Chloroplasts are specialised organelles found in plant cells and some algae. Photosynthesis, the process by which light energy is converted into chemical energy, resulting in the production of oxygen and energy-rich organic compounds, takes place in chloroplasts. The number of chloroplasts per plant cell can vary widely, ranging from one in unicellular algae to up to 100 in plants like Arabidopsis and wheat. Chloroplasts have a unique transcription machinery that is more complex than their cyanobacterial ancestors. The plastid-encoded RNA polymerase (PEP) is a multi-subunit complex crucial for transcribing chloroplast genes, which are essential for photosynthesis and plant growth. Despite its importance, the roles of many PEP-associated proteins (PAPs) are poorly understood. Researchers from the John Innes Centre and Diamond Light Source aimed to study the structure of PEP to better understand its composition, assembly, and function. They used cryo-Electron Microscopy (cryo-EM) at the electron Bio Imaging Centre (eBIC) to achieve this goal, providing a detailed view of the PEP complex and its interactions with DNA and RNA. Their work was recently published in Cell.
Before this groundbreaking study, the understanding of the plant plastid-encoded RNA polymerase (PEP) was limited. Researchers knew that PEP is a multi-subunit complex composed of four plastid-encoded subunits similar to bacterial RNA polymerases (RNAPs) and several nuclear-encoded PEP-associated proteins (PAPs). This is the first striking difference compared to bacteria: the complex is significantly larger, as the PAPs seem to be present only in chloroplasts. Furthermore, the exact composition of the complex and roles of these subunits were poorly defined. Previous studies had identified PEP subunits are essential to chloroplast biogenesis, but the detailed structural and functional insights were lacking.
In this study, researchers purified PEP complex using chromatographic separation. It showed that the PEP is a huge complex of 1.1 MDa, more than twice the size of its bacterial counterpart. 19 unique subunits were identified using liquid chromatography techniques coupled with mass spectroscopy: the four subunits that form the core polymerase, and 15 PAPs. The purified PEP samples were functional in transcribing RNA from DNA in assays. This first step was crucial to ensure that the samples used for cryo-EM represents an active complex.
To visualise the PEP complex at high resolution, the researchers used cryo-EM at eBIC. Cryo-EM is ideal for studying large protein complexes in their native state, allowing the researchers to capture the intricate details of PEP and its associated proteins.
Dr Michael Webster commented:
It was a pleasure to work with eBIC scientist Dr Vinod Vogirala on this project. Vinod’s exceptional skills in cryo-EM meant that a project I anticipated might take us several years was instead realised in a matter of months. The team are very grateful to the ongoing support provided by eBIC, which has greatly enabled our efforts to address long-standing questions of how plants make their photosynthetic proteins.
They discovered that the core polymerase of the PEP shares structural similarities with the cyanobacterial RNAP. Also, PAPs encase the core polymerase, forming extensive interactions that likely promote complex assembly and stability. The precise composition of PEP and the position of the PAP subunits with respect to the polymerase were not known prior to this work. The PAP subunits add new capability to the core polymerase. PAP1 and PAP2 add DNA binding and RNA binding, and several PAPs add enzymatic functions. Interestingly, if any single PAP subunit is missing, the polymerase will not function efficiently. Future work will be needed to understand the relationship between all the different PAPs and the core polymerase and what is the precise role of each PAPs.
This research paves the way for further studies on the functional roles of PAPs and their contributions to chloroplast transcription. The potential applications of this research are vast. Understanding the structure and function of PEP can lead to advancements in agricultural biotechnology. For instance, manipulating the PEP complex could enhance photosynthetic efficiency and stress tolerance in crops, leading to higher yields and better resilience to environmental changes. Additionally, this knowledge can be applied to synthetic biology in plants, where designing artificial transcription systems could benefit from the insights gained from PEP's structure and function.
Joint first author Dr Ángel Vergara-Cruces said:
By revealing mechanisms of chloroplast transcription, our study offers insight into its role in plant growth and adaptation and response to environmental conditions. Now that we have a structural model the next step is to confirm the role of the chloroplast transcription proteins using genetic experiments in plants.
Furthermore, the research opens new avenues for studying the evolution of transcription mechanisms. By comparing the chloroplast transcription machinery with its bacterial and cyanobacterial counterparts, scientists can gain a deeper understanding of how complex transcription systems evolved in eukaryotic cells. This can also shed light on the integration of prokaryotic gene expression systems into eukaryotic host cells, providing a broader perspective on cellular evolution.
Dr Michael Webster:
Heat, drought, and salinity limit a plant's ability to perform photosynthesis. Plants that can produce photosynthetic proteins reliably in the face of environmental stress may control chloroplast transcription differently. We look forward to seeing our work used in the important effort to develop more robust crops.
To find out more about eBIC or discuss potential applications, please contact eBIC director Peijun Zhang: [email protected].
Vergara-Cruces A, Pramanick I, Pearce D, Vogirala VK, Byrne MJ, Low JKK, Webster MW. Structure of the plant plastid-encoded RNA polymerase. Cell 187 1145 – 1159. (2024) DOI: 10.1016/j.cell.2024.01.036
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