ClockworkGreen

       Funded by Advanced Grant No 293926  of the European Research Council (ERC) 2012 - 2017

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).

Late flowering phenotypes of NaTOC1 silencing N. attenuata

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.

Inducible gene silencing in N. attenuata in its native habitat
Inducible NaLHY gene overexpression in N. attenuata

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.

Preparation of laboratory silicone tubing pieces (STs), and trapping applications

Publication: Kallenbach, M., Veit, D., Eilers, E. J., Schuman, M. C. (2015). Application of silicone tubing for robust, simple, high-throughput, and time-resolved analysis of plant volatiles in field experiments. Bio-protocol, 5(3): e1391. doi:10.21769/BioProtoc.1391.

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.

Publications:

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.

Size: 8 m x 24 m, 4 m high; Space: for 70 plants spaced 2 m apart

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).

Tracking vertical movement in N. attenuata flowers

Past research questions

A. Signaling in plant-herbivore interactions:

  1. How are fatty acid-amino acid conjugates (FACs) -specific elicitors in the oral secretions in lepidopteran herbivores- perceived by the plant?
  2. What signal transduction mechanisms are switched on very early after FAC perception to specify responses against insects?
  3. How is the plant response to insect herbivores affected after specific silencing of components of these signal transduction mechanisms?
  4. What cellular mechanisms activate oxylipin biosynthesis immediately after wounding and enhance their production after herbivory?
  5. Do different plant species (e.g. Nicotiana attenuata and Solanum nigrum) utilize different regulatory mechanisms to tune oxylipin production and metabolism after wounding and herbivory?
  6. How does the plant control jasmonate metabolism after herbivory? Do some of the JA-modified forms have a specific role in inducing systemic signaling after herbivory?
  7. How does oxylipin metabolism affect the selection of plants for feeding by insects?
  8. How is the glycerolipase NaGLA1 regulating the activation of JA biosynthesis in N. attenuata leaves during herbivory?
  9. Is NaGLA1 supplying substrates to the divinylether biosynthesis pathway in roots of infected N. attenuata plants?
  10. What other biosynthesis pathways involved in the generation of oxylipins (or not) depend on the activity of NaGLA1 in N. attenuata?
  11. Which are the mechanisms affected by NaGLA1 that induce a reduction in growth in plants silenced in its expression (ir-gla1) in natural environments but not in the glasshouse?
  12. Are these changes in growth caused by a differential colonization of roots by associated microorganisms? Or alternatively, are abiotic conditions affecting the growth of ir-gla1 plants compared to WT N. attenuata plants?
  13. By which mechanisms is the rapidly OS-inducible lectin-receptor like-kinase 1 (NalecRLK1) regulating responses of N. attenuata to herbivory?
  14. By which mechanisms is the rapidly OS-inducible putative nematode-resistance protein 1 (NaPNRP1) regulating responses of N. attenuata plants to herbivory?
  15. Is NaPNRP1 controlling the association of N. attenuata roots with specific endosymbionts? Is this differential association (compared to WT) affecting the response of N. attenuata plants silenced in PNRP1 expression (ir-pnrp) to herbivory?
  16. Are lipoxygenases (LOX) catalyzing the biosynthesis of a wider range of oxylipins than previously thought? Are some of these LOX-reactions “recycling” self-made products to generate novel signal molecules?
  17. How is the family of HD-Zip transcription factors regulating processes that integrate biotic and abiotic stresses with changes in growth and development of N. attenuata plants?
  18. How is NaHD20 regulating changes in flowering time and flower transitions upon periods of water deficiency and recovery? Are these changes connected to changes in ABA biosynthesis or transport into flowers?
  19. How is NaHD20 controlling the biosynthesis/release of benzylacetone in night flowers of N. attenuata plants?
  20. Which classes of transcription factors and other regulatory proteins contribute to regulation of plant defenses in N. attenuata against herbivores? Specifically, what are the are the roles of:

    • MYB transcription factors in defense and biosynthesis of secondary metabolites (phytoalexins) during herbivory?
    • herbivory-induced WRKY transcription factors in defense response?
    • a novel putative calmodulin-binding protein from tobacco in regulation of defense against herbivores?

