Chloroplast protein acetyltransferases – novel players in the regulation of photosynthesis

Post-translational modifications (PTMs) of proteins such as phosphorylation have been shown to play pivotal roles in the regulation of photosynthesis. However, the study of these small modifications has long been hindered by methodological limitations. In recent years, advances in mass spectrometry methods have enabled the identification of a myriad of PTMs affecting proteins in all subcellular compartments. Especially interesting is the high prevalence of protein acetylation in the chloroplast, and more specifically in photosynthetic proteins. The acetylation machinery in the chloroplast consists of seven acetyltransferase enzymes that belong to the General control non-repressible 5-related N-acetyltransferase (GNAT) superfamily. The chloroplast-localized GNATs (GNAT1-5, GNAT7 and GNAT10) have been shown to catalyse two types of protein acetylation reactions: the addition of an acetyl group to the free N-terminus and the acetylation of an internal lysine residue of a protein. In addition, GNAT1 and GNAT2 function as metabolite acetyltransferases in the biosynthesis of melatonin. The presence of such a versatile group of plastid-localized GNAT acetyltransferases points to the importance of this PTM in the chloroplast. In this thesis, I have focused on elucidating the physiological role(s) of a group of the newly identified chloroplast GNAT acetyltransferases with a special focus on photosynthesis. My work has shown that GNAT2 is required for the regulation of excitation energy distribution between the photosystems through state transitions. Specifically, the formation of the PSI-LHCII complex is hindered in the gnat2 mutant, although no defects were detected in LHCII phosphorylation, which was previously considered to be the main determinant of state transitions. Additionally, GNAT2 was shown to be essential for the dynamic responses of the thylakoid membrane to changes in light conditions. GNAT1, GNAT2, GNAT4, GNAT7 and GNAT10 have a marked effect on the metabolome of Arabidopsis thaliana, especially on the accumulation of oxylipins, lipids and two acetylated amino acids. Finally, I have characterized two previously unknown thylakoid membrane proteins acetylated by GNAT2 and revealed their involvement in the dynamic adjustment of the light harvesting antenna of photosystem II.-- Entsyymien aktiivisuutta sekä proteiinien lokalisaatiota ja vuorovaikutuksia säädellään erilaisten proteiinien entsymaattisten muokkausten avulla. Fotosynteesireaktioiden säätelyssä proteiinien fosforylaatio on tärkein tunnettu proteiinimuokkaus, jonka on osoitettu olevan muun muassa valohaaviproteiinien ja valoenergian keräyksen säätelyn edellytys nopeasti muuttuvissa valo-olosuhteissa. Proteiinimuokkausten tutkimus on ollut menetelmällisesti haastavaa, mutta massaspektrometriamenetelmien kehitys viime vuosina on mahdollistanut lukuisten uusien proteiinimuokkausten tunnistamisen solun eri osissa. Proteiinien asetylaatiota esiintyy erityisen runsaasti kasvin viherhiukkasissa, mutta sen mahdollinen rooli fotosynteesin säätelijänä tunnetaan huonosti. Viherhiukkasessa asetylaatioreaktioita katalysoi seitsemän General control non-repressible 5-related N-asetyyltransferaasien (GNAT) -ryhmään kuuluvaa entsyymiä: GNAT1-5, GNAT7 sekä GNAT10. Tämän tutkimuksen tavoitteena on ollut selvittää viherhiukkasen GNAT-asetyylitransferaasien fysiologista roolia kasvissa, sekä erityisesti niiden vaikutusta fotosynteesireaktioihin. Väitöskirjatyössäni osoitan, että GNAT2-asetyyltransferaasi osallistuu fosforylaation ohella valon keräyksen säätelyyn ja viritysenergian tasapainotukseen. GNAT2 vaikuttaa myös merkittävästi tylakoidikalvoston rakenteeseen ja kalvoston rakenteen dynamiikkaan muuttuvissa valo-olosuhteissa. Osoitan myös, että proteiinien asetylaation lisäksi GNAT1-, GNAT2-, GNAT4-, GNAT7- ja GNAT10-entsyymit vaikuttavat huomattavasti lituruohon metabolomiin. Erityisesti oxylipiinien, antioksidanttina toimivan askorbaatin ja eräiden kalvolipidien määrät ovat muuttuneet tutkituissa gnat-mutanttilinjoissa. Lisäksi osoitan, että kaksi aiemmin tuntematonta kloroplastiproteiinia, ALM-A ja ALM-B, jotka ovat GNAT2-entsyymin mahdollisia kohdeproteiineja, osallistuvat fotosysteemi II:n valohaavin koon säätelyyn

