Engineering photosynthesis

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 (i) 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 (ii) to control that expression over the day-night cycle.

Photosynthesis sets maximum crop yield but despite millions of years of natural selection is not optimised for either current atmospheric conditions or agricultural practices. The majority of photosynthetic organisms, including crops of global importance such as wheat, rice and potato use the C3 photosynthesis pathway in which Ribulose-Bisphosphate Carboxylase Oxygenase (RuBisCO) catalyses the primary fixation of CO2. However, carboxylation by RuBisCO is competitively inhibited by oxygen binding the active site. In some plants, evolution has led to a modified system known as the C4 pathway in which RuBisCO is limited to a specific compartment where CO2 is concentrated. In both C3 and C4 plants, leaves contain multiple distinct cell-types. However, our understanding of how gene expression is controlled in these cell-types is limited. Our aim is to provide a collection of DNA parts that allow targeted engineering of photosynthesis and other traits in specific cells of leaves.

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.

Focus has been on two promoters that drive specific expression in bundle sheath cells of leaves. By combining functional testing via production of truncations with computational analysis, we have identified one positive regulator in cis that is necessary and sufficient to drive cell specific expression in leaves, and one negative regulator that represses expression in mesophyll and veinal cells. Both are ready for public release.

Transcription factors and cis-elements

By combining RNA-SEQ datasets from defined cell-types of the Arabidopsis leaf with the transcription factor footprinting that is generated from DNaseI-SEQ, OpenPlant research aims to identify, characterise and release of a collection of transcription factors that bind defined cis-elements for engineering coordinated expression of synthetic pathways in leaves.

The Hibberd lab has compiled a list of transcription factors that are preferentially expressed in bundle sheath cells of A. thaliana, and identified three transcription factors of particular interest. Of these, one interacts directly with the positive regulatory DNA element identified mentioned above. Thus, these parts could be used for coordinate expression of genes in designated cells. These parts are ready for public release.

Specific expression of chloroplast genes

We aim 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). The first group of Marchantia resources have been recently published in ACS SynBio (Sauret-Gueto et al, 2020).

Circadian control  in the chloroplast

OpenPlant aims to develop circadian-controlled synthetic promoters for expression at defined phases in the day-night cycle in plants.

The genetic architecture of the circadian system in Marchantia has been analysed by Lukas Muller (Webb/Haseloff labs). The Marchantia genome lacks homologs to CCA1 and LHY and contains only one homolog to the PRR5/7/9 family in Arabidopsis. This has informed the choice of genes for synthesis of DNA parts. Promoter regions (3kb upstream of 5’UTR) of putative circadian clock genes and putative clock output genes were identified in the Marchantia genome, domesticated and equipped with the respective cloning tags (following the common syntax) for Loop assembly. Transgenic lines were generated in order to run high-throughput circadian assays in Marchantia. Different successful transformants are being screened for the transgenic trait and a protocol is developed to compare Marchantia to Arabidopsis using instruments available in the Webb lab.

Protein scaffolds for targeted expression

The Hibberd lab has been developing artificial protein scaffolds from bacteria that 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), and have been tested in Arabidopsis thaliana and shown to interact via Bimolecular Fluroescence Complementation (BiFC) coupled with Confocal Laser Scanning Microscopy.

Publications

Müller LM, Mombaerts L, Pankin A, Davis SJ, Webb AAR, Goncalves J, von Korff M (2020) Differential Effects of Day/Night Cues and the Circadian Clock on the Barley Transcriptome. Plant Physiol. Jun; 183(2): 765–779. doi: 10.1104/pp.19.01411

Dickinson, P., Kneřová, J., Szecówka, M., Stevenson, S.R., Burgess, S.J., Mulvey, H., Bågman, A-M., Gaudinier, A., Brady, S.M., Hibberd, J.M. (2020). A bipartite transcription factor module controlling expression in the bundle sheath of Arabidopsis thaliana. Nature Plants 6:1468–1479 https://doi.org/10.1038/s41477-020-00805-w

Burgess, S.J. Reyna-Llorens, I., Stevenson, S.R., Singh, P., Jaeger, K., Hibberd, J.M. (2019) Genome-wide transcription factor binding in leaves from C3 and C4 grasses. The Plant Cell 31(10):2297-2314. doi: 10.1105/tpc.19.00078.

Borba AR, Serra TS, Górska A, Gouveia P, Cordeiro AM, Reyna-Llorens I, Kneřová J, Barros PM, Abreu IA, Oliveira MM, Hibberd JM, Saibo NJM (2018). Synergistic binding of bHLH transcription factors to the promoter of the maize NADP-ME gene used in C4 photosynthesis is based on an ancient code found in the ancestral C3 state. Mol Biol Evol. 35(7):1690-1705. doi: 10.1093/molbev/msy060.

Kneřová, J., Dickinson, P.J., Szecówka, M., Burgess, S.J. Mulvey, M., Bågman, A-M., Gaudinier, A., Brady, S.M. and Hibberd, J.M. (2018) A single cis­-element that controls cell-type specific expression in Arabidopsis. BioRxiv doi: https://doi.org/10.1101/380188.

Reyna-Llorens I, Burgess SJ, Reeves G, Singh P, Stevenson SR, Williams BP, Stanley S, Hibberd JM (2018). Ancient duons may underpin spatial patterning of gene expression in C4 leaves. Proc Natl Acad Sci U S A. 115(8):1931-1936. doi: 10.1073/pnas.1720576115.

Burgess, S.J., Reyna-Llorens, I., Jaeger, K., Hibberd, J.M. (2017). A transcription factor binding atlas for photosynthesis in cereals identifies a key role for coding sequence in the regulation of gene expression. BioRxiv pre-print doi: https://doi.org/10.1101/165787

Kümpers BM, Burgess SJ, Reyna-Llorens I, Smith-Unna R, Boursnell C, Hibberd JM (2017). Shared characteristics underpinning C4 leaf maturation derived from analysis of multiple C3 and C4 species of Flaveria. J Exp Bot. 68(2):177-189. doi: 10.1093/jxb/erw488.

Reyna-Llorens I, Hibberd JM (2017). Recruitment of pre-existing networks during the evolution of C4 photosynthesis. Philos Trans R Soc Lond B Biol Sci. 372(1730). pii: 20160386. doi: 10.1098/rstb.2016.0386.

Aubry S, Aresheva O, Reyna-Llorens I, Smith-Unna RD, Hibberd JM, Genty B (2016). A Specific Transcriptome Signature for Guard Cells from the C4 Plant Gynandropsis gynandra. Plant Physiol. 170(3):1345-57. doi: 10.1104/pp.15.01203.

Hibberd JM, Furbank RT (2016). In retrospect: Fifty years of C4 photosynthesis. Nature 538(7624):177-179. doi: 10.1038/538177b.

Hibberd JM, Furbank RT (2016). Wheat genomics: Seeds of C4 photosynthesis. Nat Plants. 2(11):16172. doi: 10.1038/nplants.2016.172.

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):454-65. doi: 10.1105/tpc.15.00570.

Boehm CR, Ueda M, Nishimura Y, Shikanai T, Haseloff J (2016). A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha. Plant Cell Physiol. 57(2):291-9. doi: 10.1093/pcp/pcv160