Defining the molecular basis of chloroplast transcription of photosynthetic genes

Plant growth is driven by photosynthesis. However, it is not well understood how plants produce their photosynthetic proteins. The chloroplast contains a genome that encodes key photosynthetic proteins and a unique molecular machinery that expresses them. Despite their importance, how the chloroplast gene expression machinery functions has not been characterised in detail.

The first stage in the production of photosynthetic proteins from chloroplast genes is their transcription to produce messenger RNAs (mRNAs). This process is performed by a large assembly of proteins known as the plastid-encoded polymerase (PEP). Plants turn green in response to light due to the activation of the transcriptional activity of PEP.

In addition, plant stresses such as drought, heat and pathogen attack affect PEP activity to allow specific genes encoding photosynthetic proteins to be turned on or off.

Despite its central role in plant development and adaptation, how PEP transcribes chloroplast genes is poorly understood. PEP is made of 19 different protein subunits that each have an essential role. PEP is remarkable amongst transcription enzymes in that it contains subunits of two evolutionary origins.

The core resembles bacterial enzymes and was inherited with the chloroplast genome from a cyanobacterial ancestor. By contrast, the twelve or more proteins that stably bind to the core are encoded in the nuclear genome. We therefore expect that these proteins, known as PAPs (PEP-associated proteins), orchestrate key regulatory processes unique to the chloroplast.

To better understand how photosynthetic proteins are produced by plants, we aim to visualise PEP as it transcribes genes. To do this, we will collect images of PEP molecules using cryogenic electron microscopy (cryo-EM). By processing these images, models of PEP at atomic resolution can be constructed.

These are expected to show how PAPs activate chloroplast transcription. The level of detail provided by modern cryo-EM is immensely valuable to developing new hypotheses, as precise modifications can be designed with predictable changes in activity. In this project we will also examine the consequences of making specific changes, using transcription reactions reconstituted with purified components and plant genetic complementation experiments.

The outcome will be a better understanding of what role each component of PEP has, how it performs it, and why these processes are essential to chloroplast development and photosynthesis.

This project is expected to deepen our fundamental understanding of the biochemical basis of transcription. Decades of detailed study have been performed on the proteins that perform transcription in the eukaryotic nucleus and bacteria. This has shown that collating information about diverse proteins is essential to inferring general principles of how gene expression is regulated.

Understanding the unique set of proteins that act on chloroplast genes therefore represents an exciting opportunity to advance this. Transcription regulation is a key component to human health and disease, and this research consequently has diverse potential uses.

Photosynthesis has a central role in producing the oxygen and energy that sustains much of life on earth. Detailed structural and biochemical studies on the photosynthetic proteins have revealed in detail how they harness solar energy, and this has provided a valuable foundation for crop improvement and development of diverse biotechnologies. By contrast, equivalent mechanistic studies of the gene expression processes that underpin production of the photosynthetic proteins are largely lacking.

This project will answer a complementary set of questions: what determines the timing and level of photosynthetic protein production, and how could we modify this to develop more robust crops and new biotechnological applications?