Workpackage F: Modules for engineering photosynthesis and leaf metabolism
Plant leaves are biofactories that can accumulate valuable products in a number of discrete compartments both within and between cells. Furthermore, they also fine tune synthetic pathways in response to environmental signals. While significant progress has been made in defining cell specific gene expression in roots, this has not been achieved in leaves. This is a bottleneck in engineering this easily harvested organ, and there is no central repository of genetic modules to facilitate this. We aim to provide a library of elements that can be used to drive expression of both nuclear and plastid encoded genes in specific compartments of specific cells of leaves, and in addition to control that expression over the day-night cycle. These modules will be registered and made available in the OpenPlant repository.
OpenPlant is developing a library of DNA parts that can be used to drive expression of both nuclear and plastid encoded genes in specific compartments of specific cells of leaves, and in addition to control that expression over the day-night cycle.
Protein scaffolds for targeted expression
The Hibberd lab has been developing artificial protein scaffolds from bacteria to assemble in planta for metabolic engineering in leaves. They are investigating whether these artificial protein scaffolds can be used to improve the efficiency of photosynthesis, and for the more general aim of engineering plant metabolism. They have designed and synthetised parts and modules according to the PhytoBrick standard (Patron et al., 2015), which have been tested in Arabidopsis thaliana and shown to interact via Bimolecular Fluroescence Complementation (BiFC) coupled with Confocal Laser Scanning Microscopy. Once verified these parts will be publicly released.
DNA motifs for synthetic promoters
Stable transgenic lines of Arabidopsis thaliana containing epitope tagged nuclei and ribosomes driven by cell specific promoters have been produced in the Hibberd lab. These are being characterised and selfed to identify lines that can be used for isolation of RNA that is available for translation, as well as for cell specific DNaseISEQ. By interrogating these datasets, we aim to identify short DNA sequences that can be used to drive expression of genes in specific cells of the leaf to enhance photosynthetic efficiency.
Transcription factors and cis-elements
Recent work in the Hibberd lab led to the identification of a cis-element that regulates the accumulation of multiple enzymes involved in C4 photosynthesis in the mesophyll cells of Gynandropsis gynandra (Williams et al., 2016). OpenPlant research involves the identification, characterisation and public release of a collection of transcription factors and target cis-elements for engineering co-ordinated expression of synthetic pathways in leaves.
Specific expression of chloroplast genes
Work starting in 2017 aims to characterise inducible and cell-specific plastid targeted systems for regulation of plastid gene expression for public release. Christian Boehm in the Haseloff lab has established plastid transformation in Marchantia, and developed refactored cyan fluorescent protein markers for plastid expression. The use of fluorescent protein markers in Marchantia chloroplasts has been highly problematic for a number of years. There have been no reports of their successful use for chloroplast transformation. Christian has successfully engineered the cyan fluorescent protein gene for use in chloroplast transformation experiments in Marchantia, providing a valuable tool for characterisation of regulatory elements (Boehm et al., 2016).
Circadian control in the chloroplast
A future OpenPlant aim toward a toolkit for engineering in leaf tissues is the development of circadian controlled synthetic promoters for expression at defined phases in the day-night cycle in plants. More information coming soon.
Boehm, C.R., Ueda, M., Nishimura, Y., Shikanai, T., and Haseloff, J., 2016. A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha. Plant Cell Physiol. 57(2), p291-9. DOI: 10.1093/pcp/pcv160
Patron, N.J., Orzaez, D., Marillonnet, S., et al., 2015. Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist, 208(1), p13-19. DOI: 10.1111/nph.13532.
Williams, B.P., Burgess, S.J., Reyna-Llorens, I., Knerova, J., Aubry, S., Stanley, S., and Hibberd, J.M., 2016. An Untranslated cis-Element Regulates the Accumulation of Multiple C4 Enzymes in Gynandropsis gynandra Mesophyll Cells. Plant Cell, 28(2), p454-65. DOI: 10.1105/tpc.15.00570.