Periodic Reporting for period 2 - DENOVO-P (De novo Development of Polarity in Plant Cells)
Période du rapport: 2020-09-01 au 2022-03-31
The polarity of the single cell from which an organism develops determines the polarity of the body axis in many species. Using Marchantia, we will discover how cell polarity develops de novo in the developing spore cell and how this, in turn, directs the specification of the first major axis in the plant. There are three high-level aims:
1. Describe the cellular and morphogenetic events that occur as the spore cell polarizes, divides asymmetrically to form cells at either end of the apical-basal axis.
2. Define the mechanism underpinning the de novo establishment of polarity using a combination of forward and reverse genetics.
3. Determine the role of auxin in transmitting the spore cell polarity that directs cell specification of at both ends of the apical-basal axis.
1. Describe the cellular and morphogenetic events that occur as the spore cell polarizes, divides asymmetrically to form cells at either end of the apical-basal axis.
2. Define the mechanism underpinning the de novo establishment of polarity using a combination of forward and reverse genetics.
3. Determine the role of auxin in transmitting the spore cell polarity that directs cell specification of at both ends of the apical-basal axis.
I: Descriptive model of the system: the timing of polarization during development of the Marchantia plant from a single spore
1. Generation of a 4-D model of early Marchantia development
This work is in progress and good progress has been made. We have a a timecourse for the development of the spore cell. We are incorporating cytology data from section I.2 into this model. We anticipate that this model will be completed at the end of the funding period and will incorporate all data generated during the course of the project.
2. When does polarization of the Marchantia spore occur and is it fixed or flexible
(i) We demonstrated that the spore is polarized by light and the developing sporeling responds to light during the first 30 hours of development. Blue light can polarize spores but no other wavelengths are active. This indicates that blue light signalling controls the development of polarity in Marchantia spores.
(ii) We demonstrated that polarity is labile until the first division occurs. This suggests that that polarity is labile throughout the one-cell stage of development.
(iii) We demonstrated that polarization of the sporeling cell is accompanied by the polarization of the microtubule cytoskeleton. Early in development cortical microtubules are dispersed throughout the cortical cytoplasm. As polarization occurs, the cortical microtubules disappear from the northern hemisphere and become restricted to the southern hemisphere. We hypothesize that this is the result of the disappearance of the polar microtubule organizer from the northern side of the nucleus and the maintenance of the microtubule organizer on the southern surface of the nucleus. By contrast, microfilaments, are not polarized at any stage during polarization of the spore cell. Similarly, the Marchantia Cdc42 homolog, MpROP is not polarized during polarization. These data demonstrate that microtubules are involved in the development of polarity and neither microfilaments nor ROP proteins are involved.
II: De novo polarity: defining the mechanism polarizing the Marchantia spore cell de novo
1.Using forward genetics to define the mechanism controlling ght epolarity of the Marchantia spore cell
We must generate isogenic lines to carry out this mutagenesis analysis. We have been generating an isogenic line in which to carry out this mutant screen over the past two years. This requires eight generations of crossing and we have completed six generations. We anticipate that the isogenic populations will be ready in the next 6-12 months – after two more generations of crossing and generation of a sequenced genome for the isogenic line. This project is on schedule.
2. Identifying genes that are expressed during polarization with previously defined polarization functions.
We carried out a transcriptomic analysis of early spore development. We used this to identify blue light receptors that are expressed during the polarization, because we discovered in section I.2(ii) that blue light is the external polarization signal. We discovered that MpCRYPTOCHROME and MpPHOTOTROPIN genes are expressed at this stage of development. We generated mutants that were defective in MpCRYPTOCHROME and MpPHOTOTROPIN gene function. Mpphototropin mutants cannont polarize in either white light or blue light, demonstrating that MpPHOTOTROPIN is required for polarization. Mpcryptochrome mutants polarized like wild type. This demonstrates that MpCRYPTOCHROME is not involved in spore cell polarisation.
III: Inherited polarity: testing the hypothesis that auxin is involved in the polar specification of apical and basal cells
1.Is auxin involved in the development of the asymmetric 2-cell Marchantia plant?
