Loss of Chloroplast GNAT Acetyltransferases Results in Distinct Metabolic Phenotypes in Arabidopsis.

Acetylation is one of the most common chemical modifications found on a variety of molecules ranging from metabolites to proteins. Although numerous chloroplast proteins have been shown to be acetylated, the role of acetylation in the regulation of chloroplast functions has remained mainly enigmatic. The chloroplast acetylation machinery in Arabidopsis thaliana consists of eight General control non-repressible 5 (GCN5)-related N-acetyltransferase (GNAT)-family enzymes that catalyze both N-terminal and lysine acetylation of proteins. Additionally, two plastid GNATs have also been reported to be involved in the biosynthesis of melatonin. Here, we have characterized six plastid GNATs (GNAT1, GNAT2, GNAT4, GNAT6, GNAT7 and GNAT10) using a reverse genetics approach with an emphasis on the metabolomes and photosynthesis of the knock-out plants. Our results reveal the impact of GNAT enzymes on the accumulation of chloroplast-related compounds, such as oxylipins and ascorbate, and the GNAT enzymes also affect the accumulation of amino acids and their derivatives. Specifically, the amount of acetylated arginine and proline was significantly decreased in the gnat2 and gnat7 mutants, respectively, as compared to the wild-type Col-0 plants. Additionally, our results show that the loss of the GNAT enzymes results in increased accumulation of Rubisco and Rubisco activase (RCA) at the thylakoids. Nevertheless, the reallocation of Rubisco and RCA did not have consequent effects on carbon assimilation under the studied conditions. Taken together, our results show that chloroplast GNATs affect diverse aspects of plant metabolism and pave way for future research into the role of protein acetylation.

High-light-inducible proteins HliA and HliB: pigment binding and protein-protein interactions.

High-light-inducible proteins (Hlips) are single-helix transmembrane proteins that are essential for the survival of cyanobacteria under stress conditions. The model cyanobacterium Synechocystis sp. PCC 6803 contains four Hlip isoforms (HliA-D) that associate with Photosystem II (PSII) during its assembly. HliC and HliD are known to form pigmented (hetero)dimers that associate with the newly synthesized PSII reaction center protein D1 in a configuration that allows thermal dissipation of excitation energy. Thus, it is expected that they photoprotect the early steps of PSII biogenesis. HliA and HliB, on the other hand, bind the PSII inner antenna protein CP47, but the mode of interaction and pigment binding have not been resolved. Here, we isolated His-tagged HliA and HliB from Synechocystis and show that these two very similar Hlips do not interact with each other as anticipated, rather they form HliAC and HliBC heterodimers. Both dimers bind Chl and ß-carotene in a quenching conformation and associate with the CP47 assembly module as well as later PSII assembly intermediates containing CP47. In the absence of HliC, the cellular levels of HliA and HliB were reduced, and both bound atypically to HliD. We postulate a model in which HliAC-, HliBC-, and HliDC-dimers are the functional Hlip units in Synechocystis. The smallest Hlip, HliC, acts as a 'generalist' that prevents unspecific dimerization of PSII assembly intermediates, while the N-termini of 'specialists' (HliA, B or D) dictate interactions with proteins other than Hlips.

Plant LHC-like proteins show robust folding and static non-photochemical quenching.

Life on Earth depends on photosynthesis, the conversion of light energy into chemical energy. Plants collect photons by light harvesting complexes (LHC)-abundant membrane proteins containing chlorophyll and xanthophyll molecules. LHC-like proteins are similar in their amino acid sequence to true LHC antennae, however, they rather serve a photoprotective function. Whether the LHC-like proteins bind pigments has remained unclear. Here, we characterize plant LHC-like proteins (LIL3 and ELIP2) produced in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Both proteins were associated with chlorophyll a (Chl) and zeaxanthin and LIL3 was shown to be capable of quenching Chl fluorescence via direct energy transfer from the Chl Qy state to zeaxanthin S1 state. Interestingly, the ability of the ELIP2 protein to quench can be acquired by modifying its N-terminal sequence. By employing Synechocystis carotenoid mutants and site-directed mutagenesis we demonstrate that, although LIL3 does not need pigments for folding, pigments stabilize the LIL3 dimer.