Periodic Reporting for period 4 - Revolution (Regulation and Evolution of C4 photosynthesis)
Période du rapport: 2021-03-01 au 2022-08-31
What is the problem/issue being addressed:
Improvements in photosynthetic efficiency during land plant evolution are associated with increased cellular specialisation within the leaf. However, mechanisms responsible for variations in gene expression between specific cell-types in leaves are not understood. Although leaves are responsible for the majority of photosynthesis on terrestrial Earth, our understanding of how they operate is very poor.
Why is it important for society:
Photosynthesis is the basis of life on earth. It also provides our food and fuel. However, photosynthesis as used by crops is not efficient, and it has now been demonstrated that it can be engineered to be more efficient. The aim of this work is to better understand how the most efficient photosynthetic pathway used by crops evolved. In so doing we aim to uncover principles allowing it to be engineered to allow increased productivity.
What are the overall objectives:
This proposal aims to understand how genes encoding components of the photosynthetic apparatus are expressed in specific cell-types of C4 plants. All other labs focus on C4 species to understand this, but I consider a better route is to first discover how gene expression is regulated in specific cells of ancestral C3 leaves. This logic is based on the fact that C4 plants have arisen over sixty times and so it seems likely that evolution made use of existing regulatory processes rather than developed new mechanisms. Therefore, in C3 leaves I wish to understand how some cell-types maintain high levels of photosynthesis gene expression whilst others remain photosynthetically repressed. In C4 leaves I wish to discover the extent to which cell-specific expression is based on pre-existing regulatory networks in the C3 leaf. The overarching hypothesis is that cell-specific gene expression in C4 leaves is mediated by pre-existing regulatory networks found in C3 species.
Conclusions of the action:
Using the proposed approach, we discovered the first transcription factors to pattern C4 gene expression to bundle sheath cells. Thus, our high-risk approach was validated. In the process, we pioneered the use of DNaseIseq to define transcription factor binding in leaves, we discovered that duons regulate gene expression in leaves, identified a number of other regulatory DNA sequences that are necessary for expression of C4 genes, and in the process of conducting this work discovered significant natural variation in C4 traits between accessions of a species.
Improvements in photosynthetic efficiency during land plant evolution are associated with increased cellular specialisation within the leaf. However, mechanisms responsible for variations in gene expression between specific cell-types in leaves are not understood. Although leaves are responsible for the majority of photosynthesis on terrestrial Earth, our understanding of how they operate is very poor.
Why is it important for society:
Photosynthesis is the basis of life on earth. It also provides our food and fuel. However, photosynthesis as used by crops is not efficient, and it has now been demonstrated that it can be engineered to be more efficient. The aim of this work is to better understand how the most efficient photosynthetic pathway used by crops evolved. In so doing we aim to uncover principles allowing it to be engineered to allow increased productivity.
What are the overall objectives:
This proposal aims to understand how genes encoding components of the photosynthetic apparatus are expressed in specific cell-types of C4 plants. All other labs focus on C4 species to understand this, but I consider a better route is to first discover how gene expression is regulated in specific cells of ancestral C3 leaves. This logic is based on the fact that C4 plants have arisen over sixty times and so it seems likely that evolution made use of existing regulatory processes rather than developed new mechanisms. Therefore, in C3 leaves I wish to understand how some cell-types maintain high levels of photosynthesis gene expression whilst others remain photosynthetically repressed. In C4 leaves I wish to discover the extent to which cell-specific expression is based on pre-existing regulatory networks in the C3 leaf. The overarching hypothesis is that cell-specific gene expression in C4 leaves is mediated by pre-existing regulatory networks found in C3 species.
Conclusions of the action:
Using the proposed approach, we discovered the first transcription factors to pattern C4 gene expression to bundle sheath cells. Thus, our high-risk approach was validated. In the process, we pioneered the use of DNaseIseq to define transcription factor binding in leaves, we discovered that duons regulate gene expression in leaves, identified a number of other regulatory DNA sequences that are necessary for expression of C4 genes, and in the process of conducting this work discovered significant natural variation in C4 traits between accessions of a species.
First, within Workpackage 1, analyses of DNaseI and RNA sequencing during de-etiolation of C3 Arabidopsis thaliana were combined with functional analysis of candidate regulators in planta. By combining this approach with in silico analysis, we identified the first transcription factor network (MYC and MYB transcription factors) to control expression in bundle sheath cells. We showed both partners are necessary and sufficient for expression in this cell type. Using A. thaliana, we also used this approach to define additional elements in cis, positioned in various gene features necessary for expression of other genes in bundle sheath cells. Moreover, we realised that incorporation of data from the distantly related C3 species Oryza sativa (rice) allowed us to identify genes likely to be expressed in bundle sheath cells of all plants derived from their last common ancestor. So, we developed a conserved blueprint associated with this cell type.
