Exploring the potential of plastid biology and biotechnology

Plastids are a family of organelles that likely originate from the endosymbiosis of cyanobacteria (Howe et al., 2008) and exhibit diverse morphologies and biochemical capabilities (Fig. 1). The chloroplast is the most well-studied plastid type and performs photosynthesis in plants and algae and is thus responsible for most of the food production on the planet. However, there are other important biochemical functions performed by plastids, including the synthesis and storage of some carbohydrates, pigment production, fatty acid synthesis and nitrogen and sulphur assimilation. Plants have specialised plastid types that carry out some of these functions, such as starch-storing amyloplasts and pigment-producing chromoplasts (Fig. 1).

The annual Plastid Preview Meeting took place on 1–2 September 2022 in Norwich (UK), jointly hosted by the John Innes Centre and the University of East Anglia. Since the 1970s, Plastid Preview has showcased cutting-edge research from PhD students and early career postdoctoral scientists investigating fundamental and applied plastid biology. Research presented throughout the years has not only addressed questions in photosynthesis but also in diverse areas such as chlorophyll biosynthesis, starch metabolism, carbon fixation, plastid gene expression and plastid transformation. In the 2022 meeting, it was apparent that significant progress is being made in addressing long-standing questions in these areas with the aid of technological developments, such as machine learning, gene editing and cryo-EM approaches to study protein structure, combined with the ability to explore previously untapped biological diversity with the rapid expansion of genome sequences.

Three major domains of plastid research were covered: the first concerned ‘understanding the plastid’, probing the fundamental mechanisms that govern plastid biology. The second concerned ‘optimising the plastid’ and focussed on processes that could increase productivity and resilience in cyanobacteria, algae and plants. The third concerned ‘utilising the plastid’, specifically the use of plastid-containing organisms in biotechnology to produce high-value proteins and metabolites. Here, we highlight some of the diverse and exciting areas of work encompassing these three domains that were covered at the meeting.

Research into fundamental plastid processes, such as biogenesis, gene expression, protein import and division, is required to understand how photosynthesis and metabolism occur and are integrated within the organelle.

Protein import is a prime example of a process that influences many other plastid functions and was an area of active discussion at the meeting. The majority of the plastid proteome is encoded in the nucleus, and proteins are imported into the organelle through recognition of plastid-localisation signals, translocation through the plastid membranes and delivery to the appropriate location within the plastid. This process heavily influences the plastid proteome and thus plastid function, and recent exciting work implicated ubiquitination in this process (Ling & Jarvis, 2015). Ling et al. (2012) discovered that the cytosolic ubiquitin-mediated protein degradation pathway regulates components of the protein import machinery. More recently, ubiquitin-mediated protein degradation has also been observed for a broad range of chloroplast proteins in Arabidopsis, including those involved in photosynthesis, oxidative stress and other metabolic pathways (Sun et al., 2022). This opens new and exciting questions about the role of ubiquitination in the regulation of these processes, as well as its relevance to a broader range of species and plastid types, including crops. We also discussed the exciting links that were emerging between fundamental plastid processes, such as between protein import and plastid division (Fang et al., 2022).

While protein import is an example of how the nucleus can influence plastid function, the plastid possesses reciprocal mechanisms to signal its status back to the nucleus, known as retrograde signalling. Many aspects of this communication are not fully understood, but several important components have been uncovered, such as tetrapyrroles and reactive oxygen species (Chan et al., 2016). Many of these components accumulate due to a defect in photosynthesis, allowing the nucleus to sense the status of the chloroplast. Since photosynthesis is sensitive to perturbations in environmental conditions, this makes the chloroplast an ideal sentinel for general cell status. As such, retrograde signalling is involved in the response to both abiotic and biotic stresses (Chan et al., 2016; Littlejohn et al., 2021). An example of emerging research discussed at the meeting is how pathogen infection can affect nuclear expression of genes encoding plastid-localised proteins that influence resistance (Corredor-Moreno et al., 2022). Uncovering how pathogen effectors hijack plastid metabolism during infection and the broader roles of the plastid in biotic interactions could lead to novel approaches to protect plants from pathogens.

Photosynthesis is a central role of chloroplasts and is the main source of energy in cyanobacteria. The inefficiencies of RuBisCo have been described in detail (Flamholz et al., 2019). It can be partly attributed to the slow evolution of the enzyme (Bouvier et al., 2022) and the requirement to balance carboxylation rates with CO₂/O₂ specificity. The meeting highlighted exciting strategies to overcome these and increase RuBisCo activity.

