Ecological Tools Developed for Nicotiana attenuata

Biotic and abiotic stresses influence plant performances under natural conditions, conditions which are not readily simulated under glasshouse conditions. Our research program examines the influence of altering the expression of key genes on plant performance in semi-natural habitats (see field site at Brigham Young University's Lytle Ranch Preserve), as well as under standardised glasshouse-conditions.

Glasshouse feeding assays

To evaluate the ecological significance of Nicotiana attenuata genotypes, standardised feeding assays are conducted. In addition to caterpillars from Manduca sexta, Spodoptera littoralis, Spodoptera exigua, or Heliothis virescens moths, we maintain colonies of myrids (Tupiocoris notatus) and aphids (Myzus persicae) all of which can be used as indicator for food quality of a plant. We conduct growth measurements (caterpillars), reproduction assays (aphids), and choice tests with all of these insects. Additional Waldbauer nutritional assays are used to obtain detailed data on food uptake and ability of the insect to processes the food.

Glasshouse growth analysis

In addition to the traditional measurements of rosette diameter, stalk length, or flower number, we use photographic plant growth analysis which allows for high throughput measurements, which can provide additional detailed data on the dynamics of plant growth. We are now also able to measure root growth continuously by using a sophisticated scanner system.

Plant performance in the native habitat

Plants in the field may be attacked by a large number of different herbivores. It is essential to determine which herbivore species are responsible for the damage on plants. By using herbivore screens we are able to correlate herbivore abundance as well as damage with gene-expression and gene-silenced plants to understand the genes which allow this native plant to cope with its enemies. We developed an easy to use database for this purpose. We standardized growth measures of plants in the field and use procedures to alter light conditions, such as UVB (UV-B opaque foil) or far-red (LED clusters) radiation.

Volatile organic compound profiling in the field
Volatile organic compounds emitted from leaves or flowers can be collected dynamically on charcoal traps with a pump or statically, with PDMS tubing,  which allows for high replicate numbers, and further analyzed by GC-ion trap-MS. Real time measurements under field conditions can be performed with a portable Z-nose GC detector.

Predation assay
Caterpillar attacked plants emit volatiles which attract predacious big-eyed-bugs. Routinely we glue Manduca sexta eggs on leaves and use the native populations of Geocoris spp. to provide readouts of the quality of the emitted volatiles for attracting predators, by counting the predation rate of the attached eggs. Caterpillars of Manduca sexta fed on different Nicotianan attenuata phenotypes are used in feeding assays with diverse predators, such as ants or lizards.

Genetic variation in native populations
We regularly search for different Nicotiana attenuata phenotypes in native populations focusing on morphological differences and also on differences in metabolism. For the later, we use native herbivore species to identify natural variation in signaling or secondary metabolites, in essence using insects as "bloodhounds" to ferret out genetic variation hidden within native Nicotiana attenuata populations.  As an example, see our recent PNAS publication in which used native Empoasca leaf hoppers to identify jasmonate mutants in native populations (link to Kallenbach et al PNAS 2012).

Video observation

With a 4-channel -video-recording system we are able to observe the herbivore community as well as floral visitors of different plants at the same time. In addition, a plant IR (infrared) field camera tracking setup, which can be used for 3D quantifications of growth and herbivore behavior is currently under development. The objective of this system is to qualitatively and quantitatively characterize the growth and the movement of different plant organs over time. Its passive infrared sensor provides the opportunity to observe pollinator interactions.

Photosynthetic ability

We developed methods to collect data on photosynthetic rates (IRGA), chlorophyll content (chlorophyll-meter), and the efficiency of Photosystem II (fluorescence-meter), under field conditions.

Nectar removal and pollination rates
A standardized protocol is available to measure nectar volume and nectar sugar concentration in flowers of Nicotiana attenuata. Nectar removal provides a measure of nectar quality or floral attraction. To quantify pollen transfer, we antherectomise flowers (removal of anthers) just before anthesis, and then expose these flowers to either night or day-active pollinators. The resulting capsule set quantifies outcrossing rates, which can be further quantified by genotyping by microsatellite analysis (link to Molecular platform) to reveal the number of different paternal genotypes that have sired the seeds.