Proteomic investigation of Synechocystis SP. PCC 6803: S/T/Y phosphorylation and response of proteins to bioengineering tools based on the copper-regulated PETJ promoter

Cyanobacteria are unique prokaryotic organisms performing oxygenic photosynthesis. Their ability to transform sunlight and CO2 to sugar, together with the flexibility of metabolism, have made cyanobacteria interesting organisms for engineering the carbon and electrons fluxes toward exogenous pathways for the production of high value compounds. Understanding the complex mechanisms regulating photosynthesis and their dynamic interactions with cellular metabolism are crucial in order to improve the productivity of engineered strains. In this Thesis, reversible O-type phosphorylation on S/T/Y amino acids, a ubiquitous posttranslational regulatory mechanism, was investigated in Synechocystis 6803 using proteomics approaches. A global discovery-driven investigation of the phosphoproteome revealed that S/T/Y phosphorylation is extensive in Synechocystis 6803, and phosphoproteins are involved in various metabolic pathways. A large share of the identified phosphoproteins participate in photosynthesis-related processes. The quantitative targeted mass spectrometry method was set-up for (phospho)peptides of photosynthesis-related proteins. It was applied for screening a collection of S/T protein kinase mutants in Synechocystis 6803. The results revealed an intricate phosphoprotein-protein kinase network, including an interplay among several kinases and auxiliary proteins. In particular, the SpkG kinase was shown to phosphorylate the Fd5 protein, while Slr051 was revealed as an auxiliary protein regulating the balance between the phosphorylated and nonphosphorylated forms of Fd5. Moreover, the deletion of the SpkG kinase caused the induction of phosphorylation in several other peptides indicating a cross talk among the participants of the protein phosphorylation network. Further, the data obtained in this Thesis showed that the induction of exogenous pathways in synthetic biology approaches might cause background proteome changes due to the acclimation of the cells to the concentration of metal ions. Particularly, the Cu2+ treatment, used to regulate the petJ promoter, irreversibly affected DNA replication, transcription and translation machineries, cell wall proteins, transporters, signaling proteins and enzymes involved in lipid biosynthesis. These changes might consequently affect the expression of a pathway or the recovery of a high value product. In summary, the results presented in this Thesis provide a better understanding of Synechocystis 6803 regulatory mechanisms, which might support the optimization of engineered strains toward maximal productivity of high value compounds

Evolutionary conservation and post-translational control of S-adenosyl-L-homocysteine hydrolase in land plants

Trans-methylation reactions are intrinsic to cellular metabolism in all living organisms. In land plants, a range of substrate-specific methyltransferases catalyze the methylation of DNA, RNA, proteins, cell wall components and numerous species-specific metabolites, thereby providing means for growth and acclimation in various terrestrial habitats. Trans-methylation reactions consume vast amounts of S-adenosyl-L-methionine (SAM) as a methyl donor in several cellular compartments. The inhibitory reaction by-product, S-adenosyl-L-homocysteine (SAH), is continuously removed by SAH hydrolase (SAHH), which essentially maintains trans-methylation reactions in all living cells. Here we report on the evolutionary conservation and post-translational control of SAHH in land plants. We provide evidence suggesting that SAHH forms oligomeric protein complexes in phylogenetically divergent land plants and that the predominant protein complex is composed by a tetramer of the enzyme. Analysis of light-stress-induced adjustments of SAHH in Arabidopsis thaliana and Physcomitrella patens further suggests that regulatory actions may take place on the levels of protein complex formation and phosphorylation of this metabolically central enzyme. Collectively, these data suggest that plant adaptation to terrestrial environments involved evolution of regulatory mechanisms that adjust the trans-methylation machinery in response to environmental cues.Peer reviewe

A Young Algaeneers' perspective: Communication and networking are key to successful multidisciplinary research

