Control of plastid biogenesis by the ubiquitin-proteasome system

Chloroplasts and mitochondria are normal components of many cells - they are sub-cellular structures called organelles. Interestingly, these two organelles evolved from bacteria that were engulfed by other cells over a billion years ago, and in many ways they still resemble free-living bacteria. Chloroplasts are found in plant cells, contain the green pigment chlorophyll, and are responsible for the reactions of photosynthesis (the process that captures sunlight energy and uses it to power the activities of the cell). Since photosynthesis is the only significant mechanism of energy-input into the living world, chloroplasts are of inestimable importance, not just to plants but to all life on Earth. Actually, chloroplasts belong to a wider family of related organelles called plastids. Other members of the family are the highly-pigmented chromoplasts in ripe fruits, and etioplasts in dark-grown plants.
Although plastids do contain DNA (a relic from their evolutionary past as free-living photosynthetic bacteria), and so can make some of their own proteins, most of the proteins needed to form a functional plastid are encoded on DNA in the cell nucleus; these proteins are made outside of the plastid in the cellular matrix known as the cytosol. As plastids are each surrounded by a double membrane, or envelope, that is impervious to the passive movement of proteins, this presents a significant problem. To overcome the problem, plastids evolved a sophisticated protein import apparatus, which uses energy (in the form of ATP) to drive the import of proteins from the cytosol, across the envelope, to the plastid interior. This import apparatus comprises two molecular machines: one in the outer envelope membrane called TOC (an abbreviation of "Translocon at the outer envelope membrane of chloroplasts"), and another in the inner envelope membrane called TIC. Each machine is made up of several different proteins which cooperate to ensure the efficiency of import.
We work on a model plant called Arabidopsis that has many advantages for research, such as an availability of numerous mutants (each one with a mutation in a specific gene). One such mutant plant, ppi1, has a defect in a TOC gene such that plastid protein import does not work efficiently. Several years ago, we identified another mutation called sp1 (this stands for "suppressor of ppi1") that counteracts the negative effects of ppi1. The gene disrupted by sp1 (the SP1 gene) encodes a type of regulatory protein called a "ubiquitin E3 ligase". These work by labelling-up unwanted proteins and targeting them for removal. Because this control mechanism was not previously known to operate in plastids, this discovery was an important breakthrough in biology. The SP1 E3 ligase carefully controls the composition of the TOC machinery so that the right proteins are always imported (this is normally good, but in the abnormal ppi1 background it is apparently a hindrance). Such control is very important when plastids need to convert from one form to another; e.g. when dark-germinated plants emerge into the light, etioplasts must change into chloroplasts so that photosynthesis can begin.
In this project we will investigate whether SP1 is important for the conversion of chloroplasts into chromoplasts in tomato fruit. If it is, then our work may have commercial, agricultural importance by enabling the manipulation of fruit ripening in crops (e.g. tomato, bell pepper, citrus). We will also study in much greater detail how SP1 and related proteins control plastid development. For example, our work may elucidate how plants respond to stresses like salinity and drought, which are major limits on crop yield across the world. Photosynthetic performance (and thus the energy available to plants for growth) is strongly affected by stress, and we suspect that SP1 is involved in this process. Thus, knowledge gained from our work may enable improved adaptation of crops to adverse environmental conditions.