  21. Do JAZ proteins -- repressors of JA-signaling -- contribute to induction of priming responses in plants under herbivory stress in the natural environment?
  22. Which jasmonic acid-amino acid conjugates trigger the de-repression of JA signaling by interacting with the E3-ubiquitin-ligase SCFCOI1 complex and degradation of JAZ proteins?
  23. Which jasmonates are responsible for individual jasmonate-elicited defense responses? Are direct and indirect defenses controlled by different conjugates of jasmonic acid? What biosynthetic steps are regulated by jasmonates?
  24. How do herbivore-specific defense responses change during ontogeny?
  25. Does UVB prime defenses against herbivores in natural environment?
  26. Are some of pathogen resistance-related genes also involved in plant-herbivore interactions?
  27. What is the role of MYC2 homologues in regulating JA-dependent defenses in N. attenuata?
  28. How and which defenses are induced by direct wounding of roots in N. attenauta?

B. Hormone crosstalk in plant-herbivore interactions:

  1. Is deconjugation of IAA-amino acid conjugates by IAR3 enzymes responsible for higher accumulation of JA-Ile in N. attenuata leaves?
  2. How does JA and ethylene crosstalk regulate accumulation of polyamine-hydroxycinnamic acid conjugates in herbivore attacked plants?

C. Herbivore behavior as function of plant defenses:

  1. Does the frequency of herbivore attack influence the ability of N. attenuata plants to elicit a jasmonic acid burst?
  2. How do multiple elicitations affect the defense response of N. attenuata: Can plants “count” herbivores and their herbivore loads and their potential fitness liabilities?
  3. How does herbivore behavior differ in response to active and deactivated plant defenses in WT versus plants in which endogenous defenses were suppressed by RNAi-mediated genetic approach?

D. Cost and benefits of direct and indirect defenses

  1. What are the benefits and the costs of protease inhibitor production in N. attenuata?
  2. Do diterpene secondary metabolites function as a defense against herbivore attack in N. attenuata, and, if so, what is their mechanism of action? Are defensive diterpenoids differently effective against specialist and generalist herbivores?
  3. How do native plants in wild N. attenuata populations vary in their herbivore- and jasmonate-elicited production of defenses? Which secondary metabolites are stable within populations, and which are highly variable? How do insect herbivore and predator communities respond to this variation?
  4. What are the pros and cons for a plant to be “apparent” and attract the attention of insects and how do the fitness consequences of herbivore “escape” and “defense” compare under field condition?
  5. What are the comparative fitness consequences of attack from the major different biotic agents found in nature: herbivores, viruses, pathogens?

E. Seed germination mechanisms

  1. What are the active smoke constituents that break secondary dormancy in N. attenuata seeds?
  2. How is germination and dormancy of N. attenuata seeds regulated on a molecular level?
  3. How do different genotypes of N. attenuata differ in their germination from long-lived seedbanks?

F. Plant-plant interactions

  1. What role do green leaf volatiles (GLVs) have in transferring information to a neighboring, conspecific plant?
  2. Is there, in addition to the known aboveground emissions, also volatiles released from the roots, and are these soil-borne VOCs recognized by roots of a neighbouring plant?
  3. What are the total effects of herbivore-inducible volatile emissions from neighbors on a plant's defensive status and fitness? Are these effects due to priming or induction of plant defenses, or to the apparency or defensive status of the neighbor?

G. Plant-pollinator interaction and mate choice mechanism

  1. What are the floral traits that mediate out-crossing in a plant?
  2. Do out-crossed seeds have a better chance to compete with their neighbors?
  3. Two ecotypes of N. attenuata (Utah and Arizona) differentially choose their mates amongst several natural genotypes. What is the basis of these selections and is this mate choice adaptive in nature? How does the non-random mate selection correlate with the survival of the offspring in long-term seed banks?
  4. In mixed pollination, mate discrimination occurred amongst non-self pollen against hygromycin-B resistance (transformation selectable marker) in WT styles and for it in transformed styles. How is this atypical mate selection related to the transgene escape through pollen-mediated gene flow for N. attenuata? How the molecular augmentations of insect-resistance (e.g. CRY) affect this selection pattern?
  5. N. attenuata discriminates amongst different pollen genotypes in mixed pollination. Moreover, post-pollination ethylene burst and pollen tube competition within different pollen genotypes are the main predictors for seed paternity. However, disruption in ethylene production/perception eliminates stylar selection of pollen tubes. What is the molecular mechanism that regulates pre-zygotic mate selection? Are there any stylar compounds that enhance/restrict pollen tube growth of specific genotypes?
  6. Which traits (genetic and environmental) do play a role in dormancy and survival of N. attenuata seed banks?