In this Letter, we report the outcomes of the third biannual Young Algaeneers Symposium (YAS) that took place in April 2016 in Qawra, Malta. We are reviewing the importance of interdisciplinary communication and benefits of discussion panels. By communicating our experience of hosting YAS2016, we would like to encourage and instill scientific exchange amongst the new generation of scientists and suggest solutions to various problems arising from a lack of mutual understanding. (C) 2016 Elsevier B. V. All rights reserved

ACONITASE 3 is part of the ANAC017 transcription factor-dependent mitochondrial dysfunction response

Mitochondria are tightly embedded within metabolic and regulatory networks that optimize plant performance in response to environmental challenges. The best-known mitochondrial retrograde signaling pathway involves stress-induced activation of the transcription factor NAC DOMAIN CONTAINING PROTEIN 17 (ANAC017), which initiates protective responses to stress-induced mitochondrial dysfunction in Arabidopsis (Arabidopsis thaliana). Post-translational control of the elicited responses, however, remains poorly understood. Previous studies linked protein phosphatase 2A subunit PP2A-B’γ, a key negative regulator of stress responses, with reversible phosphorylation of ACONITASE 3 (ACO3). Here we report on ACO3 and its phosphorylation at Ser91 as key components of stress regulation that are induced by mitochondrial dysfunction. Targeted mass spectrometry-based proteomics revealed that the abundance and phosphorylation of ACO3 increased under stress, which required signaling through ANAC017. Phosphomimetic mutation at ACO3-Ser91 and accumulation of ACO3S91D-YFP promoted the expression of genes related to mitochondrial dysfunction. Furthermore, ACO3 contributed to plant tolerance against UV-B or antimycin A-induced mitochondrial dysfunction. These findings demonstrate that ACO3 is both a target and mediator of mitochondrial dysfunction signaling, and critical for achieving stress tolerance in Arabidopsis leaves.</p

In the lycophyte Selaginella martensii is the "extra-qT" related to energy spillover? Insights into photoprotection in ancestral vascular plants

Lycophytes are early diverging vascular plants, representing a minor group as compared to the dominating euphyllophytes, mostly angiosperms. Having maximally developed in a CO2-rich atmosphere, extant lycophytes are characterized by a low carbon fixing capacity, which is compensated by a marked ability to induce the non-photochemical quenching of chlorophyll fluorescence (NPQ). Different kinetic components contribute to NPQ, in particular the fast relaxing high-energy quenching qE, the middle relaxing qT, and the slowly relaxing qI. Unlike angiosperms, lycophytes enhance the qT component under high light, originating from an "extra-qT". In this research, we analyze whether in Selaginella martensii the extra-qT can reflect a photosystem (PS) I-based quenching mechanism activated upon saturation of qE capacity. From comparative analyses of fluorescence quenching parameters, carbon fixation, in vivo low- and room-temperature fluorescence spectroscopy, and thylakoid protein phosphorylation, it is proposed that the extra-qT is not mechanistically separate from the ordinary qT. The results suggest a relationship between qT and photoprotective energy spillover to PSI, which is activated upon sensing the excitation energy pressure inside PSII and is possibly facilitated by phosphorylation of Lhcb6, a minor antenna protein of PSII. Energy spillover emphasizes 77K fluorescence emission from PSI core (F714) and becomes more relevant at irradiance levels corresponding to the CO2-limited, potentially photoinhibiting phase of photosynthesis. At the highest irradiances, when Lhcb6 phosphorylation potential has been saturated, the major LHCII increases in turn its phosphorylation level, probably leading to the full exploitation of PSI as a safe excitation sink. It is suggested that the low photosynthetic capacity of lycophytes could allow an easier experimental access to the use of PSI as a safe excitation quencher for PSII, a debated, emerging issue about thylakoid photoprotection in angiosperms

Study of <i>O</i>‑Phosphorylation Sites in Proteins Involved in Photosynthesis-Related Processes in <i>Synechocystis</i> sp. Strain PCC 6803: Application of the SRM Approach

