Elucidating the multifaceted functions of protein acetyltransferases in plant stress response and regulation of metabolism. – KatNat

Jointly, the skill set of our five groups will allow a unique, cutting-edge, interdisciplinary approach to obtain a multi-scale description of protein acetyltransferases in cellular stress response and the regulation of plant physiology. Consortium members have suitable and necessary expertise on the topic, international visibility, and shared focus on diverse aspects of protein acetylation. Each team utilises highly complementary approaches that will bring added value to the KatNat project. A particular strength lies in the mass-spec capacity and expertise of several partners for the global quantitation and mapping of K- (IF) and Nt-acetylation (CG). Thus, key components of the research program are not dependent on third party data generation, but rather will exploit cutting-edge in-house technologies and skills. This also offers exceptional value for money since these global proteomic analyses are comparatively much more expensive if outsourced. In addition to advanced mass-spec capabilities, consortium members have demonstrable biochemical expertise for elucidating enzymatic functions (IF, CG, MW, DG), photosynthetic analysis (IF, PM) and the mechanistic study of protein degradation/ubiquitination (DG, MW, CG).

1) We standardized the growth conditions to allow direct analysis and comparison of the set of tissues samples by all teams.
2) We characterized the cellular localization of GNAT enzymes and we could confirm a plastid localization for 8 out of the 10 plastid enzymes; with GNAT9 and 10 found at the cytoplasm level despite a transit peptide was predicted. A deeper characterization showed thylakoid membrane localization for GNAT5 and 10. Finally, I. Finkemeier group used bisubstrate analog of Lys-CoA and could show that GNAT1 was found in young but not in old leaves, while GNAT7 is present in all tissues except roots. NAA50 was found active in all tissues except roots, while NAA10 was present in roots as well.
3) We used our in-house developed GAP assay to assay the NTA activity of all putative plastid GNATs in addition to the cytosolic NAA50 and NAA60. In parallel, the K-activity of these enzymeswas performed.
4) We generated KO mutants for each single enzyme and we compared the K- and Nt-acetylome changes induced by the absence of NAA60, NAA50, GNAT1, GNAT2 and GNAT4 with the reference acetylomes of the wild type.
5) To investigate the dynamics of plastid K- and Nt- protein acetylation during normal, low and high light we have started a whole omics analysis (transcriptomics, metabolomics, proteomics, N-terminomics, K-acetylomics) of WT vs GNAT1, GNAT2 and GNAT4 KO lines grown at the 3 different conditions.
6) Sensitivity to lincomycin of all available mutants was also screened 7) We analyzed the transcriptome and metabolome of NAA50, NAA60, GNAT1, GNAT2 and GNAT4 mutant lines.
8) We also created an amiNAA50 line in order to be able to assess NAA50 – an essential gene in plants - for stress sensitivities.
9) We also established the fFT-Protein age determination system in planta.
10) We checked all GNAT mutants available for effect on photosynthesis.
11) Characterization of the function and specificity of specialized E3 ligases (Not4 and DOA10).

Protein modifications represent an emerging area of study with the potential to impact agriculture, as it is becoming increasingly apparent that signal-responsive modulation of protein activity plays a fundamental role instructing developmental decisions, including regulation of metabolism and stress tolerance. Protein acetylation is one such modification, yet research in this area is severally lacking. KatNat is therefore extremely timely in its aim to investigate new links between protein acetylation, plant signalling, metabolism and physiology. Crucially, our work seeks to place our fundamental findings into an agronomic context (e.g., photosynthetic efficiency, impacting productivity, and abiotic stress resilience).
As the global population rapidly rises, and weather patterns become increasingly unpredictable due to climate change, there is a pressing need to identify new targets for manipulating crop performance to increase food security. A critical aim of KatNat is therefore to create new knowledge that could pave the way for manipulating diverse processes associated with plant productivity, environmental-adaptation and stress-resilience. This does not solely include crops that are constitutively tolerant to a particular stress, but rather the investment in flexible crop varieties that can adapt to fluctuating conditions whilst maintaining yield. Since KatNat will use Arabidopsis as a model, the societal, economic and environmental outputs will not be direct. It is anticipated, however, that key outputs of the KatNat project will have significant impact on fundamental plant science and the potential to impact agriculture in the future by providing new targets for breeders and biotechnologists to develop a new generation of crops. In addition, the PhD students and postdocs of KatNat will acquire both cutting-edge technical and transferable skills that will enable them to contribute to the economy and society in a wide spectrum of future careers.

