Danny Kessler

Field experiments with transformed plants reveal the sense of floral scents

Plants use many means to attract pollinators, including visual cues and odor. We investigated how nonpigment floral chemistry influences nectar removal, floral visitation, florivory, rates of outcrossing, and fitness through both male and female functions. We blocked expression of biosynthetic genes of the dominant floral attractant [benzyl acetone (Nachal1)] and nectar repellent [nicotine (Napmt1/2)] in all combinations in the native tobacco Nicotiana attenuata and measured their effects on plants in their native habitat. Both repellent and attractant were required to maximize capsule production and seed siring in emasculated flowers and flower visitation by native pollinators, whereas nicotine reduced florivory and nectar robbing.

Changing pollinators as a means of escaping herbivores.

All animal-pollinated plants must solve the problem of attracting pollinators while remaining inconspicuous to herbivores, a dilemma exacerbated when voracious larval-stage herbivores mature into important pollinators for a plant [1]. Herbivory is known to alter pollination rates, by altering flower number [2], size [3, 4], nectar production [5], seasonal timing of flowering [6], or pollinator behavior [7]. Nicotiana attenuata, a night-flowering tobacco that germinates after fires in the Southwestern United States, normally produces flowers that open at night and release benzyl acetone (BA) to attract night-active hawkmoth pollinators (Manduca quinquemaculata and M. sexta), which are both herbivores and pollinators. When plants are attacked by hawkmoth larvae, the plants produce flowers with reduced BA emissions that open in the morning and are preferentially pollinated by day-active hummingbirds. This dramatic change in flower phenology, which is elicited by oral secretions (OSs) from feeding hawkmoth larvae and requires jasmonate (JA) signal transduction, causes the majority of outcrossed seeds to be produced by pollinations from day-active hummingbirds rather than night-active hawkmoths. Because oviposition and nectaring are frequently coupled behaviors in hawkmoths, we propose that this OS-elicited, JA-mediated change in flower phenology complements similarly elicited responses to herbivore attack (direct defenses, indirect defenses, and tolerance responses) that reduce the risk and fitness consequences of herbivory to plants.

Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9

Angiosperms developed floral nectaries that reward pollinating insects. Although nectar function and composition have been characterized, the mechanism of nectar secretion has remained unclear. Here we identify SWEET9 as a nectary-specific sugar transporter in three eudicot species: Arabidopsis thaliana, Brassica rapa (extrastaminal nectaries) and Nicotiana attenuata (gynoecial nectaries). We show that SWEET9 is essential for nectar production and can function as an efflux transporter. We also show that sucrose phosphate synthase genes, encoding key enzymes for sucrose biosynthesis, are highly expressed in nectaries and that their expression is also essential for nectar secretion. Together these data are consistent with a model in which sucrose is synthesized in the nectary parenchyma and subsequently secreted into the extracellular space via SWEET9, where sucrose is hydrolysed by an apoplasmic invertase to produce a mixture of sucrose, glucose and fructose. The recruitment of SWEET9 for sucrose export may have been a key innovation, and could have coincided with the evolution of core eudicots and contributed to the evolution of nectar secretion to reward pollinators.

Petunia flowers solve the defence/apparency dilemma of pollinator attraction by deploying complex floral blends

Flowers recruit floral visitors for pollination services by emitting fragrances. These scent signals can be intercepted by antagonists such as florivores to locate host plants. Hence, as a consequence of interactions with both mutualists and antagonists, floral bouquets likely consist of both attractive and defensive components. While the attractive functions of floral bouquets have been studied, their defensive function has not, and field-based evidence for the deterrence of floral-scent constituents is lacking. In field and glasshouse experiments with five lines of transgenic Petunia x hybrida plants specifically silenced in their ability to release particular components of their floral volatile bouquet, we demonstrate that the emission of single floral-scent compounds can dramatically decrease damage from generalist florivores. While some compounds are used in host location, others prevent florivory. We conclude that the complex blends that comprise floral scents are likely sculpted by the selective pressures of both pollinators and herbivores.

Solanum nigrum: a model ecological expression system and its tools.

Plants respond to environmental stresses through a series of complicated phenotypic responses, which can be understood only with field studies because other organisms must be recruited for their function. If ecologists are to fully participate in the genomics revolution and if molecular biologists are to understand adaptive phenotypic responses, native plant ecological expression systems that offer both molecular tools and interesting natural histories are needed. Here, we present Solanum nigrum L., a Solanaceous relative of potato and tomato for which many genomic tools are being developed, as a model plant ecological expression system. To facilitate manipulative ecological studies with S. nigrum, we describe: (i) an Agrobacterium‐based transformation system and illustrate its utility with an example of the antisense expression of RuBPCase, as verified by Southern gel blot analysis and real‐time quantitative PCR; (ii) a 789‐oligonucleotide microarray and illustrate its utility with hybridizations of herbivore‐elicited plants, and verify responses with RNA gel blot analysis and real‐time quantitative PCR; (iii) analyses of secondary metabolites that function as direct (proteinase inhibitor activity) and indirect (herbivore‐induced volatile organic compounds) defences; and (iv) growth and fitness‐estimates for plants grown under field conditions. Using these tools, we demonstrate that attack from flea beetles elicits: (i) a large transcriptional change consistent with elicitation of both jasmonate and salicylate signalling; and (ii) increases in proteinase inhibitor transcripts and activity, and volatile organic compound release. Both flea beetle attack and jasmonate elicitation increased proteinase inhibitors and jasmonate elicitation decreased fitness in field‐grown plants. Hence, proteinase inhibitors and jasmonate‐signalling are targets for manipulative studies.

