Challenging the role of plastid cotranslational N-terminal modifications upon stress response – CANMORE

Objective 1

Coordinators: Team1 (CG, France) will be the coordinator of the overall objective 1 and WP1 and WP3, whereas Team3 (IF, Germany) will be responsible for WP2.
Main methodology: structural, biochemical and in-house proteomics techniques
Objective 2

Coordinators: Team3 (IF, Germany) will be the coordinator of the overall objective 2 and WP1.
Main methodology: molecular biology, enzymology, cell-biochemistry and in-house proteomics techniques applied to in vitro and in vivo systems.
Objective 3

Coordinators: Team2 (BG, Germany) will coordinate the overall objective 3 as well as WP1 and WP2.
Main methodology: molecular biology, genetics, physiology, metabolomics, proteomics, cell biology, biochemistry, growth chambers and green house facilities

In the frame of task1 of WP1, CG team has completed the enzymatic characterization of the 3 pMetAPs, providing a detailed knowledge of enzyme properties and substrate specificity of plastid MetAPs.N-Terminomics characterization and a global quantitative proteomics of different MetAP mutant lines produced by BG team were made in CG team. On 6 biological replicates. The overall N-terminomics analysis revealed as expected that the global N-terminal acetylation was not changed in any of the MetAP mutant lines. Focusing on the N-terminal Met, 22 proteins have N-ter that were observed in two distinct forms: with or without the iMet in position 1. 14 are plastid encoded proteins. We primarly focused on the plastid encoded proteins and we checked their N-termini and relative abundance (average spectral intensity of each N-terminal form) in the different pMetAP conditions. From this analysis, we clear observed that some plastid encoded proteins are strictly dependent on one or the other MetAPs, whereas other plastid proteins are still taking in charge even when the 3 pMetAP level is low.

Summary N-terminomics for N-termini obtained in all samples:
1) NAD(P)H-quinone oxidoreductase subunit K (ATCG00430, MNSIKFPILDR/NSIKFPILDR): MetAP1C/MetAP1D
2) ATP synthase subunit beta (ATCG00480, *MRTNPTTSNPE/ TNPTTSNPEV): MetAP1B
3) 50S, L33 (ATCG00640.1, MAKGKDVRVTI/AKGKDVRVTI): MetAP1C
4) Cytochrome b6 (thylakoid membrane) petB (ATCG00720.1, MSKVYDWFEER/SKVYDWFEER): MetAP1D
5) Cytochrome b6-f- complex S4 petD (thylakoid membrane) (ATCG00730.1, MGVTKKPDLND/GVTKKPDLND): MetAP1C
6) 30S rps15 S15 (ATCG01120.1, MIKNIVISFEE/IKNIVISFEE): MetAP1C
7) RBCL (ATCG00490.1, MSPQTETKASVG/*SPQTETKASVG/ *PQTETKASVG): MetAP1C.

Summary of CG global proteome made on 4 of the 6 biological replicates:
• Proteome overall very stable in every condition
• In the double KO metap1b x metap1d + VIGS GFP:
? Increases: AT4G02520.1/AT2G02930.1; glutathione transferase; 2.3 fold change (t-test ++)
• In the double KO metap1b x metap1d:
? Increases: ATCG00720.1; Cytochrome b6; 2. 3 fold change
? Decreases: AT3G59970.3; Methylenetetrahydrofolate reductase 1; 0.4 fold change (++)
AT3G61470.1; Photosystem I chlorophyll a/b-binding protein 2; 0.4 fold change
AT3G10060.1; Peptidyl-prolyl cis-trans isomerase FKBP16-4; 0.5 fold change
• In the triple KO metap1b x metap1d + VIGS MetAP1C:
? Increases: AT4G25130.1; Peptide methionine sulfoxide reductase A4; 2.1 fold change (++)
AT5G26742.3; DEAD-box ATP-dependent RNA helicase 3; 2.1 fold change (++)
AT4G23600.3; Cystine lyase CORI3; 2.6 fold change (++)
? Decreases: AT5G54270.1; Chlorophyll a-b binding protein 3; 0.5 fold change (++)
• Mostly chloroplastic proteins affected.
WP2: Characterization of plastid GNATs involved in RuBisCO acetylations
Task 1: Searching for plastid NAT(s) responsible for N-terminal RbcL acetylation
Several strategies were adopted by CG team to identify the GNAT responsible of N-terminal Proline acetylation of RbcL
From this overall characterization using combinatory investigation tools, the CG team has now gained extremely robust proofs about the characterization of the plastid GNAT responsible for NTA of RbcL Pro, strongly suggesting that GNAT7 is the major even the only one in cellulo, and to less extend also GNAT1 in vitro). GNAT 2,4,6 or 10. Are not involved.

Scientific production is underway

Chloroplasts primarily function as bioreactors converting light into chemical energy. In addition, they also act as regulatory hub for intracellular communication and mediation of environmental impacts to regulate nuclear gene expression. Chloroplast proteins are encoded either by the plastid or nuclear genome. Several chloroplast multi-protein complexes, such as RuBisCO (D-ribulose 1,5-bisphosphate carboxylase/oxygenase), which is the major enzyme of the Calvin-Benson cycle and the most abundant protein on earth, are assembled from plastid- as well as nuclear-encoded protein subunits. All nuclear and plastid-encoded chloroplast-localized proteins undergo many co- and post-translational modifications (CTMs and PTMs), which have important roles in controlling stability, accumulation, activity, assembly, and compartmentalisation of these proteins. However, CTMs and PTMs of plastid proteins and their catalytic modifiers have not intensively been explored up to date and the number of modifications continuously increases. Over the last years, the collaborative work among the three teams (Gif, Münster and Berlin) have brought multiple preliminary results highlighting unique properties of plastid N-terminal modifiers together with specific and essential modulated functions of the catalysed NPMs, which now build the research hypothesis and objectives of the CANMORE project. The overarching aim of CANMORE is to elucidate the role(s) of specific plastid N-terminal modifications (NPMs) and their regulatory interdependency with other PTMs. Via three interconnecting objectives, we will perform a structural and mechanistic characterization of the plastid modifiers involved in NPMs and related PTMs. Strong emphasis will be given to the unconventional and enigmatic N-terminal maturation of the RuBisCO large subunit. Hence, a detailed understanding of the protein modifications occurring on RuBisCO, especially under adverse environmental conditions, will be necessary for our understanding and possible future improvement of RuBisCO activity and photosynthesis in general. In this context, we will provide a thorough investigation of the plastid N-terminome and acetylome in response to temperature stress, which is particularly important for RuBisCO activity. Collectively, the CANMORE project will not only provide a comprehensive analysis of the plastid NPM machinery, but will also significantly enhance our understanding of the consequences of NPMs on specific plastid activities and during changing environmental conditions. For this project, a synergy of complementary approaches involving proteomics, biochemistry, structural biology, genetics and cell biology will be proposed. The CANMORE project will bring together three highly complementary groups [Carmela Giglione, Team1 (France); Bernhard Grimm, Team2 (Germany); and Iris Finkemeier, Team3, (Germany)] with interests and world lead in both CTMs/PTMs and chloroplast physiology.