The first strategy was based on harnessing pre-existing diversity in RuBisCo. There is large variation in RuBisCo kinetics between phototrophs (Savir et al., 2010) that could potentially be exploited to replace native enzymes with more efficient versions, if localised CO₂ concentrations could be increased. Extensive modelling shows that replacing the wheat RuBisCo with that from a C₄ species could significantly increase the efficiency of carbon uptake (Iqbal et al., 2021), and experimentally implementing these predictions may validate this route to increasing photosynthetic efficiency. Furthermore, there are many species where RuBisCo kinetics have not been reported, particularly in nonmodel species. As machine learning becomes increasingly prevalent, it may be employed to predict RuBisCo sequences with the potential to improve photosynthesis.

The second strategy is to improve RuBisCo efficiency via carbon concentrating mechanisms (CCMs). CCMs can be biophysical, like algal pyrenoids and cyanobacterial carboxysomes, or biochemical, as in C₄ plants. Recent work has focussed on developing a mechanistic understanding on how these CCMs form and function. One breakthrough was the discovery of the Chlamydomonas reinhardtii protein EPYC1, which physically links RuBisCo to the pyrenoid (Mackinder et al., 2016). This not only enhanced our understanding of pyrenoid assembly but also opened the possibility of engineering pyrenoids into plants (Adler et al., 2022). Similar work to transfer carboxysomes of cyanobacteria into plants may follow, benefitting from recent insights into the structure of the α-carboxysome (Ni et al., 2022).

Beyond photosynthesis in chloroplasts, there are many metabolic pathways that could be manipulated to increase crop quality, from carotenoid storage in chromoplasts to fatty acid synthesis in elaioplasts (Fig. 1). Specifically, exciting progress has been made on starch synthesis in amyloplasts of wheat, which produce two distinct types of starch granules, large A-type and small B-type granules. The discovery that the protein MRC regulates the formation of B-type granules can be exploited to introduce desirable changes in starch granule size properties for the food and milling industries (Chen et al., 2022). Further understanding of these processes may come from exploiting intra- and interspecies diversity and will drive improvements in crop nutritional and functional quality in a similar way to improvements in photosynthesis.

Biotechnological approaches to using the plastid as a biofactory have been a key theme of recent Plastid Preview meetings. The plastid provides a distinct environment from the rest of the cell for producing valuable proteins and metabolites. Much attention has fallen on cyanobacteria and algae, especially newly discovered fast-growing species like Synechococcus sp. PCC 11901 (Włodarczyk et al., 2020), for which large volumes of cells can be cultivated with ease and in a smaller space compared with plants. There are numerous examples of C. reinhardtii chloroplast engineering to produce vaccine subunits, antibacterial compounds and antibodies (Dyo & Purton, 2018).

A primary focus of this meeting was to explore new techniques and approaches to improve cyanobacterial, algal and plant biotechnology. Regulators of chloroplast transgenes are of great interest, to allow robust control and easy on/off switching of expression. Riboswitches, for instance the thiamine pyrophosphate riboswitch, are a potential solution (Mehrshahi et al., 2020), and the discovery of further novel switches and using these in tandem can expand our existing toolkit. Another area highlighted was targeting heterologous proteins to distinct plastid subcompartments, like the chloroplast thylakoid lumen. This would expand the range of proteins that can be produced to those that are not stable in the stroma (Chin-Fatt & Menassa, 2021).

To ensure that such exciting biotechnological approaches can be realised, a key priority is to expand our toolkit of plastid transformation technologies. This includes improved synthetic biology tools, for example, MoClo cloning systems that are compatible across a range of phototrophs (Vasudevan et al., 2019). We expect that progress will come from developing novel Cas enzymes, beyond Cas9, for efficient function in the plastid and adapting base editing technology for efficient plastome editing. An additional challenge when engineering organisms with multiple plastids per cell is achieving homoplasmy, and exciting new techniques are emerging to overcome this (Okuzaki et al., 2020). These methods could result in the widespread adoption of plastid transformation and utilisation of plastids as biofactories in cyanobacteria, algae and plants.

The Plastid Preview Meeting remains guided by its fundamental goal of shining a light on plastid function, both in photosynthesis and beyond. This meeting highlighted diversity in the plastid field – from the variety of plastid types studied, to intra- and interspecies differences in mechanisms of plastid function. However, much natural diversity remains unexplored and technologies continue to evolve, particularly through adopting and combining techniques from different fields. Exploiting these will be essential to understand, improve and utilise the plastid further, and we have no doubt that this will be covered at future Plastid Previews.

RM and ÁV-C contributed equally to this work.