A characteristic feature of most plants is their ability to perform photosynthesis, which ultimately provides energy and organic substrates to most life. Photosynthesis dominates chloroplast physiology but represents only a fraction of the tightly interconnected metabolic network that spans the entire cell. Here, we explore how photosynthetic activity affects the energy physiological status in cell compartments beyond the chloroplast. We develop precision live monitoring of subcellular energy physiology under illumination to investigate pH, MgATP2- and NADH/NAD+ dynamics at dark-light transitions by confocal imaging of genetically encoded fluorescent protein biosensors in Arabidopsis thaliana leaf mesophyll. We resolve the in vivo signature of stromal alkalinisation resulting from photosynthetic proton pumping and observe a similar pH signature also in the cytosol and the mitochondria suggesting that photosynthesis triggers an 'alkalinisation wave' that affects the pH landscape of large parts of the cell. MgATP2- increases in the stroma at illumination, but no major effects on MgATP2- concentrations in the cytosol were resolved. Photosynthetic activity triggers a signature of substantial NAD reduction in the cytosol that is driven by photosynthesis-derived electron export. Strikingly, cytosolic NAD redox status was deregulated in mutants of chloroplastic NADP- and mitochondrial NAD-dependent malate dehydrogenases even at darkness, pinpointing the participation of the chloroplasts and mitochondria in shaping cytosolic redox metabolism in vivo with a dominant function of malate metabolism. Our data illustrate how profoundly and rapidly changes in photosynthetic activity affect the physiological and metabolic landscape throughout green plant cells.
Post-translational modifications (PTMs) of proteins play important roles in the acclimation of plants to environmental stress. Lysine acetylation is a dynamic and reversible PTM, which can be removed by histone deacetylases. Here we investigated the role of lysine acetylation in the response of Arabidopsis leaves to 1 week of salt stress. A quantitative mass spectrometry analysis revealed an increase in lysine acetylation of several proteins from cytosol and plastids, which was accompanied by altered histone deacetylase activities in the salt-treated leaves. While activities of HDA14 and HDA15 were decreased upon salt stress, HDA5 showed a mild and HDA19 a strong increase in activity. Since HDA5 is a cytosolic-nuclear enzyme from the class II histone deacetylase family with yet unknown protein substrates, we performed a lysine acetylome analysis on hda5 mutants and characterized its substrate proteins. Next to histone H2B, the salt stressresponsive transcription factor GT2L and the dehydration-related protein ERD7 were identified as HDA5 substrates. In addition, in protein-protein interaction studies, HDA18 was discovered, among other interacting proteins, to work in a complex together with HDA5. Altogether, this study revealed the substrate proteins of HDA5 and identified new lysine acetylation sites which are hyperacetylated upon salt stress. The identification of specific histone deacetylase substrate proteins, apart from histones, will be important to unravel the acclimation response of Arabidopsis to salt stress and their role in plant physiology.
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