Arabidopsis SP1 encodes a RING-type E3 ligase of the chloroplast outer membrane. It targets plastid protein import machinery components (i.e. TOC proteins) for ubiquitination and degradation. SP1 regulates which proteins are imported, and this in turn controls the plastid's proteome, development and functions. Thus, the ubiquitin-proteasome system (UPS) directly regulates plastids. Here we will elucidate the roles of SP1 and UPS in plastid biogenesis, using tomato and Arabidopsis.
SP1 is important for developmental transitions in which plastids convert from one form to another (e.g. de-etiolation). A commercially important example is fruit ripening, when chloroplasts change into chromoplasts; we will study SP1's role in this by altering its expression in transgenic tomato plants. This may eventually enable the manipulation of fruit ripening in crops.
E3 ligases are typically regulated by post-translational modification (PTM). We found that SP1 is developmentally controlled, that it can be ubiquitinated, that it is likely phosphorylated, and that some of its targets are potentially sumoylated. We will study these PTMs to elucidate how SP1 is regulated to optimize plastid protein import. We will also study SP1's role in stress responses, which may involve reorganizing the plastid proteome to optimize photosynthetic activity against photo-oxidative damage.
As chaperones aid extraction of UPS targets from ER and mitochondrial membranes, we propose a similar role for them in plastids. We will test for plastid localization of such chaperones, and assess the effects of their inactivation in planta on TOC protein levels and polyubiquitination, and plastid protein import.
Finally, we will seek new factors involved in the UPS-control of plastid proteins. We will study Arabidopsis SP1 homologues by assessing the effects of gene inactivation in planta, and by using TAP tagging to identify their targets and regulators. We will also use informatics to seek novel factors.

Academic impact will be large due to the work's interdisciplinarity as detailed in the Academic Beneficiaries section. This will manifest itself in several ways: 1) The work will contribute to scientific advancement, providing new knowledge with relevance in several overlapping fields and disciplines. 2) The work will stimulate international collaboration via the link with Prof Grimm of Humboldt University and via the involvement of Dr Ling as RA. Dr Ling maintains strong links with the prestigious Shanghai institute where he completed his PhD, and may eventually return to China as an independent research leader (whereupon we would expect to maintain collaborative links). 3) The work will contribute to the health of UK plant science due to publicity surrounding the project, the interactions it will foster, and by our hosting of visitors from schools as this will generate enthusiasm for plant biology. 4) The work will deliver highly-trained individuals who will also contribute to the health of UK plant science. Training will result not only from the direct involvement of the research staff but also from Prof Jarvis' supervision of PhD and MSc students who will work on projects related to the proposed work and have daily interaction with the staff.

Our work may have agricultural, commercial and societal impact. It may enable the manipulation of chromoplast development during fruit ripening in crops like tomato, bell pepper and citrus, which are rich in carotenoid pigments of dietary importance. By altering SP1 expression, we might accelerate or inhibit fruit ripening or otherwise alter fruit properties. Thus a preliminary patent application has been filed. We will begin to explore these possibilities using tomato as a model. We also plan to apply for Pathfinder and Follow-On Funding to further develop these commercial ideas. In the longer term our work may facilitate improved adaptation of crops to the environment, as preliminary data suggest that SP1 acts in stress responses, perhaps through effects on photosynthetic performance. Stresses like salinity and drought are major limits on crop yield across the world, which may become more prominent in the future due to climate change.
Unforeseen benefits may arise in the future due to the fundamental importance of the area in which the project is focused: chloroplasts are the site of photosynthesis and synthesize a diversity of products (e.g. starch, amino acids, fatty acids), many of which are vital in mammalian diets. Plastids offer many possibilities for industrial exploitation. Raw materials for biofuel production are derived largely via the actions of plastids. Chloroplasts dominate plant cells enabling foreign proteins to be expressed at very high levels in plastids; greater understanding of plastid biology may facilitate their use as bioreactors. New knowledge on plastids may even have medical applications, as apicomplexan parasites (e.g. malaria) contain a relict plastid. Moreover, our work on SP1 may elucidate related processes in mitochondrial biogenesis; significantly, mitochondria and the ubiquitin proteasome system are both implicated in ageing and neurodegenerative diseases.

The general public and schools will benefit as we will engage with them in various ways. We will develop a schools engagement activity on Chloroplast Biology as part of a two-day event entitled Dynamic DNA organized by GENIE, a Centre for Excellence in Teaching and Learning at the University. Through the University's Botanic Garden (which receives 40k visitors annually) we will contribute to a publicly-circulated newsletter, deliver a public lecture on project-related topics, prepare a display board for placement at the gardens, and contribute to well-attended educational activities for local schools. Finally, we will continue to accept visitors into our lab via different schemes (e.g. sixth-formers funded by the Nuffield Foundation) and engage the media via the University Press Office.