Armbruster L, et al (2020) NAA50 is an enzymatically active N(a)-acetyltransferase that is crucial for development and regulation of stress responses. Plant Physiol 183

Bailey M, et al (2021) The Arabidopsis NOT4A E3 ligase promotes PGR3 expression and regulates chloroplast translation. Nat Commun 12

Bienvenut WV, et al (2020) Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acetylation. Mol Syst Biol 16: e9464


Giglione C, Meinnel T (2021) Evolution-driven versatility of N terminal acetylation in photoautotrophs. Trends Plant Sci in press, 10.1016/j.tplants.2020.11.012

Huber M, et al (2020) NatB-mediated N-terminal acetylation affects growth and abiotic stress responses. Plant Physiol 182: 792-806

Koskela MM, et al (2020) Comparative analysis of thylakoid protein complexes in state transition mutants nsi and stn7: focus on PSI and LHCII. Photosynth Res 145

Linster E, er al (2020) The Arabidopsis Na-acetyltransferase NAA60 locates to the plasma membrane and is vital for the high salt stress response. New Phytol 228

Linster E,et al (2021) The ribosome-associated N-terminal acetyltransferase A regulates proteome stability in plants in preparation

Miklánková P, et al (2021) HYPK affects proteostasis by facilitating co-translational imprinting of the proteome HYPK affects proteostasis by facilitating co-translational imprinting of the proteome with acetylation marks. In preparation

Weidenhausen J, et aI (2020) Structural and functional characterization of the N-terminal acetyltransferase Naa50. Structure in press

Westrich LD, et al (2020) The versatile interactome of chloroplast ribosomes revealed by affinity purification mass spectrometry. Nucleic Acids Res in press

Plants must constantly respond to a wide range of signals, including stresses, in order to coordinate their development and survival within a dynamic environment. One way in which this is achieved is through chemical modifications of proteins, allowing flexible and rapid changes of the proteome to alter cellular and physiological outputs. Protein acetylation is one such modification, which occurs on the N-termini (Nt) and internal lysines (K) of many proteins. Despite its prevalence, and in contrast to other wellstudied modifications (e.g., phosphorylation), our knowledge of: (i) the regulation, specificity and plasticity of protein acetylation, and (ii) its downstream functional consequences on protein activity and physiology are severely lacking, especially in plants. It is therefore extremely timely to elucidate the multifaceted functions of protein acetylation and open up this new area of plant molecular biology, in which Europe has the capacity to take a world lead through strategic ERA-CAPS funding.
The overarching aim of the KatNat project is to provide a mechanistic understanding of protein acetylation in plants, with a particular focus on investigating the enzymes that catalyze this modification (Nt- and K-acetyltransferases) and the resultant effects on proteostasis, photosynthesis, and metabolism. Crucially, this work will be carried out within the context of agronomically relevant stresses. KatNat consists of four interrelated objectives that will answer broad questions: (1) How does abiotic stress regulate the global Nt- and K-acetylome?; (2) What are the specificities, targets and stress-responsive dynamics of the acetyltransferases?; (3) How does protein acetylation impact protein stability and turnover?; (4) How does protein acetylation in plastids regulate photosynthesis and metabolism? By answering these connected questions, KatNat will not only shift the forefront of the field but will provide regulatory mechanisms and fundamental new insight into how plants sense and respond to environmental changes, which may become entered into textbooks. Last but not least, the obtained information will identify key new targets for the future development of superior crops.
The KatNat consortium brings together five European groups who all have a significant, demonstrable interest in the study of protein acetylation, and who have the highly complementary expertise in mass spectrometry, protein biochemistry and molecular plant biology required to carry out this original and competitive research at the highest international level. Consortium members already have a world lead in this field, and several members currently collaborate informally. The synergistic value of our collaboration will be the development and exploitation of an understudied area of in plant science, with key importance to agriculture. The proposed research is highly innovative, aligns closely with ERA-CAPS priority themes and has measurable and impactful outcomes that will shed light onto this emerging, exciting and important new area of plant molecular biology.