How scent and nectar influence floral antagonists and mutualists.

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 tobaccos 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 plants 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. DOI: http://dx.doi.org/10.7554/eLife.07641.001

Pithy protection: Nicotiana attenuata's jasmonic acid-mediated defenses are required to resist stem-boring weevil larvae.

Folivory is the best studied plant-herbivore interaction, but it is unclear whether the signaling and resistance traits important for the defense of leaves are also important for other plant parts. Larvae of the tobacco stem weevil, Trichobaris mucorea, burrow into stems of Nicotiana attenuata and feed on the pith. Transgenic N. attenuata lines silenced in signaling and foliar defense traits were evaluated in a 2-year field study for resistance against attack by naturally occurring T. mucorea larva. Plants silenced in early jasmonic acid (JA) biosynthesis (antisense [as]-lipoxygenase3 [lox3]; inverted repeat [ir]-allene oxide cyclase), JA perception (as-coronatine insensitive1), proteinase inhibitors (ir-pi), and nicotine (ir-putrescine methyl-transferase) direct defenses and lignin (ir-cad) biosynthesis were infested more frequently than wild-type plants. Plants unable to emit C6 aldehydes (as-hpl) had lower infestation rates, while plants silenced in late steps in JA biosynthesis (ir-acyl-coenzyme A oxidase, ir-opr) and silenced in diterpene glycoside production (ir-geranylgeranyl pyrophosphate synthase) did not differ from wild type. Pith choice assays revealed that ir-putrescine methyl-transferase, ir-coronatine insensitive1, and ir-lox3 pith, which all had diminished nicotine levels, were preferred by larvae compared to wild-type pith. The lack of preference for ir-lox2 and ir-cad piths, suggest that oviposition attraction and vascular defense, rather than pith palatability accounts for the higher attack rates observed for these plants. We conclude that traits that influence a plant’s apparency, stem hardness, and pith direct defenses all contribute to resistance against this herbivore whose attack can be devastating to N. attenuata’s fitness.

Back to the past for pollination biology

Manipulations of the interactions between plants and their floral visitors remain the most successful path to an understanding of floral traits, which may have been shaped by both herbivores and pollinators. By using genetic tools in combination with old-fashioned field work the dual protective/advertisement functions of floral traits are being realized. The distinction between wanted and unwanted floral visitors is blurring, and plants with specialized pollination systems are being found capable of using alternative pollinators if the specialized pollinators fail to perform.

Context dependency of nectar reward-guided oviposition

Nectar is the most common floral reward used to recruit pollination services. Changes in nectar volume may affect not only pollination services, but also the attraction of antagonists such as herbivores, especially if the same insect species acts as herbivore and pollinator. Plants compete with each other for the best pollination services, at the same time employing various strategies to avoid herbivory. Datura wrightii Regel and Nicotiana attenuata Torr. ex Watson, two sympatric solanaceous species, compete for the same hawkmoth pollinator, Manduca quinquemaculata (Haworth) (Lepidoptera: Sphingidae), although standing nectar volume per flower differs 50‐fold (70 vs. 1.3 μl, respectively) between these species. This large difference may also result in differences in oviposition rates. I conducted a detailed analysis of diurnal changes in nectar volume and sugar concentration in field‐ and glasshouse‐grown D. wrightii and N. attenuata plants, and tested how well nectar production is buffered against the loss of large amounts of foliar tissue that frequently occurs due to M. quinquemaculata larval herbivory. I examined the influence of nectar volume on herbivore damage in the field and compared the results with previously published data from N. attenuata which were collected simultaneously. Oviposition by M. quinquemaculata moths increased significantly in D. wrightii plants whose nectar volume had been experimentally increased five‐fold compared to untreated control plants, and correlated with the numbers of flowers per plant in native populations. The results suggest that a hawkmoth mother may use standing nectar volume of a potential host plant to estimate its size, and possibly health, to make the optimal decision for her progeny. This mode of assessment, however, is apparently not used with another plant species, as other more herbivory‐related cues, such as olfaction or vision, are more influential in determining oviposition rates on other plant species. Yet within a plant species, regulating nectar volume strongly influences future herbivory.

A new acyclic diterpene glycoside from Nicotiana attenuata with a mild deterrent effect on feeding Manduca sexta larvae

Abstract To investigate the role of secondary metabolites in the feeding behavior of Manduca sexta larvae feeding on Nicotiana attenuata, an aqueous acetone extract of the aerial parts of the plant was subjected to feeding-performance bioassay-guided fractionation. We isolated three 20- hydroxygeranyllinalool glycosides from the leaves of N. attenuata, which acted as mild deterrents to the feeding herbivore M. sexta. One of the diterpenoid glycosides, attenoside (3), is a novel natural product. The structures of the compounds were determined using APCI mass spectrometry and 1- and 2D-NMR spectroscopy.