Delphine Charif

Motivation: Resource allocation modeling—as the Resource Balance Analysis (RBA) framework—provides a way to understand and predict how limited cellular resources (e.g. energy, proteins, etc.) in cells are distributed among competing cell processes within a limited cellular space. Currently, resource allocation modeling for eukaryotes remains limited due to the lack of software capable of generating calibrated RBA models for these types of cells, unlike prokaryotes, which benefit from the software tools such as RBApy, RBAtools, and the RBAml format for model encoding. Results: Here, we extended the RBA toolkit (RBApy, RBAtools, and RBAml) to account for specific aspects of eukaryotic cells growing in complex environments such as varying temperature, light, or nutritional conditions. We used them to generate and simulate RBA models of both prokaryotic (Escherichia coli) and eukaryotic (Arabidopsis thaliana) cells for varying temperatures. The resulting models show excellent prediction capabilities when benchmarked against published experimental datasets. The upgraded RBA toolkit will pave the way to creating, calibrating, and running resource allocation models for crops, livestock, or humans for a wide range of medical, biotechnological, or agricultural applications in the future. Availability and implementation: RBApy and RBAtools are available via PyPI and at https://github.com/RBAgroup.

Cytoplasmic male sterility (CMS) is of major agronomical relevance in hybrid breeding. In gametophytic CMS, abortion of pollen is determined by the grain genotype, while in sporophytic CMS, it is determined by the mother plant genotype. While several CMS mechanisms have been dissected at the molecular level, gametophytic CMS has not been straightforwardly accessible. We used the gametophytic Sha-CMS in Arabidopsis to characterize the cause and process of pollen abortion by implementing in vivo biosensing in single pollen and mitoTALEN mutagenesis. We obtained conclusive evidence that orf117Sha is the CMS-causing gene, despite distinct characteristics from other CMS-genes. We measured the in vivo cytosolic ATP content in single pollen, followed pollen development and analyzed pollen mitochondrial volume in two genotypes that differed only by the presence of the orf117Sha locus. Our results show that the Sha-CMS is not triggered by ATP deficiency. Instead, we observed desynchronization of a pollen developmental program. Pollen death occurred independently in pollen grains at diverse stages and was preceded by mitochondrial swelling. We conclude that pollen death is grain-autonomous in Sha-CMS and propose that mitochondrial permeability transition, which was previously described as a hallmark of developmental and environmental-triggered cell death programs, precedes pollen death in Sha-CMS.

Cytoplasmic male sterility (CMS) is of major agronomical relevance in hybrid breeding. In gametophytic CMS, abortion of pollen is determined by the grain genotype, while in sporophytic CMS, it is determined by the mother plant genotype. While several CMS mechanisms have been dissected at the molecular level, gametophytic CMS has not been straightforwardly accessible. We used the gametophytic Sha-CMS in Arabidopsis to characterize the cause and process of pollen abortion by implementing in vivo biosensing in single pollen and mitoTALEN mutagenesis. We obtained conclusive evidence that orf117Sha is the CMS-causing gene, despite distinct characteristics from other CMS-genes. We measured the in vivo cytosolic ATP content in single pollen, followed pollen development and analyzed pollen mitochondrial volume in two genotypes that differed only by the presence of the orf117Sha locus. Our results show that the Sha-CMS is not triggered by ATP deficiency. Instead, we observed desynchronization of a pollen developmental program. Pollen death occurred independently in pollen grains at diverse stages and was preceded by mitochondrial swelling. We conclude that pollen death is grain-autonomous in Sha-CMS and propose that mitochondrial permeability transition, which was previously described as a hallmark of developmental and environmental-triggered cell death programs, precedes pollen death in Sha-CMS. Highlight: The Arabidopsis CMS-causing gene orf117Sha does not limit pollen ATP supply. Pollen-centered approaches show desynchronization of development and mitochondrial swelling before pollen death, which occurred at diverse stages.

CRISPR and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR–Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted, inPhyscomitrium patens, a comprehensive genome-wide investigation of off-target mutations using either a CRISPR–Cas9 or a TALEN strategy. We observed a similar number of differences for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and did not lead to transgene integration. The PEG treatment, a well-established biotechnological method, in itself, was the main source of mutations found in the edited plants.