<i>O</i>-Phosphorylation has been shown in photosynthesis-related proteins in a cyanobacterium <i>Synechocystis</i> sp. strain PCC 6803 (thereafter <i>Synechocystis</i> 6803), suggesting that phosphorylation of S, T, and Y residues might be important in photosynthesis-related processes. Investigation of biological roles of these phosphorylation events requires confident knowledge of the phosphorylated sites and prospects for their individual assessment. We performed phosphoproteomic analysis of <i>Synechocystis</i> 6803 using TiO₂ enrichment of the phosphopeptides, followed by LC–MS/MS, and discovered 367 phosphorylation sites in 190 proteins participating in various cellular functions. Furthermore, we focused on the large group of phosphoproteins that are involved in light harvesting, photosynthesis-driven electron flow, photoprotection, and CO₂ fixation. The SRM approach was applied to verify/improve assignments of phosphorylation sites in these proteins and to investigate possibilities for analysis of phosphopeptide isomers. The SRM assays were designed for peptides comprising 45 phosphorylation sites. The assays contain peptide iRT values and Q1/Q3 transitions comprising those discriminating between phosphopeptide isoforms. The majority of investigated phosphopeptides and phosphorylated isoforms could be individually assessed with the SRM technique. The assays could be potentially used in future quantitative studies to evaluate an extent of phosphorylation in photosynthesis-related proteins in <i>Synechocystis</i> 6803 cells challenged with various environmental stresses

ACONITASE 3 is part of the ANAC017 transcription factor-dependent mitochondrial dysfunction response

Mitochondria are tightly embedded within metabolic and regulatory networks that optimize plant performance in response to environmental challenges. The best-known mitochondrial retrograde signaling pathway involves stress-induced activation of the transcription factor NAC DOMAIN CONTAINING PROTEIN 17 (ANAC017), which initiates protective responses to stress-induced mitochondrial dysfunction in Arabidopsis (Arabidopsis thaliana). Posttranslational control of the elicited responses, however, remains poorly understood. Previous studies linked protein phosphatase 2A subunit PP2A-B'gamma, a key negative regulator of stress responses, with reversible phosphorylation of ACONITASE 3 (ACO3). Here we report on ACO3 and its phosphorylation at Ser91 as key components of stress regulation that are induced by mitochondrial dysfunction. Targeted mass spectrometry-based proteomics revealed that the abundance and phosphorylation of ACO3 increased under stress, which required signaling through ANAC017. Phosphomimetic mutation at ACO3-Ser91 and accumulation of ACO3(S91D)-YFP promoted the expression of genes related to mitochondrial dysfunction. Furthermore, ACO3 contributed to plant tolerance against ultraviolet B (UV-B) or antimycin A-induced mitochondrial dysfunction. These findings demonstrate that ACO3 is both a target and mediator of mitochondrial dysfunction signaling, and critical for achieving stress tolerance in Arabidopsis leaves.Peer reviewe

Light-dependent reversible phosphorylation of the minor photosystem II antenna Lhcb6 (CP24) occurs in lycophytes

Evolution of vascular plants required compromise between photosynthesis and photodamage. We analyzed representative species from two divergent lineages of vascular plants, lycophytes and euphyllophytes, with respect to the response of their photosynthesis and light-harvesting properties to increasing light intensity. In the two analyzed lycophytes, Selaginella martensii and Lycopodium squarrosum, the medium phase of non-photochemical quenching relaxation increased under high light compared to euphyllophytes. This was thought to be associated with the occurrence of a further thylakoid phosphoprotein in both lycophytes, in addition to D2, CP43 and Lhcb1-2. This protein, which showed light intensity-dependent reversible phosphorylation, was identified in S. martensii as Lhcb6, a minor LHCII antenna subunit of PSII. Lhcb6 is known to have evolved in the context of land colonization. In S. martensii, Lhcb6 was detected as a component of the free LHCII assemblies, but also associated with PSI. Most of the light-induced changes affected the amount and phosphorylation of the LHCII assemblies, which possibly mediate PSI-PSII connectivity. We propose that Lhcb6 is involved in light energy management in lycophytes, participating in energy balance between PSI and PSII through a unique reversible phosphorylation, not yet observed in other land plants