Project Outline

The rotation of the Earth creates periodic day-night changes in environmental conditions such as light and temperature. Organisms are able to adapt to these predictable diurnal cycles through their internal timekeeping system, the circadian clock, which has a periodicity of approximately 24 hours. The circadian clock enables plants to synchronise their metabolism and development with environmental rhythms to maximise fitness. This is reflected in the observation that at least 30% of Arabidopsis thaliana transcripts exhibit circadian rhythmicity and includes genes involved in flowering time, germination, photosynthesis, gas exchange, phytohormone synthesis and signalling, cellular redox status and plant immunity. Thus, circadian rhythmicity occurs at multiple levels from whole organs to individual organelles.

The endoplasmic reticulum (ER) is the gateway to the plant secretory pathway acting as the major platform for secretory protein production, transport, folding and quality control. The ER forms an architecturally intricate network of tubules and cisternae which is constantly remodelling in response to internal and external cues. Such ER reorganisation occurs through tubule extension and sliding and the concomitant formation and closure of polygonal regions; expansion and contraction of cisternae which also results in a shifting of the ratio of tubules to cisternae, and bulk ER streaming along actin filaments. Collaboration between the Frigerio lab (Warwick) and the Fricker lab (Oxford) has enabled us to develop and utilise a quantitative multivariate analysis tool (AnalyzER) (1) which extracts morphological, topological and dynamic features of the ER network and captures even subtle changes in ER structure or behaviour with statistical confidence.

Through our characterisation of the plant ER as part of a wider BBSRC-funded project on ER-mediated plant immunity, we have observed significant changes in the morphology and dynamics of the ER in Arabidopsis leaf epidermal cells associated with different times in the light-dark cycle. Alterations in ER form are likely intrinsically linked to changes in ER function, suggesting that protein secretion may also be rhythmic. Such behaviour would presumably enable the plant to prime the ER machinery for anticipated increases in protein demand thus avoiding ER stress. In animal cells, the unfolded protein response was recently shown to be activated in a rhythmic fashion under the control of the circadian clock. Disrupted clock function was associated with ER stress, with potential consequences for metabolic disorders. However, nothing is known about the impact of circadian rhythmicity on the plant secretory pathway.

Objectives/ Biological questions

1. Is the plant secretory pathway under the control of the circadian clock? Does protein flux through the ER exhibit rhythmic behaviour?
2. Can we manipulate the morphology and/or dynamics of the ER to modulate protein secretion? This would have multiple potential beneficial impacts including biotechnological applications and breeding plants with increased resistance to plant pathogens

Methods

1. Characterise further the rhythmic changes in ER morphology and dynamics of both wild-type plants and known clock mutants using confocal microscopy with subsequent image analysis.
2. Identify which, if any, components of the ER machinery (eg chaperones, folding enzymes, heat shock proteins, redox regulators, unfolded protein response pathway) exhibit rhythmic changes in expression to support the hypothesis that ER activity is circadian regulated.
3. Perform functional analysis of known clock mutants to assess impact on secretory protein levels, in terms of, for example, seed storage protein profiles and/or antimicrobial protein production and pathogen susceptibility

Project management

This project will be a joint collaboration between the Frigerio/Breeze group who specialise in plant cell biology and plant pathology, and the Carré group who have extensive expertise in circadian biology. As such the successful candidate will have the opportunity to take the project in several different directions depending on their own biological interests and results. They will also receive training in a variety of cell biology and molecular biology techniques

References

1. C. Pain, V. Kriechbaumer, M. Kittelmann, C. Hawes, M. Fricker, Quantitative analysis of plant ER architecture and dynamics. Nat Commun 10, 984 (2019)

Techniques

Analysis of confocal microscopy images will form a significant part of the project. This requires some familiarity with not only ImageJ but also command line coding in the R environment in order to further develop or adapt the existing analysis tool to make it fit for purpose for the images being obtained during the project.