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Identification of a highly efficient chloroplast-targeting peptide for plastid engineering [1]

['Chonprakun Thagun', 'Department Of Material Chemistry', 'Graduate School Of Engineering', 'Kyoto University', 'Kyoto-Daigaku-Katsura', 'Kyoto', 'Center For Bioscience Research', 'Education', 'Utsunomiya University', 'Tochigi']

Date: 2024-09

Plastids are pivotal target organelles for comprehensively enhancing photosynthetic and metabolic traits in plants via plastid engineering. Plastidial proteins predominantly originate in the nucleus and must traverse membrane-bound multiprotein translocons to access these organelles. This import process is meticulously regulated by chloroplast-targeting peptides (cTPs). Whereas many cTPs have been employed to guide recombinantly expressed functional proteins to chloroplasts, there is a critical need for more efficient cTPs. Here, we performed a comprehensive exploration and comparative assessment of an advanced suite of cTPs exhibiting superior targeting capabilities. We employed a multifaceted approach encompassing computational prediction, in planta expression, fluorescence tracking, and in vitro chloroplast import studies to identify and analyze 88 cTPs associated with Arabidopsis thaliana mutants with phenotypes linked to chloroplast function. These polypeptides exhibited distinct abilities to transport green fluorescent protein (GFP) to various compartments within leaf cells, particularly chloroplasts. A highly efficient cTP derived from Arabidopsis plastid ribosomal protein L35 (At2g24090) displayed remarkable effectiveness in chloroplast localization. This cTP facilitated the activities of chloroplast-targeted RNA-processing proteins and metabolic enzymes within plastids. This cTP could serve as an ideal transit peptide for precisely targeting biomolecules to plastids, leading to advancements in plastid engineering.

Funding: K. N. was financially supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Data Creation and Utilization-type MaTerial R&D project: https://dxmt.mext.go.jp/en/about ; Grant Number JPMXP1122714694, Japan Science and Technology Corporation, Exploratory Research for Advanced Technology (JST-ERATO): https://www.jst.go.jp/erato/en/ ; Grant Number JPMJER1602, and Japan Science and Technology Corporation, Program on open innovation platform for industry-academia co-creation (COI-NEXT): https://www.jst.go.jp/pf/platform/ ; Grant Number JPMJPF2114. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we classified the plastid-targeting efficiencies of transit peptides derived from nucleus-encoded proteins related to chloroplast phenotypes in Arabidopsis (Arabidopsis thaliana) mutants [ 29 , 30 ]. We fused computationally predicted cTPs to GFP and compared the plastid-targeting abilities of 89 different cTP-GFPs by in planta expression analysis. Surprisingly, the cTP-GFPs localized to distinct cellular compartments, including chloroplasts, mitochondria, and the cytosol, regardless of their predicted subcellular localizations. Our data emphasize the importance of cTP length and cleavage sites in determining the efficiency of protein translocation into chloroplasts. Among the cTPs, a superior cTP that showed approximately 10 times higher chloroplast-targeting efficiency than the widely used Arabidopsis AtRbcS cTP was identified. This outstanding cTP exhibited a greater ability than native cTPs to import functionalized proteins into plastids for the engineering of RNA processing and metabolic flux. This newly identified cTP has the potential to revolutionize plastid engineering and expand our ability to engineer plastids for biotechnological applications.

In the field of plastid biotechnology, various cTPs have been employed to direct the localization of recombinantly expressed cargo proteins, such as green fluorescent protein (GFP) [ 14 , 15 ], metabolic enzymes [ 16 – 18 ], protective agents [ 19 ], plastid-membrane transporters [ 20 ], and RNA-processing proteins to plastids [ 21 ]. Moreover, the integration of cTPs into plastid-targeted DNA-editing proteins such as zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR-type deaminase has led to selective plastome modifications in plants [ 22 – 25 ]. cTPs derived from plant Rubisco small subunit proteins (RbcS) have been used as biorecognition modules on nanoparticles to specify biomolecule delivery to chloroplasts [ 26 ]. In addition, manipulating peptide sequences improved the targeting abilities of cTPs [ 13 , 18 ]. Until now, the comparison of import efficiencies among numerous cTPs has been experimentally performed using various techniques, including in vitro import assays, in vivo protoplast transfection, and expression studies in transgenic plant cells [ 27 , 28 ]. Thus, superior cTPs from available genetic resources remain to be identified and analyzed.

cTPs regulate both the efficiency and specificity of preprotein import into plastids. N-terminal cTPs typically encompass the first 20 to 100 amino acids of the preproteins [ 7 ]. These cTPs do not exhibit distinctive characteristics except for the presence of an abundance of hydroxylated serine/proline residues [ 4 , 5 ]. Cytosolic scaffold proteins such as 14-3-3, HSP70, and HSP90 stabilize unfolded preproteins by interacting with amino acids in the cTP domains that are in the appropriate conformation [ 8 ]. Once preproteins reach the plastid envelope, cTPs guide preproteins into plastids via an energy-dependent process. This import is mediated by translocons located on the outer and inner envelope membranes (TOC/TIC complexes). Subsequently, stromal processing peptidases remove the cTP portions of preproteins that function within the plastid stroma (the protein-rich fluid inside plastids) at specific cleavage sites [ 6 ]. Additionally, various plastidial proteins are transported to the thylakoid membrane and lumen. These thylakoid-localized proteins normally contain bipartite chloroplast/thylakoid-targeting sequences in tandem with 2 distinct cleavage sites [ 9 , 10 ]. The mutation and depletion of cTP domains lead to the mislocalization and malfunctioning of plastidial proteins in plant cells [ 11 – 13 ].

Plastids are DNA-containing endosymbiotic organelles that are the sites of photosynthesis and supply chemical energy to plant cells. Plastids contain plastidial proteins encoded by numerous circular plastid-genome DNAs (plastomes) and by DNA from the nuclear genome [ 1 ]. Approximately 120 of these plastidial proteins are encoded by the plastome, whereas the majority are encoded by the nuclear genome [ 2 ]. Nucleus-encoded plastidial proteins are synthesized as preproteins (i.e., precursors) in the cytosol and posttranslationally imported into plastids and subplastidial compartments such as the thylakoids [ 3 ]. In general, preprotein import to plastids is directed by transit peptides known as chloroplast-targeting peptides (cTPs) located at either the amino (N)- or carboxyl (C)-terminus of the protein [ 4 , 5 ]. After successful translocation into plastids, the cTP domain is irrevocably excised by plastidial signal peptidases, leaving a mature protein to function in its related organellar process [ 4 – 6 ].

Results

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002785

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