Second, in Workpackage 2, analogous sequencing analysis of C4 Gynandropsis gynandra has also been combined with functional analysis to test candidate regulators in cis and trans. This allowed us identify duons within C4 genes, and to provide functional evidence for a critical role of these duons in patterning gene expression to the bundle sheath. We also identified elements in promoter regions and untranslated regions of C4 genes that restrict expression to bundle sheath cells. In collaboration with international partners we developed a chromosome level genome assembly for G. gynandra, comprehensive data for transcripts and therefore gene models, as well as genome-wide data on transcription factor binding in vivo. Much of this was instrumental in our findings from Workpackage 3.
Third, in Workpackage 3, which aimed to combine insights from the first two Workpackages we made progress in two main areas. First, this analysis showed that the amplitude in the response of C4 genes to light increases in C4 compared with C3 leaves. Thus, C4 genes are already part of photosynthesis gene regulatory networks in the ancestral C3 state, but their response to these networks is amplified in the C4 state. We used functional testing to show that this increased responsiveness is associated with cis-elements known to be bound by transcription factors that respond to light. Different C4 genes have acquired distinct cis-elements, but they are all bound by light responsive transcription factors. Second, we have now identified multiple examples of components of gene regulatory networks that operate in the ancestral C3 state, and have been co-opted by additional genes in the C4 state to restrict their expression to specific cells. The MYC-MYB module that patterns gene expression to bundle sheath cells of A. thaliana (WP1) is also found in C4 genes of G. gynandra. Moreover, we also identified the sequences bound by these transcription factors in the GLDP gene from C3-C4 intermediate species. A switch from ubiquitous GLDP gene expression in leaves of C3 plants to bundle sheath expression is considered one of the early events in the evolution of photosynthesis, but the molecular events underpinning this event were not clear. We were able to therefore provide the first mechanistic insight into how this key evolutionary event took place. In addition, we have also discovered a short sequence of DNA and its cognate transcription factor family, which causes a domain switch from vascular cells of C3 A. thaliana to mesophyll cells of C4 leaves. This is the first example of a molecular mechanism underpinning a domain shift during the evolution of C4 photosynthesis.
Second, in Workpackage 2, analogous sequencing analysis of C4 Gynandropsis gynandra has also been combined with functional analysis to test candidate regulators in cis and trans. This allowed us identify duons within C4 genes, and to provide functional evidence for a critical role of these duons in patterning gene expression to the bundle sheath. We also identified elements in promoter regions and untranslated regions of C4 genes that restrict expression to bundle sheath cells. In collaboration with international partners we developed a chromosome level genome assembly for G. gynandra, comprehensive data for transcripts and therefore gene models, as well as genome-wide data on transcription factor binding in vivo. Much of this was instrumental in our findings from Workpackage 3.
Third, in Workpackage 3, which aimed to combine insights from the first two Workpackages we made progress in two main areas. First, this analysis showed that the amplitude in the response of C4 genes to light increases in C4 compared with C3 leaves. Thus, C4 genes are already part of photosynthesis gene regulatory networks in the ancestral C3 state, but their response to these networks is amplified in the C4 state. We used functional testing to show that this increased responsiveness is associated with cis-elements known to be bound by transcription factors that respond to light. Different C4 genes have acquired distinct cis-elements, but they are all bound by light responsive transcription factors. Second, we have now identified multiple examples of components of gene regulatory networks that operate in the ancestral C3 state, and have been co-opted by additional genes in the C4 state to restrict their expression to specific cells. The MYC-MYB module that patterns gene expression to bundle sheath cells of A. thaliana (WP1) is also found in C4 genes of G. gynandra. Moreover, we also identified the sequences bound by these transcription factors in the GLDP gene from C3-C4 intermediate species. A switch from ubiquitous GLDP gene expression in leaves of C3 plants to bundle sheath expression is considered one of the early events in the evolution of photosynthesis, but the molecular events underpinning this event were not clear. We were able to therefore provide the first mechanistic insight into how this key evolutionary event took place. In addition, we have also discovered a short sequence of DNA and its cognate transcription factor family, which causes a domain switch from vascular cells of C3 A. thaliana to mesophyll cells of C4 leaves. This is the first example of a molecular mechanism underpinning a domain shift during the evolution of C4 photosynthesis.
The main progress beyond the state-of-the-art was identification of small and discrete DNA regulators of C4 gene expression combined with cognate transcription factors that bind them. Previous to our work no transcription factors regulating the spatial patterning of C4 genes had been reported. From this key finding, we have also been able to generate a molecular model that predicts how the patterns of gene expression seen in so called C2 species (intermediate between C3 and C4 species) came about. This provides the first mechanistic insight into events thought to have facilitated early events in the evolution of the complex C4 phenotype. Moreover, we have been able to build gene regulatory networks for separate cell types of both C3 Arabidopsis thaliana and C4 Gynandropsis gynandra. In addition, we also realised during the action that there was significant natural variation in C4 traits in Gynandropsis gynandra, and so built a population such that in the future we can use forward genetics to dissect regulators of the C4 pathway. We also serendipitously discovered that monocotyledons could be grafted and the ability to generate somatic hybrids between C3 and C4 plants is also completely novel and has not been achieved before.