Chloroplast Acetyltransferase GNAT2 is Involved in the Organization and Dynamics of Thylakoid Structure

Higher plants acclimate to changes in light conditions by adjusting the thylakoid membrane ultrastructure. Additionally, excitation energy transfer between photosystem II (PSII) and photosystem I (PSI) is balanced in a process known as state transition. These modifications are mediated by reversible phosphorylation of Lhcb1 and Lhcb2 proteins in different pools of light-harvesting complex (LHCII) trimers. Our recent study demonstrated that chloroplast acetyltransferase NUCLEAR SHUTTLE INTERACTING (NSI)/GNAT2 (general control non-repressible 5 (GCN5)-related N-acetyltransferase 2) is also needed for the regulation of light harvesting, evidenced by the inability of the gnat2 mutant to perform state transitions although there are no defects in LHCII phosphorylation. Here, we show that despite contrasting phosphorylation states of LHCII, grana packing in the gnat2 and state transition 7 (stn7) mutants possesses similar features, as the thylakoid structure of the mutants does not respond to the shift from darkness to light, which is in striking contrast to wild type (Wt). Circular dichroism and native polyacrylamide gel electrophoresis analyses further revealed that the thylakoid protein complex organization of gnat2 and stn7 resembles each other, but differ from that of Wt. Also, the location of the phosphorylated Lhcb2 as well as the LHCII antenna within the thylakoid network in gnat2 mutant is different from that of Wt. In gnat2, the LHCII antenna remains largely in grana stacks, where the phosphorylated Lhcb2 is found in all LHCII trimer pools, including those associated with PSII. These results indicate that in addition to phosphorylation-mediated regulation through STN7, the GNAT2 enzyme is involved in the organization and dynamics of thylakoid structure, probably through the regulation of chloroplast protein acetylation.</p

Root-type ferredoxin-NADP(+) oxidoreductase isoforms in Arabidopsis thaliana : Expression patterns, location and stress responses

In Arabidopsis, two leaf-type ferredoxin-NADP(+) oxidoreductase (LFNR) isoforms function in photosynthetic electron flow in reduction of NADP(+), while two root-type FNR (RFNR) isoforms catalyse reduction of ferredoxin in non-photosynthetic plastids. As the key to understanding, the function of RFNRs might lie in their spatial and temporal distribution in different plant tissues and cell types, we examined expression of RFNR1 and RFNR2 genes using beta-glucuronidase (GUS) reporter lines and investigated accumulation of distinct RFNR isoforms using a GFP approach and Western blotting upon various stresses. We show that while RFNR1 promoter is active in leaf veins, root tips and in the stele of roots, RFNR2 promoter activity is present in leaf tips and root stele, epidermis and cortex. RFNR1 protein accumulates as a soluble protein within the plastids of root stele cells, while RFNR2 is mainly present in the outer root layers. Ozone treatment of plants enhanced accumulation of RFNR1, whereas low temperature treatment specifically affected RFNR2 accumulation in roots. We further discuss the physiological roles of RFNR1 and RFNR2 based on characterization of rfnr1 and rfnr2 knock-out plants and show that although the function of these proteins is partly redundant, the RFNR proteins are essential for plant development and survival.Peer reviewe

Comparative analysis of thylakoid protein complexes in state transition mutants nsi and stn7: focus on PSI and LHCII

