Periodic Reporting for period 2 - BSLchlamy (Understanding the essential function of the conserved plant-specific protein phosphatase family BSL)
Reporting period: 2020-12-11 to 2021-12-10
What is the problem/issue being addressed?
Accurate control of when and where cells divide is critical to development and tissue patterning in multicellular organisms. In plants, cell division research has focused primarily on universally conserved eukaryotic genes, which have previously been studied yeast and animals. Many plant-specific factors that influence cell division likely remain to be uncovered. Here, we focus on one plant-specific gene, the BSL1 phosphatase, that is critical for cell division in the green alga Chlamydomonas, a simple unicellular representative of the plant kingdom. We extend the results to the role of BSL1 homologs on cell division in Arabidopsis, a reference dicot plant. Our results generate new insight into regulation of plant cell division.
Why is it important for society?
(1) Chlamydomonas is a model system for plant cell biology that can inform studies in other plants, including crops, which are themselves less amenable to experimentation. As one example, mutations in a rice BSL1 phosphatase homolog is responsible for increased grain size. Our study will help uncover the function of BSL1 proteins in plants, which may guide rational breeding strategies toward improved yield.
(2) Microalgae, including Chlamydomonas, are emerging as potential cost-effective production platforms for value-added compounds. Numerous bioactive molecules, most recently the receptor-binding domain of the SARS-CoV-2 virus, have been produced in Chlamydomonas. Moreover, a recent toxicological study concluded that consumption of Chlamydomonas present no health concerns (GRAS designation by the US FDA), making it possible to use its biomass either directly for human nutrition, or for the development of “edible” vaccines and other orally administered therapeutics. In recent years there has been rapid developments of diverse synthetic biology tools, enabling targeted genetic engineering of both the nuclear and chloroplast genomes. However, yield is still limited due, in part, to a lack of understanding of factors that restrict cell growth and division under different culturing conditions. The results from the project may help define biological constraints on microalgal biomass production, an important step towards realization of the biotechnological potential of this diverse group of organisms.
Conclusions of the Action
(i) In Chlamydomonas, the BSL1 phosphatase activity is required for timing of basal body segregation and proper orientation of the mitotic spindle.
(ii) In Arabidopsis, BSL family protein activity is required for maintenance of the root meristem.
(iii) A novel biotin ligase (BioID) variant, in combination with global phosphoproteomic profiling has been used to identify candidate BSL1 substrates.
Accurate control of when and where cells divide is critical to development and tissue patterning in multicellular organisms. In plants, cell division research has focused primarily on universally conserved eukaryotic genes, which have previously been studied yeast and animals. Many plant-specific factors that influence cell division likely remain to be uncovered. Here, we focus on one plant-specific gene, the BSL1 phosphatase, that is critical for cell division in the green alga Chlamydomonas, a simple unicellular representative of the plant kingdom. We extend the results to the role of BSL1 homologs on cell division in Arabidopsis, a reference dicot plant. Our results generate new insight into regulation of plant cell division.
Why is it important for society?
(1) Chlamydomonas is a model system for plant cell biology that can inform studies in other plants, including crops, which are themselves less amenable to experimentation. As one example, mutations in a rice BSL1 phosphatase homolog is responsible for increased grain size. Our study will help uncover the function of BSL1 proteins in plants, which may guide rational breeding strategies toward improved yield.
(2) Microalgae, including Chlamydomonas, are emerging as potential cost-effective production platforms for value-added compounds. Numerous bioactive molecules, most recently the receptor-binding domain of the SARS-CoV-2 virus, have been produced in Chlamydomonas. Moreover, a recent toxicological study concluded that consumption of Chlamydomonas present no health concerns (GRAS designation by the US FDA), making it possible to use its biomass either directly for human nutrition, or for the development of “edible” vaccines and other orally administered therapeutics. In recent years there has been rapid developments of diverse synthetic biology tools, enabling targeted genetic engineering of both the nuclear and chloroplast genomes. However, yield is still limited due, in part, to a lack of understanding of factors that restrict cell growth and division under different culturing conditions. The results from the project may help define biological constraints on microalgal biomass production, an important step towards realization of the biotechnological potential of this diverse group of organisms.
Conclusions of the Action
(i) In Chlamydomonas, the BSL1 phosphatase activity is required for timing of basal body segregation and proper orientation of the mitotic spindle.
(ii) In Arabidopsis, BSL family protein activity is required for maintenance of the root meristem.
(iii) A novel biotin ligase (BioID) variant, in combination with global phosphoproteomic profiling has been used to identify candidate BSL1 substrates.
We have performed time-lapse imaging of wild-type and bsl1 loss-of-function mutant Chlamydomonas cells. These results show that BSL1 phosphatase activity is required for timing of mitotic events and proper alignment of the mitotic spindle. We have also localized the BSL protein in dividing cells by time-lapse microscopy, and shown strong localization to the basal bodies (BB) from early mitosis to metaphase. We conclude that dephosphorylation of one or more substrates, probably near the BBs, is required for faithful progression through mitosis in Chlamydomonas.
