Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - Revolution (Regulation and Evolution of C4 photosynthesis)

Teaser

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, the mechanisms responsible for variations in gene expression between specific...

Summary

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, the 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 are 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 in crops. 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 that allow it to be engineered into less efficient crops 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 has made use of existing regulatory processes rather than developed new mechanisms each time. 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.

Work performed

To date, progress has been made in three main areas. One additional area of relevance to the overarching aims of the project has been identified and work in this has been integrated into the overall programme.

First, within Workpackage 1, analyses of DNaseI sequencing and RNA sequencing during de-etiolation of C3 Arabidopsis thaliana have been combined with functional analysis of candidate regulators in planta. In particular, upstream of a transcription factor that is preferentially expressed in the A. thaliana bundle sheath, we have identified an eight base pair motif that is necessary and sufficient for this patterning. This sequence is therefore part of an ancestral mechanism that controls gene expression in this cell type in this C3 species. Our focus now is on the identification of trans-factors that bind this region and that are therefore responsible for restricting gene expression to this cell type. Through analysis of a second transcription factor that is also strongly expressed in the A. thaliana bundle sheath, we identified two additional regions of sequence that are necessary for this gene expression. Our next focus is on identifying the regions that are also sufficient for this behaviour.

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 in trans. In this case, our current focus is on two motifs that appear to be acting co-operatively to repress gene expression in mesophyll cells and in so doing restrict it to the C4 bundle sheath. Although the sequence is present in the ancestral C3 state, it appears not to be active in repressing expression in the mesophyll implying a change in trans has occurred during the evolution of C4 photosynthesis. Our current focus here is on identifying the protein partners that bind these sequences.

Third, in Workpackage 3, which aimed to combine insights from the data collected in the first two Workpackages, we have made progress in two main areas. First, we have identified a number of DNA sequences that we consider strong candidates for enhancing the expression of genes of the C4 pathway in C4 compared with C3 plants. We are continuing our functional analysis to identify these motifs and show they are both necessary and sufficient for this key phenotype associated with the evolution of C4 photosynthesis. Second, we have a clear hypothesis that lays out how the gene regulatory networks have altered as C4 photosynthesis evolved from the ancestral C3 state. This hypothesis is being tested through analysis of existing mutant alleles and/or CRISPR/Cas9 in key components of the network in the ancestral C3 state.

Lastly, alongside this work, we have developed a system by which C3 and C4 plants can routinely be hybridised for the first time. This work should therefore allow analysis of how genomes from each type of plant interact and regulate each other – ie what regulatory mechanisms are dominant and which are recessive. This offers an exciting new technology to study how the regulation of gene expression has evolved.

Final results

The main progress beyond the state of the art relates to our identification of small and discrete DNA regulators of gene expression. Moreover, we have been able to build gene regulatory networks for separate cell types of both C3 Arabidopsis thaliana and C4 Gynandropsis thaliana. This has never been done before. From these networks, we are testing the importance of key components to validate their architecture.
The ability to generate somatic hybrids between C3 and C4 plants is also completely novel and has not been achieved before.