The photosynthetic machinery of plants can acclimate to changes in light conditions by balancing light-harvesting between the two photosystems (PS). This acclimation response is induced by the change in the redox state of the plastoquinone pool, which triggers state transitions through activation of the STN7 kinase and subsequent phosphorylation of light-harvesting complex II (LHCII) proteins. Phosphorylation of LHCII results in its association with PSI (state 2), whereas dephosphorylation restores energy allocation to PSII (state 1). In addition to state transition regulation by phosphorylation, we have recently discovered that plants lacking the chloroplast acetyltransferase NSI are also locked in state 1, even though they possess normal LHCII phosphorylation. This defect may result from decreased lysine acetylation of several chloroplast proteins. Here, we compared the composition of wild type (wt), stn7 and nsi thylakoid protein complexes involved in state transitions separated by Blue Native gel electrophoresis. Protein complex composition and relative protein abundances were determined by LC–MS/MS analyses using iBAQ quantification. We show that despite obvious mechanistic differences leading to defects in state transitions, no major differences were detected in the composition of PSI and LHCII between the mutants. Moreover, both stn7 and nsi plants show retarded growth and decreased PSII capacity under fluctuating light as compared to wt, while the induction of non-photochemical quenching under fluctuating light was much lower in both nsi mutants than in stn7.</p

Root-type ferredoxin-NADP(+) oxidoreductase isoforms in Arabidopsis thaliana: Expression patterns, location and stress responses

In Arabidopsis, two leaf-type ferredoxin-NADP(+) oxidoreductase (LFNR) isoforms function in photosynthetic electron flow in reduction of NADP(+), while two root-type FNR (RFNR) isoforms catalyse reduction of ferredoxin in non-photosynthetic plastids. As the key to understanding, the function of RFNRs might lie in their spatial and temporal distribution in different plant tissues and cell types, we examined expression of RFNR1 and RFNR2 genes using beta-glucuronidase (GUS) reporter lines and investigated accumulation of distinct RFNR isoforms using a GFP approach and Western blotting upon various stresses. We show that while RFNR1 promoter is active in leaf veins, root tips and in the stele of roots, RFNR2 promoter activity is present in leaf tips and root stele, epidermis and cortex. RFNR1 protein accumulates as a soluble protein within the plastids of root stele cells, while RFNR2 is mainly present in the outer root layers. Ozone treatment of plants enhanced accumulation of RFNR1, whereas low temperature treatment specifically affected RFNR2 accumulation in roots. We further discuss the physiological roles of RFNR1 and RFNR2 based on characterization of rfnr1 and rfnr2 knock-out plants and show that although the function of these proteins is partly redundant, the RFNR proteins are essential for plant development and survival.</p

The Arabidopsis NOT4A E3 ligase promotes PGR3 expression and regulates chloroplast translation

Chloroplast function requires the coordinated action of nuclear- and chloroplast-derived proteins, including several hundred nuclear-encoded pentatricopeptide repeat (PPR) proteins that regulate plastid mRNA metabolism. Despite their large number and importance, regulatory mechanisms controlling PPR expression are poorly understood. Here we show that the Arabidopsis NOT4A ubiquitin-ligase positively regulates the expression of PROTON GRADIENT REGULATION 3 (PGR3), a PPR protein required for translating several thylakoid-localised photosynthetic components and ribosome subunits within chloroplasts. Loss of NOT4A function leads to a strong depletion of cytochrome b6f and NAD(P)H dehydrogenase (NDH) complexes, as well as plastid 30 S ribosomes, which reduces mRNA translation and photosynthetic capacity, causing pale-yellow and slow-growth phenotypes. Quantitative transcriptome and proteome analysis of the not4a mutant reveal it lacks PGR3 expression, and that its molecular defects resemble those of a pgr3 mutant. Furthermore, we show that normal plastid function is restored to not4a through transgenic PGR3 expression. Our work identifies NOT4A as crucial for ensuring robust photosynthetic function during development and stress-response, through promoting PGR3 production and chloroplast translation.</p

Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acetylation

Protein acetylation is a highly frequent protein modification. However, comparatively little is known about its enzymatic machinery. N-alpha-acetylation (NTA) and epsilon-lysine acetylation (KA) are known to be catalyzed by distinct families of enzymes (NATs andKATs, respectively), although the possibility that the sameGCN5-relatedN-acetyltransferase (GNAT) can perform both functions has been debated. Here, we discovered a new family of plastid-localizedGNATs, which possess a dual specificity. All characterizedGNATfamily members display a number of unique features. Quantitative mass spectrometry analyses revealed that these enzymes exhibit both distinctKAand relaxedNTAspecificities. Furthermore, inactivation ofGNAT2 leads to significantNTAorKAdecreases of several plastid proteins, while proteins of other compartments were unaffected. The data indicate that these enzymes have specific protein targets and likely display partly redundant selectivity, increasing the robustness of the acetylation processin vivo. In summary, this study revealed a new layer of complexity in the machinery controlling this prevalent modification and suggests that other eukaryoticGNATs may also possess these previously underappreciated broader enzymatic activities

