Abstract:In biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue-types and/or at specific times often precludes co-expression of multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator-like effectors (dTALEs) and cognate synthetic TALE-ac… Show more
“…The coding sequence for mTurquoise2 was obtained from Luginbuehl et al, 2020. The promoter sequence from Zoysia japonica PHOSPHOENOLPYRUVATE CARBOXYKINASE in combination with the dTALE STAP4 system was obtained from Danila et al, 2022. The coding sequence of Arabidopsis thaliana H2B (At5g22880) was used as an N-terminal signal for targeting mTurquoise2 to the nucleus.…”
In multicellular systems changes to the patterning of gene expression drive modifications in cell function and trait evolution. One striking example is found in more than sixty plant lineages where compartmentation of photosynthesis between cell types allowed evolution of the efficient C4 pathway from the ancestral C3 state. The molecular events enabling this transition are unclear. We used single nuclei sequencing to generate a cell level expression atlas for C3 rice and C4 sorghum during photomorphogenesis. In both species a conserved cistrome was identified for each cell type and initiation of photosynthesis gene expression was conditioned by cell identity. Photosynthesis genes switching expression from mesophyll in rice to bundle sheath in sorghum acquire hallmarks of bundle sheath identity. The sorghum bundle sheath has also acquired gene networks associated with C3 guard cells. We conclude C4 photosynthesis is based on rewiring in cis that exapts cell identity networks of C3 plants.
“…The coding sequence for mTurquoise2 was obtained from Luginbuehl et al, 2020. The promoter sequence from Zoysia japonica PHOSPHOENOLPYRUVATE CARBOXYKINASE in combination with the dTALE STAP4 system was obtained from Danila et al, 2022. The coding sequence of Arabidopsis thaliana H2B (At5g22880) was used as an N-terminal signal for targeting mTurquoise2 to the nucleus.…”
In multicellular systems changes to the patterning of gene expression drive modifications in cell function and trait evolution. One striking example is found in more than sixty plant lineages where compartmentation of photosynthesis between cell types allowed evolution of the efficient C4 pathway from the ancestral C3 state. The molecular events enabling this transition are unclear. We used single nuclei sequencing to generate a cell level expression atlas for C3 rice and C4 sorghum during photomorphogenesis. In both species a conserved cistrome was identified for each cell type and initiation of photosynthesis gene expression was conditioned by cell identity. Photosynthesis genes switching expression from mesophyll in rice to bundle sheath in sorghum acquire hallmarks of bundle sheath identity. The sorghum bundle sheath has also acquired gene networks associated with C3 guard cells. We conclude C4 photosynthesis is based on rewiring in cis that exapts cell identity networks of C3 plants.
“…In addition, DNA assembly systems (e.g. Loop assembly; Pollak et al., 2019) with application across broad phylogenies (Pollak et al., 2020), and tuneable expression of multigene constructs (Danila et al., 2022), opens up opportunities to test expression constructs in higher throughput photosynthetic systems such as Chlamydomonas reinhardtii ( Chlamydomonas ; Kafri et al., 2023) before application to crop plants.…”
Section: Ccm Components and Their Requirements For Synthetic Reconstr...mentioning
SUMMARYThe introduction of the carboxysome‐based CO2 concentrating mechanism (CCM) into crop plants has been modelled to significantly increase crop yields. This projection serves as motivation for pursuing this strategy to contribute to global food security. The successful implementation of this engineering challenge is reliant upon the transfer of a microcompartment that encapsulates cyanobacterial Rubisco, known as the carboxysome, alongside active bicarbonate transporters. To date, significant progress has been achieved with respect to understanding various aspects of the cyanobacterial CCM, and more recently, different components of the carboxysome have been successfully introduced into plant chloroplasts. In this Perspective piece, we summarise recent findings and offer new research avenues that will accelerate research in this field to ultimately and successfully introduce the carboxysome into crop plants for increased crop yields.
“…We recently demonstrated that the synthetic transcription activator-like effector (dTALE)/synthetic TALE-activated promoter (STAP) system (Brückner et al 2015 ; Danila et al 2022 ) provides a potential solution to the problems outlined above (Fig. 5 ).…”
“…5 ). An added advantage of this system is that it has been observed that the dTALE-STAP system can substantially amplify the strength of a weak promoter (Danila et al 2022 ), potentially also addressing the problem of sufficient high-level expression in the correct cell type in the C 4 rice prototype. Fig.…”
“… Fig. 5 Cell specific multiplexing strategy using synthetic promoters and transcriptional activators (Danila et al 2022 ). Two separate dTALE transitional activators are used, each capable of activating as set of STAP synthetic promoters.…”
Traditional “Green Revolution” cereal breeding strategies to improve yield are now reaching a plateau in our principal global food crop rice. Photosynthesis has now become a major target of international consortia to increase yield potential. Synthetic biology is being used across multiple large projects to improve photosynthetic efficiency. This review follows the genesis and progress of one of the first of these consortia projects, now in its 13th year; the Bill and Melinda Gates funded C4 Rice Project. This project seeks to install the biochemical and anatomical attributes necessary to support C4 photosynthesis in the C3 crop rice. Here we address the advances made thus far in installing the biochemical pathway and some of the key targets yet to be reached.
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