The ubiquitin-proteasome system regulates chloroplast biogenesis.

In a recently published work, we identified the first chloroplast-localized E3 ligase, and showed that it degrades TOC translocon proteins by the ubiquitin-proteasome system (UPS). This regulation was found to be critical for plastid developmental transitions, such as the conversion of etioplasts to chloroplasts. Here we discuss the importance of SP1 for plants under different circumstances, and speculate about SP1 function and its possible application in agriculture. We anticipate that further work in this area may reveal additional roles of the UPS system, through action on other chloroplast OEPs with different roles in plastid biogenesis and function.

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In eukaryotic organisms, selective protein breakdown by the ubiquitin-proteasome system (UPS) is widely used to regulate development and other processes. In general, the function of the UPS pathway is to conjugate ubiquitin (a 76 amino acid protein) to Lys residues within substrate proteins by the consecutive action of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3), thus targeting them for degradation by the 26S proteasome. In the model plant Arabidopsis thaliana, nearly 6% of proteins are related to the UPS system, and they are found to contribute significantly to a wide range of processes, including embryogenesis, morphogenesis, hormone signaling and responses to environmental challenges. Approximately 90% of these UPS proteins are E3 ubiquitin ligases, which confer substrate specificity to the pathway., The principal sites of action of the UPS are the cytosol and nucleus.

Chloroplasts in plant cells originate from the endosymbiosis of a cyanobacteria-like prokaryote. During the ensuing evolution, the host cell and the endosymbiont became well integrated, with many endosymbiont genes being transferred to the nuclear genome. Thus, many chloroplast proteins are synthesized in the cytosol as preproteins, which are then imported into the chloroplast through translocons at the outer and inner envelope membranes of chloroplasts (TOC and TIC, respectively). The TOC complex is composed of the Toc75 channel and the Toc159 and Toc34 receptor subunits that recognize the preproteins. In Arabidopsis (and other species), there are multiple isoforms of the receptors which recognize different sets of preproteins: atToc159 and atToc33 are the major receptor isoforms in chloroplasts, and these mediate the import of the highly-abundant, photosynthesis-related proteins; by contrast, atToc132/atToc120 and atToc34 are minor isoforms that mediate the import of non-photosynthetic, housekeeping proteins.

It is very interesting that the first chloroplast-localized E3 ligase to be identified is a regulator of the TOC translocon. Using genetic screening for second-site suppressors of plastid protein import1 (ppi1; an Arabidopsis atToc33 mutant), we identified SUPPRESSOR OF PPI1 LOCUS1 (SP1), a RING-type E3 ligase protein. Using in vitro and in vivo assays, the SP1 protein was shown to ubiquitinate all tested TOC proteins, but not other chloroplast outer envelope proteins (OEPs). Moreover, abundance of ubiquitinated TOC proteins depended on proteasomal activity, indicating that TOC proteins are targeted by SP1 for UPS-mediated degradation. Interestingly, sp1 single mutant plants are defective in greening (de-etiolation) and leaf senescence. Further analysis showed that SP1 selectively regulates TOC protein levels in response to developmental and environmental cues. During de-etiolation, atToc132/atToc120 (which are specialized for non-photosynthetic protein import) are degraded by SP1 through the UPS pathway. This may allow the released Toc75 channels to associate with atToc159 and atToc33, which are specialized for the import of photosynthetic proteins.

By analyzing public microarray data (Genevestigator), we found that in Arabidopsis the mRNA expression levels of the atTOC132, atTOC120 and atTOC159 genes are quite stable during de-etiolation. This further supports the importance of the UPS regulation of TOC proteins in this process. Compared with transcriptional regulation of gene expression, such post-transcriptional regulation can control protein amounts more promptly and precisely in response to different cues, especially when the proteins are unwanted. Post-transcriptional regulation of the import machinery by the UPS enables the rapid development of etioplasts into chloroplasts in response to a dark-to-light shift. It should be noted that de-etiolation and photomorphogenesis are very important processes for plants, and several key regulators such as COP1, HY5 and PIF1/PIF3 have been well studied.- COP1, another RING-type E3 ligase, together with the SPA complex, functions as a negative regulator for photomorphogenesis by destabilizing HY5 (a nuclear transcription factor that activates downstream light-inducible genes) through the UPS system in the dark. PIF1 and PIF3 negatively regulate chlorophyll synthesis and chloroplast development when seedlings are in the dark. These regulatory events prevent the premature development of chloroplasts in darkness.

Given that the sp1 single mutant displays no obvious abnormal phenotypes under optimal, controlled growth conditions, the SP1-mediated TOC reorganization perhaps allows fine-tuning in response to more challenging conditions, which are common in nature. For example, as we have shown, SP1 aids seedling survival after prolonged dark stress upon germination. Besides, TOC reorganization may also occur during other plastid transition processes, such as during senescence when chloroplasts transform into gerontoplasts, and perhaps also fruit ripening when the green chloroplasts convert into carotenoid-pigmented chromoplasts. Whether SP1 can control fruit ripening will be particularly interesting. The answer to this question will have major implications for food quality and shelf life, and many aspects of agricultural food production.

Since the 26S proteasome only exists in the cytosol and nucleus, most likely only the chloroplasts’ OEPs are potential targets of the UPS. Our discovery opens an interesting new door to the regulatory mechanisms that govern chloroplast OEPs. So far, besides the TOC proteins, only a small number of OEPs have been identified, and even fewer have had their functions elucidated., Examples include: the DGD1/2, MGD2/3 and LACS9 proteins that are involved in lipid metabolism, and SFR2 which is a galactolipid remodeling enzyme and is essential for freezing tolerance in Arabidopsis;- PDV1/2 which are related to chloroplast division,; CHUP1 which is responsible for chloroplast redistribution in different light regimes; and a chloroplast outer envelope-bound PHD transcription factor, PTM, that mediates chloroplast retrograde signals to the nucleus. Other OEPs include OEP16, OEP21, OEP24 and OEP37, which are probably cation-selective channels.

In our study, we identified homologs of SP1 in the Arabidopsis genome, and data showed that the encoded proteins are also located in the chloroplast envelope. Intriguingly, SP1’s closest relative does not share redundancy with SP1. Perhaps these other E3 ligases target other OEPs, which act in different aspects of chloroplast biogenesis. An important challenge to be addressed in future experiments is the identification of the substrates of these, and perhaps other, E3 ligases in chloroplasts; this will not be trivial, as it cannot be done in silico, and so multiple experimental assays will be necessary to identify their functions. In addition, other basic questions concerning the function of SP1 itself need to be answered. Although most TOC proteins are substrates of SP1, how they are selectively degraded under different circumstances remains unclear. Since SP1 itself can be ubiquitinated, and degraded by the UPS, this is one possible mechanism; but there may also be other mechanisms controlling SP1 dynamics, to be identified in future experiments.