Clockwork Green is bringing state-of-the-art analytical chemistry and molecular biology tools into field ecological research, allowing us to understand the ecological consequences for a plant of having an internal circadian clock which is “not in tune” with its environment at each stage in its life cycle.
Establishment of 7 toolboxes for the ClockworkGreen project (2012-2013.9)
1. Creating Nicotiana attenuata plants that are silenced in the expression of their circadian clock genes
We silenced several key components of N. attenuata’s endogenous clock by RNAi using inverted repeat (IR) constructs in isogenic plants. We are currently focusing on the characterization of the silenced lines of LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1 (TOC1), and ZEITLUPE (ZTL).
Publication: Yon, F., Seo, P. J., Ryu, J. Y., Park, C.-M., Baldwin, I. T., Kim, S.-G. (2012). Identification and characterization of circadian clock genes in a native tobacco, Nicotiana attenuata. BMC Plant Biology, 12: 172.
2. A ‘Real-time’ gene manipulation system
We developed inducible gene manipulation system for analyzing the importance of the plant´s ability to adjust its circadian clock to changing environmental conditions in its native habitat. We used the chimeric LhGR-N>>pOp6 system, which is extremely sensitive to minute amounts of the chemical inducer, dexamethasone (DEX). Separating the genomic location of the inducer (35S:LhGR-N) and the reporter (pOp6:xxx) constructs by independent transformation, creates plants with almost silent promoter activity in the absence of DEX. We have transformed N. attenuata plants with LhGR-N and pOp6 reporter constructs to test both, inducible overexpression and gene silencing. Overexpression is tested by using a pOp6:Atipt (Agrobacterium tumefaciens isopentenyltransferase) reporter that leads to local morphological defects when activated. To screen the effectiveness of inducible gene silencing we are using pOp6:irNaPDS (Nicotiana attenuata phytoenedesaturase), a reporter that is frequently used in our group as a visual control for transient silencing experiments because NaPDS-silencing generates photobleaching in silenced tissues (e.g. Wu et al. 2007, The Plant Cell 19 pp. 1069-1121; Meldau et al. 2011, New Phytologist 189, 1143-1156). The rapid appearance of the visual phenotypes of both constructs allowed us to test their effectiveness under field conditions.
Publication: Schäfer, M., Brütting, C., Gase, K., Reichelt, M., Baldwin, I. T., Meldau, S. (2013). “Real time” genetic manipulation: a new tool for ecological field studies. The Plant Journal. doi:10.1111/tpj.12301.
3. ‘Real-time’ volatile measurement system in the field
Plant volatiles (PVs) mediate interactions between plants and arthropods, microbes, and other plants, and are involved in responses to abiotic stresses. However, PVs are usually sampled in the artificial environments of laboratories or climate chambers. Sampling of PVs in natural environments is difficult, limited by requirements to transport, maintain, and power instruments, or to employ expensive sorbent devices in replicate. We developed a method for collecting PVs under the field conditions by using polydimethysiloxane (PDMS), a sorbent commonly used for PV sampling. Coupling with thermal desorption (TD)-GC-MS analysis – a 40-year-old and widely available technology – yields reproducible, sensitive, spatiotemporally resolved, quantitative data from headspace samples taken in natural environments.
Manuscript in preparation: Mario Kallenbach, Youngjoo Oh, Daniel Veit, Ian T. Baldwin, Meredith C. Schuman. A robust, simple, high-throughput technique for time-resolved plant volatile analysis in field experiments.
4. New method for network analysis
We created a 66K full-transcriptome microarray (GEO accession number: GPL13527) and UPLC-TOF-MS method (Kim et al., in submission) to identify circadian maker genes and metabolites, respectively. Total 134 chips (44K) and 76 chips (66K) were used to analyze circadian rhythm of transcriptome in three different tissues (GEO accession GSE30287). Ca. 900 runs on an UPLC-ToF-MS were performed to analyze circadian rhythms in the metabolomes of different plant tissues.
To examine the roles of the circadian clock for plant defense, we first designed a novel approach of combining an extended Self-Organizing Maps (SOM) based dimensionality reduction method with bootstrap-based non-parametric ANOVA models to identify the onset and context of signaling and metabolic pathway activations.
Kim, S.-G., Yon, F., Gaquerel, E., Gulati, J., Baldwin, I. T. (2011). Tissue specific diurnal rhythms of metabolites and their regulation during herbivore attack in a native tobacco, Nicotiana attenuata. PLoS One, 6(10), e26214.
Gulati, J., Kim, S.-G., Baldwin, I. T., Gaquerel, E. (2013). Deciphering herbivory-induced gene-to-metabolite dynamics in Nicotiana attenuata tissues using a multifactorial approach. Plant Physiology, 162(2), 1042-1059.
5. Genome sequencing of N. attenuata
The sequencing of the Nicotiana attenuata (accession Utah WT) genome was approved for funding in May, 2011 and is being conducted at the MPG Sequencing Center at the MPI for Molecular Genetics in Berlin. The project has been started with Illumina paired end sequencing. Full assembly of the genome is expected to be finished by the end 2013.
6. Tent project
The main objective of the tent is to investigate pollination and oviposition behavior of Manduca sexta (Sphingidae), and other hawkmoths, by manipulating different plant traits of Nicotiana attenuata (Solanaceae) including the circadian rhythms in flowers. Unlike in the field, we are able to measure ecological interactions of one single insect species and a line of transgeneic plants at a time, which allows for deeper insights in the evolution of certain plant traits and helps to bring the important plant-insect interactions to light. The large size and the air-permeable cover of the tent permits the study of plant-insect interactions in a simulated natural environment. The permanent air flow through the mesh avoids volatile accumulation in the tent, unlike in a glasshouse. As a consequence, M. sexta shows oviposition and nectaring behaviors comparable to those observed in the field – only one egg per plant is laid. Experiments in the tent will be used to complement field research in Utah and allow for the rigor dissection of these complicated plant-pollinator interactions under conditions under which the pollinator behavior matches those of pollinators in the field.
7. High-Resolution digital imaging
We are developing high-resolution digital imaging system to characterize several circadian rhythms in N. attenuata. For this work, we are collaborating with Prof. Dr. Thomas Altmann (the Leibniz Institute of Plant Genetics and Crop Plant Research, IPK).