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Ecological performance of arrhythmic plants in nature

Final Report Summary - CLOCKWORKGREEN (Ecological performance of arrhythmic plants in nature)

The overarching aims of the Clockwork Green project have been addressed, and deeper insights are being pursued into the association of phenotypes and their regulatory factors. We continue to establish techniques and instrumentation in order to address the next goals.
Aim 1: Creating permanently and inducibly “jet-lagged” Nicotiana attenuata plants by silencing different clock components under constitutive and dexamethasone-inducible promotors.
This aim has been accomplished: circadian clock genes have been identified and cloned into stable silencing inverted repeat constructs (Yon et al. 2012), DEX-inducible constructs (Schäfer et al. 2013), the latter for the core clock components, and a more accurate bioinformatics platform has been established for identifying oscillating genes and metabolites (Walter et al. 2014). The characterization of these clock-silenced lines has been performed under glasshouse and field conditions (Great Basin Desert, Utah-USA), comprising transcript expression analysis for evaluating endogenous rhythms, coupled with vegetative and reproductive key traits. To identify and quantify “jet-lagged” lines, measurements comprise: growth data (image analysis), floral traits (image analysis), phytohormone/defense profiles (HPLC-MS approach), vegetative and floral volatile profiling (now with the PDMS-TDU-GC-MS approach), and photosynthesis sensitivity and responsiveness; these data are in functional publications and publications in preparation or review.
Aim 2: With the plants generated in aim 1, systematically examine the importance of entrained endogenous rhythms separately at all stages in the plant’s life.
This applies aim 1 methodologies to all developmental stages and this goal has been partially achieved. Seed bank experiments have been completed but we are waiting for the results of QTL analysis to publish these data. Rosette and elongation stages have been characterized as continuous tracking and single points using aim 1 methodologies plus the integration of microarray and metabolomics platforms (see aim 3). Further studies including the analysis of abiotic stress responses and competition, and also trophic interactions such as predation of herbivores by Geocoris sp. (tritrophic interactions) and Trichobaris spp. pith infestation. The flowering stage, meaning all relevant floral traits, has been examined not only physiologically, but functionally by combining plant-pollinator interactions, such as glasshouse/field outcrossing and wind-tunnel studies of hawkmoth behavior. Studies of the gene and protein actors during flower development are being conducted with DEX-inducible lines to pinpoint the spatial and temporal key events. The functional aspect of the senescence stage has been addressed in studies manipulating cytokinin activity and metabolism.
Aim 3: Conduct integrated kinetic analyses of plants’ volatile and non-volatile metabolomes and transcriptomes with a high degree of temporal resolution to analyze time metabolic responses and new output.
The use of UPLC-Q-TOFMS and microarrays has been implemented on a large scale. The combination of gene and metabolite analyses has allowed the discovery of networks and interactions, in control and defense induction, in a temporal scope (Gaquerel et al. 2014, Gulati et al. 2014). This was conducted under light/dark and continuous light conditions in order to distinguish circadian from diurnal patterns and further in progress to analyze field vs glasshouse samples. Transcript expression kinetics have yielded candidate oscillating genes from different gene ontology groups, which are being associated with morphological and/or physiological phenotypes. These publications are currently in preparation .