H. Plant-microbial endophyte interactions

  1. What role do bacterial endophytes play in N. attenuata and S. nigrum’s performance? When these endophytes alter plant growth, do they do so by altering the plant’s phytohormone status? Do the endophytes have other means of altering plant growth?
  2. What role do mycorrhizal fungi of the genus Glomus play in N. attenuata’s ecological interactions?
  3. How different are the bacterial communities in different plant developmental stages?
  4. What are the main factors important for endophyte recruitment?
  5. How important are endophytic bacteria as natural biocontrols against phytopathogenic fungi?
  6. Do some bacteria play a role in the tritrophic interaction against herbivores?

I. Effect of polyploidy on the evolution of anti-herbivore defense system in Nicotiana attenuata 

  1. The genus Nicotiana is ideally suited to study polyploidy because of its robust phylogenetic framework and the genus contains a large number of polyploid species (approx 40% are allopolyploids).  Nicotiana attenuata is a diploid species which is thought to be involved in the formation of several of these allopolyploids, particularly in the formation of N. bigelovii and N. clevelandii (allotetraploid descendants of the ancestors of N. attenuata and N. trigonophylla). Our research focuses on understanding how the anti-herbivore defense system, as well as pollination, germination and growth systems modified after neo-polyploidization and polyploidy speciation, using synthetic and natural polyploids.

J. Plant-herbivore interactions: the herbivore side

  1. What are the transcriptional responses of specialist (Manduca sexta) and generalist herbivores (Heliothis virescens) to feeding on N. attenutata host plants transformed to silence different secondary metabolites and defense signaling? From these analyses we hope to understand how herbivores adjust to the defenses that their feeding elicits in their host plants.
  2. How do specialist or generalist herbivores (e.g. M. sexta and Heliothis virescens) cope with different host plants? What are the differences in the transcriptomes of these herbivore species when feeding on N. attenuata?
  3. How do Corimelaena extensa seed predators find  their host plant, N. attenuata, in it's natural habitat and do plant traits mediate this herbivore's preferences and performance?
  4. What are the molecular mechanisms underlying M. sexta’s high nicotine tolerance? Does the dietary nicotine play a role in M. sexta’s interactions with its parasites and predators? Are the nicotine detoxification strategies of the generalist lepidopteran herbivores of N. attenuata different than that of specialist herbivore M. sexta? How M. sexta copes with the other specialized metabolites (than nicotine) of N. attenuata?

K. Genome evolution in N. attenuata and N. obtusifolia

Two genome sequencing projects are going on in our department: N. attenuata, our premier model system, and N. obtusifolia. Both species are diploid, but they have different genome sizes (2.5 vs. 1.5 Gb). They also display contrasting resistance to pathogens and herbivores and differences in life history traits. Our questions are:

  1. How can we use these genome data to learn about interspecies genome evolution in Nicotiana spp.?
  2. Particularly, what role are promoters playing in the adaptation to different modes of reponse to herbivory?
  3. What is the cause for the difference in genome sizes?
    We have obtained transcriptome data (Illumina Hi-seq) for different N. attenuata ecotypes (Utah and Arizona). Also from different tissues, timepoints and induction with OS.
  4. What can we learn about low abundant smRNAs, alternative splicing and their role in response to herbivory?
  5. Are there transcripts specific to ecotypes, certain conditions, at a certain times?

L. The ecological functions of the circadian clock

  1. Does the N. attenuata circadian clock orchestrate the plants' interactions with herbivores and microbes? Does shifting or abolishing the plant clock or the herbivore clock change the outcome of these interactions?
  2. How does the clock influence plant-pollinator interactions?
  3. What is the ecological importance of clock-regulated plant movement?
  4. How, and to what extent are plant specialized metabolites regulated by the clock? Are they coordinated with general metabolism so as to optimize biosynthetic efficiency, or with external events so as to optimize functional efficiency?
  5. What role does the clock play in the life history of the plant: in the plant's ability to successfully complete each stage of its growth and development in nature?
  6. Do herbivores co-opt or alter the plant's circadian rhythm? If so, is this to the benefit of the plant or of the herbivore?