We charted the proximity proteome of BSL1 using a novel version of the biotin ligase BioID. To identify potential substrates of BSL1, we performed a global phosphoproteomic screen in wild type and bsl mutant cells. A dual-specific kinase was identified by proximity labeling, and was hyperphosphorylated in bsl1-1 mutant cells, suggesting that it may be a direct BSL1 substrate. The interaction between BSL1-kinase interaction was verified by immunoprecipitation using a FLAG-tagged version of the kinase. The kinase gene was deleted by CRISPR-Cas9; dyrkp mutant cells completed a normal mitosis, but displayed a defect in hatching, the final step in the cell cycle. These results imply that the dual-specific kinase is not a key mitotic substrate of BSL1, and suggests additional roles for BSL1 beyond mitosis.
We tested the hypothesis that BSL-family proteins are involved in cell division in land plants by analyzing the effects of conditional BSL knock-down in Arabidopsis. The results showed that BSL is required for cell proliferation in the root meristem. Independent evidence supporting this result was obtained with a hypomorphic bsl2 mutant allele. Time-lapse imaging showed that BSL-family proteins localize specifically to the cell plate during phragmoplast formation and expansion. By combining BSL knock-down with genetic and pharmacological manipulation of the brassinosteroid (BR) signaling pathway, we demonstrated that the meristem maintenance function of BSL is unlikely to result primarily from downregulation of BR signaling.
In summary, our results demonstrate specific roles for BSL1 in mitosis in Chlamydomonas, and suggest a function in cell cycle regulation and cytokinesis in land plants. These results from the Action will be disseminated through two publications (in addition to an already published methods paper on Chlamydomonas mating). The first paper will focus on the phenotypic characterization of BSL-family proteins in Chlamydomonas and Arabidopsis. The second paper will cover the biochemical characterization of BSL1 substrates in Chlamydomonas by proximity labeling and phosphoproteomics. Both papers will be published as open access.
We charted the proximity proteome of BSL1 using a novel version of the biotin ligase BioID. To identify potential substrates of BSL1, we performed a global phosphoproteomic screen in wild type and bsl mutant cells. A dual-specific kinase was identified by proximity labeling, and was hyperphosphorylated in bsl1-1 mutant cells, suggesting that it may be a direct BSL1 substrate. The interaction between BSL1-kinase interaction was verified by immunoprecipitation using a FLAG-tagged version of the kinase. The kinase gene was deleted by CRISPR-Cas9; dyrkp mutant cells completed a normal mitosis, but displayed a defect in hatching, the final step in the cell cycle. These results imply that the dual-specific kinase is not a key mitotic substrate of BSL1, and suggests additional roles for BSL1 beyond mitosis.
We tested the hypothesis that BSL-family proteins are involved in cell division in land plants by analyzing the effects of conditional BSL knock-down in Arabidopsis. The results showed that BSL is required for cell proliferation in the root meristem. Independent evidence supporting this result was obtained with a hypomorphic bsl2 mutant allele. Time-lapse imaging showed that BSL-family proteins localize specifically to the cell plate during phragmoplast formation and expansion. By combining BSL knock-down with genetic and pharmacological manipulation of the brassinosteroid (BR) signaling pathway, we demonstrated that the meristem maintenance function of BSL is unlikely to result primarily from downregulation of BR signaling.
In summary, our results demonstrate specific roles for BSL1 in mitosis in Chlamydomonas, and suggest a function in cell cycle regulation and cytokinesis in land plants. These results from the Action will be disseminated through two publications (in addition to an already published methods paper on Chlamydomonas mating). The first paper will focus on the phenotypic characterization of BSL-family proteins in Chlamydomonas and Arabidopsis. The second paper will cover the biochemical characterization of BSL1 substrates in Chlamydomonas by proximity labeling and phosphoproteomics. Both papers will be published as open access.
Progress beyond the state of the art
1. Successful application of novel biotin ligase (BioID) in Chlamydomonas. Biotin ligase-based proximity labeling has emerged as a promising technology for proteome and protein-protein interaction studies in many organisms but has not been successfully implemented in Chlamydomonas before. We predict that this technology will have broad impact on many areas of Chlamydomonas research.
2. Our study expands the scope of physiological functions of BSL family proteins to root meristem maintenance, and reports describes for the first time the cell plate-specific localization of BSL1 proteins.
Potential impact
Proper regulation of root development is critical to plant growth and productivity. Since the BSL family is highly conserved throughout the plant kingdom, our finding that BSL activity contributes to maintenance of the root meristem may have implications for breeding or rational engineering of root traits in crop species.
1. Successful application of novel biotin ligase (BioID) in Chlamydomonas. Biotin ligase-based proximity labeling has emerged as a promising technology for proteome and protein-protein interaction studies in many organisms but has not been successfully implemented in Chlamydomonas before. We predict that this technology will have broad impact on many areas of Chlamydomonas research.
2. Our study expands the scope of physiological functions of BSL family proteins to root meristem maintenance, and reports describes for the first time the cell plate-specific localization of BSL1 proteins.
Potential impact
Proper regulation of root development is critical to plant growth and productivity. Since the BSL family is highly conserved throughout the plant kingdom, our finding that BSL activity contributes to maintenance of the root meristem may have implications for breeding or rational engineering of root traits in crop species.