Dr Alex Webb
Find out more about Alex Webb and his Group or email Alex.Webb@plantsci.cam.ac.uk
Project titles:
- Molecular Dissection of the Input and Outputs of the Circadian Ca2+ Signalling Network
- Bioinformatic and Functional Analysis of Circadian Signalling Networks
- Chemical Genomics to Identify New Components of the Circadian Signalling Network
- Circadian Gating of Extracellular Stimuli
- The Circadian Regulation of Photosynthesis
- Mathematical Modelling of the Circadian Ca2+ Signalling Network
Project Title: Molecular Dissection of the Input and Outputs of the Circadian Ca2+ Signalling Network
Supervisor: Dr Alex Webb
Project Outline:
|
|
Figure 1 Circadian oscillations of Ca2+ measured in whole seedlings of Arabidopsis using aequorin (Love et al., 2004)
The circadian clock is a 24 h timekeeper that regulates a huge array of biological process. We demonstrated that this confers considerable advantage to the plant (Dodd et al. 2005 Science 309, 630 – 633). We are particularly interested in the role of the signalling molecule Ca2+ in the circadian signalling network (Love et al., Plant Cell 16, 956 – 966). The cytosolic free Ca2+ concentration ([Ca2+]cyt) oscillates with a circadian period (Figure 1). We have identified that circadian oscillations of [Ca2+]cyt are driven by oscillations in the concentration of the small signalling molecule cyclic ADP ribose. We have also identified some of the genes that regulate circadian oscillations of [Ca2+]cyt and this has allowed us to conclude that the circadian clock that regulates [Ca2+]cyt is subtley different from that which regulates other processes (Figure 2). We wish to understand further the structure of this new circadian clock and also find the molecular targets for the circadian oscillations of [Ca2+]cyt.
|
|
Figure 2 A model of the genes regulating circadian oscillations of [Ca2+]cyt (Xu et al., submitted)
In this project you will unravel the hierarchy of the plant circadian Ca2+ signalling network. You will use reverse genetics, photon counting imaging of luciferase and aequorin, T-DNA insertion mutants, biochemical assays of cyclic ADP ribose concentration, pharmacology, promoter reporter gene fusions and RT-PCR to analyse the pathways by which the circadian clock regulates circadian oscillations of [Ca2+]cyt.
See Research or email: Alex.Webb@plantsci.cam.ac.uk
Project Title: Bioinformatic and Functional Analysis of Circadian Signalling Networks
Supervisor: Dr Alex Webb
Project Outline:
|
|
Figure 3 We use the firefly luciferase gene to follow circadian regulation of gene promoters
Recently, we have used microarrays to analyse the circadian regulation of the entire genome in leaf cells (Gardner et al., Unpublished). This is a unique resource that allows us to delve into the signalling networks by which the circadian clock regulates gene expression. Using this approach we have determined that 25 % of all circadian-regulated genes are regulated by cADPR. Further bioinformatic and functional analyses of the signalling pathway has demonstrated that cADPR is responsible for driving circadian oscillations of [Ca2+]cyt and that cADPR is part of the mechanism that regulates clock period.
You will extend this unique analysis technique to identify molecular targets for Ca2+ in the circadian clock. You will identify Ca2+-regulated promoter sequences in circadian regulated genes. Once these sequences have been identified you will make novel promoter::luciferase (Figure 3) constructs containing the sequences you have identified to test the regulation of the sequences by Ca2+, light and the circadian clock. Additionally, you will use site-directed-mutagenesis to alter potentially Ca2+-sensitive regulatory domains in circadian clock genes and assess the effect on circadian function of the mutated genes. Tools will include molecular biology, cloning, bioniformatics, photon counting imaging, real time imaging, biochemical assays and physiological approaches developed in our lab.
See Research or email: Alex.Webb@plantsci.cam.ac.uk
Project Title: Chemical Genomics to Identify New Components of the Circadian Signalling Network
Supervisor: Dr Alex Webb
Project Outline:
An important new approach is the use of small chemical molecules to identify new genetic components of signalling networks. You will begin a genetic screen to identify mutants that have aberrant responses to a range of small molecules that we have identified to regulate clock period. This screen will use photon counting imaging of CAB2::LUCIFERASE (Figure 3) expression and will identify major new components of the circadian signalling network. Once the mutants have been isolated you will identify the genes and characterise their role in the circadian signalling network using the whole range of techniques available in the laboratory.
