Engineering chloroplast development in rice through cell‐specific control of endogenous genetic circuits

Abstract

Summary The engineering of C4 photosynthetic activity into the C3 plant rice has the potential to nearly double rice yields. To engineer a two‐cell photosynthetic system in rice, the rice bundle sheath (BS) must be rewired to enhance photosynthetic capacity. Here, we show that BS chloroplast biogenesis is enhanced when the transcriptional activator, Oryza sativa Cytokinin GATA transcription factor 1 (OsCGA1), is driven by a vascular specific promoter. Ectopic expression of OsCGA1 resulted in increased BS chloroplast planar area and increased expression of photosynthesis‐associated nuclear genes (PhANG), required for the biogenesis of photosynthetically active chloroplasts in BS cells of rice. A further refinement using a DNAse dead Cas9 (dCas9) activation module driven by the same cell‐type specific promoter, directed enhanced chloroplast development of the BS cells when gRNA sequences were delivered by the dCas9 module to the promoter of the endogenous OsCGA1 gene. Single gRNA expression was sufficient to mediate the transactivation of both the endogenous gene and a transgenic GUS reporter fused with OsCGA1 promoter. Our results illustrate the potential for tissue‐specific dCas9‐activation and the co‐regulation of genes needed for multistep engineering of C4 rice.

Chloroplast development in green plant tissues: the interplay between light, hormone, and transcriptional regulation

“…Moreover, constitutive expression of ZmGLK genes in rice enhanced leaf chlorophyll levels and pigment‐antenna complexes leading to improved light harvesting efficiency, possibly due to improved repair and increased photosynthesis and vegetative biomass and grain yield (Li et al ., 2020). Similarly, overexpression of CGA1 in rice resulted in an increase in chloroplast numbers and chlorophyll levels thus providing another potential target for engineering crops with improved photosynthesis (Hudson et al ., 2013; Ermakova et al ., 2020; Lee et al ., 2021). It may well be that tuning these responses such that they take place at specific developmental stages or under particular environmental conditions will lead to further improvements in photosynthesis and yield.…”

CRISPR–Cas-mediated transcriptional control and epi-mutagenesis

“…The development of CRISPR–dCas systems for targeted transcriptional control and epi-mutagenesis is still in its infancy and there are many ways in which these tools can be improved (see Outstanding Questions). Their recent development means that these tools have only been utilized in a few studies outside of the ones creating or optimizing them; however, examples are available and demonstrate the usefulness of these systems in answering basic questions and bioengineering ( Lee et al, 2021 ; Leydon et al, 2021 ). The further development of these tools provides us with additional ways to target specific transcriptional or epigenetic manipulations in plants, allowing us to collect more direct evidence for the function of epigenetic marks and genes which can then be applied to the benefit of agriculture.…”

“…In plants, targeted manipulation of transcriptional control and epimutagenesis have already been used to increase resistance to drought, manipulate plant developmental phenotypes, increase our understanding of interactions between essential proteins, and further our understanding on how plants add, maintain and use DNA methylation ( Johnson et al, 2014 ; Harris et al, 2018 ; Gallego-Bartolomé et al, 2019 ; Papikian et al, 2019 ; Roca Paixão et al, 2019 ; Ichino et al, 2021 ; Lee et al, 2021 ; Leydon et al, 2021 ; Liu et al, 2021 ; Tang et al, 2021 ; Xue et al, 2021 ). As we continue to develop these tools by improving their efficiency and expanding the available effector domains, more possible applications in both research and industry arise.…”

HY5 regulates GLK transcription factors to orchestrate photomorphogenesis inArabidopsis thaliana

“…These phenotypes should be related to the accumulation of positive regulators of photomorphogenesis in darkness, but the specific regulators and their influence on the phenotypes are not clear. It has been reported that long-term ABA treatment activates the activity of COP1, which then interacts with GLK1 protein to mediate its ubiquitination and degradation (Tokumaru et al, 2017; Lee et al, 2021). Further, brassinolide or darkness induces GLK1 degradation by inhibiting the BIN2 phosphorylation of GLK1, and it was speculated that BIN2-mediated phosphorylation prevents the interaction of GLK1 and E3 ligases (Zhang et al, 2021).…”