Localization of root-type ferredoxin-NADP+ oxidoreductases in Arabidopsis thaliana

Arabidopsis thaliana (Arabidopsis) genome encodes four plant-type ferredoxin-NADP+ oxidoreductases (FNRs) that mainly function in photosynthesis and anabolic metabolism. The two leaf-type FNRs present in chloroplasts are well characterized and operate in the last step of photosynthetic linear electron transfer by oxidizing ferredoxin and reducing NADP+ to NADPH. Two distinct root-type FNR isoforms, RFNR1 and RFNR2, function in the opposite direction producing reduced ferredoxin, which is further utilized in e.g. assimilation of nitrogen. Apart from a recent study showing that RFNR2 is required for nitrite detoxification, the functional specificities of the RFNR isoforms remain elusive. Determining the localization of the RFNR isoforms in distinct cell types and tissues could provide further insight into the biological roles of the isoforms. In this study, immunoblot analysis was used to determine tissue specific localization of the RFNR isoforms as well as to characterize mutants lacking functional RFNR1 or RFNR2. Additionally, tissue and cell specific localization of RFNR isoforms was studied by stably expressing YFP-tagged RFNRs in Arabidopsis. To achieve stable expression, binary vectors encoding YFP-tagged RFNRs were constructed and delivered into Arabidopsis by Agrobacterium tumefaciens mediated transformation. We have confirmed the presence of both RFNR isoforms in the roots and specific tissues of the shoot. Confocal imaging of Arabidopsis leaves expressing YFP-tagged RFNRs under a constitutively active 35S-promoter revealed chloroplastic localization of the fusion proteins suggesting that RFNRs are targeted to the plastids. Immunoblot analysis of samples from the rfnr mutant and wild type plants revealed no differences in the accumulation of proteins involved in nitrogen and carbon assimilation (GOGAT, NR, Rubisco, Rubisco activase)

Chloroplast Acetyltransferase GNAT2 is Involved in the Organization and Dynamics of Thylakoid Structure

Higher plants acclimate to changes in light conditions by adjusting the thylakoid membrane ultrastructure. Additionally, excitation energy transfer between photosystem II (PSII) and photosystem I (PSI) is balanced in a process known as state transition. These modifications are mediated by reversible phosphorylation of Lhcb1 and Lhcb2 proteins in different pools of light-harvesting complex (LHCII) trimers. Our recent study demonstrated that chloroplast acetyltransferase NUCLEAR SHUTTLE INTERACTING (NSI)/GNAT2 (general control non-repressible 5 (GCN5)-related N-acetyltransferase 2) is also needed for the regulation of light harvesting, evidenced by the inability of the gnat2 mutant to perform state transitions although there are no defects in LHCII phosphorylation. Here, we show that despite contrasting phosphorylation states of LHCII, grana packing in the gnat2 and state transition 7 (stn7) mutants possesses similar features, as the thylakoid structure of the mutants does not respond to the shift from darkness to light, which is in striking contrast to wild type (Wt). Circular dichroism and native polyacrylamide gel electrophoresis analyses further revealed that the thylakoid protein complex organization of gnat2 and stn7 resembles each other, but differ from that of Wt. Also, the location of the phosphorylated Lhcb2 as well as the LHCII antenna within the thylakoid network in gnat2 mutant is different from that of Wt. In gnat2, the LHCII antenna remains largely in grana stacks, where the phosphorylated Lhcb2 is found in all LHCII trimer pools, including those associated with PSII. These results indicate that in addition to phosphorylation-mediated regulation through STN7, the GNAT2 enzyme is involved in the organization and dynamics of thylakoid structure, probably through the regulation of chloroplast protein acetylation