated by JA in leaves. By RNA-seq, we found a JAZ gene, NaJAZi, specifically expressed in early-stage floral tissues. Gene silencing revealed that NaJAZi functions as a flower-specific jasmonate repressor that regulates JAs, (E)-α-bergamotene, TPIs, and a defensin. Flowers silenced in NaJAZi are more resistant to tobacco budworm attack, a florivore. When the defensin was ectopically expressed in leaves, performance of Manduca sexta larvae, a folivore, decreased. NaJAZi physically interacts with a newly identified NINJA-like protein, but not the canonical NINJA. This NINJA-like recruits the corepressor TOPLESS that contributes to the suppressive function of NaJAZi on floral defenses. This study uncovers the defensive function of JA signaling in flowers, which includes components that tailor JA signaling to provide flower-specific defense

“…As previously reported, constitutively produced defense metabolites such as nicotine, rutin, and chlorogenic acid exhibit comparatively low degrees of natural variation. Not only is nicotine a direct defense compound effective against attack by leaf herbivores but its occurrence in the nectar also affects pollinator visits and outcrossing rates (37,39); as such, stronger selection pressures are expected to erode large quantitative variations in nicotine levels. However, recent work has shown that yet to be identified molecular mechanisms generate high variations in nectar nicotine concentrations that surpass variations found in vegetative tissues (37).…”

Section: Resultsmentioning

confidence: 99%

“…Not only is nicotine a direct defense compound effective against attack by leaf herbivores but its occurrence in the nectar also affects pollinator visits and outcrossing rates (37,39); as such, stronger selection pressures are expected to erode large quantitative variations in nicotine levels. However, recent work has shown that yet to be identified molecular mechanisms generate high variations in nectar nicotine concentrations that surpass variations found in vegetative tissues (37). This previous result suggests that independent mechanisms may control the variability of nicotine concentrations in these two compartments of a plant.…”

Section: Resultsmentioning

confidence: 99%

Navigating natural variation in herbivory-induced secondary metabolism in coyote tobacco populations using MS/MS structural analysis

Baldwin

Gaquerel

2015

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Natural variation can be extremely useful in unraveling the determinants of phenotypic trait evolution but has rarely been analyzed with unbiased metabolic profiling to understand how its effects are organized at the level of biochemical pathways. Native populations of Nicotiana attenuata, a wild tobacco species, have been shown to be highly genetically diverse for traits important for their interactions with insects. To resolve the chemodiversity existing in these populations, we developed a metabolomics and computational pipeline to annotate leaf metabolic responses to Manduca sexta herbivory. We selected seeds from 43 accessions of different populations from the southwestern United Statesincluding the well-characterized Utah 30th generation inbred accession-and grew 183 plants in the glasshouse for standardized herbivory elicitation. Metabolic profiles were generated from elicited leaves of each plant using a high-throughput ultra HPLC (UHPLC)-quadrupole TOFMS (qTOFMS) method, processed to systematically infer covariation patterns among biochemically related metabolites, as well as unknown ones, and finally assembled to map natural variation. Navigating this map revealed metabolic branch-specific variations that surprisingly only partly overlapped with jasmonate accumulation polymorphisms and deviated from canonical jasmonate signaling. Fragmentation analysis via indiscriminant tandem mass spectrometry (idMS/MS) was conducted with 10 accessions that spanned a large proportion of the variance found in the complete accession dataset, and compound spectra were computationally assembled into spectral similarity networks. The biological information captured by this networking approach facilitates the mining of the mass spectral data of unknowns with high natural variation, as demonstrated by the annotation of a strongly herbivory-inducible phenolic derivative, and can guide pathway analysis.plant-insect interactions | metabolomics | mass spectrometry | natural variation E lucidating the structure of metabolites underlying complex traits and the factors that maintain their variation in natural populations are important challenges in plant ecological studies (1). Many studies have notably shown that stress-responsive pathways that produce secondary metabolites are sporadically found across different plant taxa with extensive diversification (2). This important diversification suggests that particular metabolic systems have been recruited through natural selection when the set of compounds that they produce address specific ecological needs. Interactions with insects are important selection pressures that have sculpted plant metabolism, and many plant metabolites protect against herbivore attack and physical damage (3-5). The timely production of particular secondary metabolites in response to insect attack benefits plants by decreasing the costs of constitutive metabolite production. Trade-offs between defense metabolite productions and the intrinsic growth-related functions of central metabolic pathways likely prov...