ClockworkGreen will bring 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.
Arrhythmic Nicotiana attenuata plants
We will silence one or more key components of N. attenuata’s endogenous clock by RNAi using inverted repeat (IR) constructs in isogenic plants, to specifically shorten the circadian rhythm of a native tobacco which grows and develops during an elongating day in its natural habitats in the Great Basin Desert of North America. For our first target, we have chosen TIMING OF CAB EXPRESSION 1 (TOC1), the evening component of the core circadian oscillator. If the silenced TOC1 does not sufficiently shorten the endogenous clock, we will silence other clock components (ZTL, LHY, ADO2, ADO3, PRRs) until we find a line suitable for the field work.
Manuscripts in preparation:
Identification and characterization of circadian clock genes in a native tobacco, Nicotiana attenuata. Felipe et al.
Circadian marker genes and metabolites in N. attenuata
Single transcriptome and metabolome measurement will be compared to a database of circadian marker genes and metabolites to characterize circadian mutants more easily and quickly (3). A 44K full-transcriptome microarray (GEO accession number: GPL13527) and UPLC-TOF-MS method (Kim et al., in submission) was used to identify circadian maker genes and metabolites, respectively. Total 134 chips 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.
Tissue specific diurnal rhythms of metabolites and their regulation during herbivore attack in a native tobacco, Nicotiana attenuata. Kim et al. 2011, PLoS ONE
Manuscript in preparation
A system biology analysis reveals independent and inter-connected transcriptomics modules mediating N. attenuata resistance and tolerance responses to Manduca sexta herbivory. Jyotasana et al.
Genome sequencing of N. attenuata
The sequencing of the Nicotiana attenuata (accession Utah WT) genome was approved for funding in May, 2011 and will be 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 within two years.
Dex-inducible promoters in N. attenuata
We will use inducible promoters for analyzing the importance of the plant´s ability to adjust its circadian clock to changing environmental conditions in its native habitat. We will use the chimeric LhGR-N>>pOp6 system, which is extremely sensitive to minute amounts of the chemical inducer, dexamethasone (DEX) (Samalova et. al.2005, The Plant Journal 41, pp. 919-935). 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 (Samalova et. al.2005, The Plant Journal 41, pp. 919-935). 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 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 will allow us to test their effectiveness under field conditions.