See Research or email: Alex.Webb@plantsci.cam.ac.uk
Project Title: Circadian Gating of Extracellular Stimuli
Supervisors: Dr Alex Webb and Dr Cahir O’Kane, Department of Genetics
Project Outline:
|
|
Figure 4 Recombinant Ca2+ reporter protein expressed in guard cells (Dodd et al., 2006)
We have recently demonstrated that the circadian clock modulates signals from the environment (Dodd et al. 2006 Plant J. 48, 962 – 973). Particularly, we have shown that the time of day cold is applied to plants influences the size and dynamics of cold-induced increases in [Ca2+]cyt and the magnitude of gene expression. You will use a recombinant ratiometric reporter of Ca2+ yellow cameleon 6.1 (YC6.1) to measure ABA-induced increases in cytosolic free Ca2+ in guard cells and other cell types, to determine the effect of time of day on Ca2+ signalling (Figure 4). You will use promoter::LUCIFERASE fusions to characterise the circadian modulation of cold and ABA signalling. In addition, you will use microarrays to analyse the effects of time of day on gene induction in wild type and circadian mutant backgrounds. The goal will be to identify time-of-day dependent transcriptional signalling networks.
See Research or email: Alex.Webb@plantsci.cam.ac.uk
Project Title: The Circadian Regulation of Photosynthesis (with Dr Julian Hibberd, Department of Plant Sciences)
Supervisors: Dr Alex Webb and Dr Julian Hibberd
Project Outline:
The circadian clock regulates many aspects of plant physiology and controls the expression of over 6 % of the genome. Surprisingly, very little is known about the competitive advantage provided by the circadian regulation of physiology. Our laboratory has made a major advance by demonstrating that the circadian clock increases the competitive advantage of plants by increasing the efficiency of photosynthesis (Dodd et al. 2005, Science 309, 630 – 633). We now wish to determine the cellular basis of the increase of photosynthetic efficiency by the circadian clock. You will use fluorescence imaging, infra red gas analysis, transcriptomics and metabolic profiling in wild type and circadian clock mutants to understand for the first time how the circadian clock makes plants fix more carbon, grow faster and out compete their neighbours.
See Research or email: Alex.Webb@plantsci.cam.ac.uk
See Research or email: Julian Hibberd@plantsci.cam.ac.uk
Project Title: Mathematical Modelling of the Circadian Ca2+ Signalling Network
Supervisors: Dr Alex Webb and Dr Jorge Goncalves, Department of Engineering, University of Cambridge
Project Outline:
|
|
Figure 5 Model architecture (Dalchau et al., unpublished)
As part of our systems approach we are looking to appoint a graduate with a degree in a mathematical subject to improve and extend our mathematical model of the circadian regulation of cytosolic free Ca2+ ([Ca2+]cyt). Ca2+ is a ubiquitous second messenger, involved in the transmission of inter- and intracellular signals. While short-term elevations of the concentration of cytosolic free [Ca2+]cyt have been studied extensively in many organisms, little is known about the circadian regulation of it’s basal level. To establish the importance of this circadian control, we are characterizing the pathways that regulate [Ca2+]cyt in the model plant Arabidopsis thaliana, both experimentally and mathematically. We have shown that the longer time-scale circadian control of [Ca2+]cyt can be described by a linear system of ordinary differential equations (ODEs), and show necessity for circadian-independent light activation (Figure 5).
|
|
Figure 6 Comparison of measured (black) and simulated (red) aequorin luminescence in constant light (Dalchau et al., unpublished).
Standard systems identification techniques were used to estimate model parameters from a single time-series experiment measuring [Ca2+]cyt and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) promoter activity in diurnal (12 h light, 12 h dark) cycles and constant darkness. The resulting model has been validated (Figure 6).
Linear systems identification has provided an accurate description of the dynamic regulation of basal [Ca2+]cyt. Our simulations have demonstrated that the transition of dark to light triggers the accumulation of Ca2+ in the cytosol, and this increase is prolonged by a slower-acting regulation by the central oscillator to achieve a peak between 7 and 9 hours after dawn.
The non-intuitive predictions of our model suggest a further combined mathematical experimental approach. You will improve and extend our model developing the mathematical approaches and also using automated high throughput data capture systems to generate the datasets required for model development. The key questions that require immediate attention are what is the nature of the molecular oscillator regulating [Ca2+]cyt, what are the advantages of the circadian and light regulation of [Ca2+]cyt and are light-mediated increases in basal [Ca2+]cyt ‘gated’ by the circadian clock? Full training in biological approaches and techniques will by provide by Dr Alex Webb in Plant Sciences whilst mathematical training will by provided by Dr Jorge Goncalves in Engineering. This is an exciting opportunity for a mathematically trained graduate to make the transition to systems biology.
See Research or email: Alex.Webb@plantsci.cam.ac.uk