2. Identification and characterization of genes involved in auxin synthesis, transport and signalling during the development of the apical and basal cells
To carry out this part of the proposed research programme, we must first develop FACs protocols to isolate nuclei from apical and basal cells. We are developing this protocol. To date, we have developed a protocol to isolate nuclei. We are refining this protocol to allow us to separate apical from basal cell. We anticipate that we will be able to isolate nuclei and sort nuclei from apical cells and basal cells by the end of 2021.
Dissemination
These data are presented at international symposia and invited seminars. The first result will likely be submitted for publication in 2022.
1. Generation of a 4-D model of early Marchantia development
This work is in progress and good progress has been made. We have a a timecourse for the development of the spore cell. We are incorporating cytology data from section I.2 into this model. We anticipate that this model will be completed at the end of the funding period and will incorporate all data generated during the course of the project.
2. When does polarization of the Marchantia spore occur and is it fixed or flexible
(i) We demonstrated that the spore is polarized by light and the developing sporeling responds to light during the first 30 hours of development. Blue light can polarize spores but no other wavelengths are active. This indicates that blue light signalling controls the development of polarity in Marchantia spores.
(ii) We demonstrated that polarity is labile until the first division occurs. This suggests that that polarity is labile throughout the one-cell stage of development.
(iii) We demonstrated that polarization of the sporeling cell is accompanied by the polarization of the microtubule cytoskeleton. Early in development cortical microtubules are dispersed throughout the cortical cytoplasm. As polarization occurs, the cortical microtubules disappear from the northern hemisphere and become restricted to the southern hemisphere. We hypothesize that this is the result of the disappearance of the polar microtubule organizer from the northern side of the nucleus and the maintenance of the microtubule organizer on the southern surface of the nucleus. By contrast, microfilaments, are not polarized at any stage during polarization of the spore cell. Similarly, the Marchantia Cdc42 homolog, MpROP is not polarized during polarization. These data demonstrate that microtubules are involved in the development of polarity and neither microfilaments nor ROP proteins are involved.
II: De novo polarity: defining the mechanism polarizing the Marchantia spore cell de novo
1.Using forward genetics to define the mechanism controlling ght epolarity of the Marchantia spore cell
We must generate isogenic lines to carry out this mutagenesis analysis. We have been generating an isogenic line in which to carry out this mutant screen over the past two years. This requires eight generations of crossing and we have completed six generations. We anticipate that the isogenic populations will be ready in the next 6-12 months – after two more generations of crossing and generation of a sequenced genome for the isogenic line. This project is on schedule.
2. Identifying genes that are expressed during polarization with previously defined polarization functions.
We carried out a transcriptomic analysis of early spore development. We used this to identify blue light receptors that are expressed during the polarization, because we discovered in section I.2(ii) that blue light is the external polarization signal. We discovered that MpCRYPTOCHROME and MpPHOTOTROPIN genes are expressed at this stage of development. We generated mutants that were defective in MpCRYPTOCHROME and MpPHOTOTROPIN gene function. Mpphototropin mutants cannont polarize in either white light or blue light, demonstrating that MpPHOTOTROPIN is required for polarization. Mpcryptochrome mutants polarized like wild type. This demonstrates that MpCRYPTOCHROME is not involved in spore cell polarisation.
III: Inherited polarity: testing the hypothesis that auxin is involved in the polar specification of apical and basal cells
1.Is auxin involved in the development of the asymmetric 2-cell Marchantia plant?
2. Identification and characterization of genes involved in auxin synthesis, transport and signalling during the development of the apical and basal cells
To carry out this part of the proposed research programme, we must first develop FACs protocols to isolate nuclei from apical and basal cells. We are developing this protocol. To date, we have developed a protocol to isolate nuclei. We are refining this protocol to allow us to separate apical from basal cell. We anticipate that we will be able to isolate nuclei and sort nuclei from apical cells and basal cells by the end of 2021.
Dissemination
These data are presented at international symposia and invited seminars. The first result will likely be submitted for publication in 2022.
Our research thus far has demonstrated how an external signal controls the polarity of a cell from which a mature plant body develops. We have also identified a gene that is required for this environmental signal to polarise this cell. This has never been achieved for developing plants and represents a conceptual breakthrough.
We predict that during the remaining course of research, the details of this mechanism will be worked out. We will also be able to hypothesise on how this mechanism originated during the course of plant evolution.
We predict that during the remaining course of research, the details of this mechanism will be worked out. We will also be able to hypothesise on how this mechanism originated during the course of plant evolution.