Buchert, F. E. | Search bioRxiv

bioRxiv2025

Advanced illumination-imaging reveals photosynthesis-triggered pH, ATP and NAD redox signatures across plant cell compartments

Photosynthesis provides energy and organic substrates to most life. In plants, 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 energy physiology within and beyond the chloroplast. We developed a new standard for the live-monitoring of subcellular energy physiology by combining confocal imaging of genetically encoded fluorescent protein biosensors with advanced on-stage illumination technology to investigate pH, MgATP2- and NADH/NAD+ dynamics at dark-light transitions in Arabidopsis mesophyll cells. Our findings reveal a stromal alkalinization signature induced by photosynthetic proton pumping, extending to the cytosol and mitochondria as an alkalinization wave. Photosynthesis leads to increased MgATP2- levels in both the stroma and cytosol. Additionally, we observed reduction of the NAD pool driven by photosynthesis-derived electron export. Arabidopsis lines defective in chloroplast NADP- and mitochondrial NAD-dependent malate dehydrogenases show more reduced cytosolic NAD redox status even in darkness, highlighting the involvement of chloroplasts and mitochondria in shaping cytosolic redox metabolism via malate metabolism. Our study sets a novel methodological standard for precision live-monitoring of photosynthetic cell physiology. Applying this technology reveals signatures of photosynthetic physiology within and beyond the chloroplast with unprecedented resolution. Those signatures link photosynthetic activity and the fundamental biochemical functions of phototrophic cells. Significance statementBy applying novel live microscopy monitoring using fluorescent protein biosensors in plant cells, we reveal that dark-light transitions trigger profound re-orchestration of subcellular pH, ATP and NAD redox physiology not limited to chloroplasts but extending into the cytosol and the mitochondria.

plant biology↗

bioRxiv2021

The algal PETC-Pro171-Leu suppresses electron transfer in the cytochrome b6f under acidic lumenal condition

Linear photosynthetic electron flow (LEF) produces NADPH and generates a proton electrochemical potential gradient across the thylakoid membrane used to synthesize ATP, both of which are required for CO2 fixation. As cellular demand for ATP and NADPH are variable, cyclic electron flow (CEF) between PSI and cytochrome b6f complex (b6f) produces extra ATP. The b6f regulates LEF and CEF via photosynthetic control, which is a pH-dependent b6f slowdown of plastoquinol oxidation at the lumenal site. This protection mechanism is triggered at more alkaline lumen pH in the pgr1 mutant of the vascular plant Arabidopsis thaliana, carrying Pro194Leu in the b6f Rieske Iron-sulfur protein. In this work, we introduced pgr1 mutation in the green alga Chlamydomonas reinhardtii (PETC-P171L). Consistent with pgr1 phenotype, PETC-P171L displayed an impaired NPQ induction along with slower photoautotrophic growth under high light conditions. Our data provides evidence that the {Delta}pH component in PETC-P171L is dependent on oxygen availability. Only under low oxygen conditions the {Delta}pH component was sufficient to trigger a phenotype in algal PETC-P171L where the mutant b6f was more restricted to oxidize the PQ pool and showed a diminished electron flow through the b6f complex. One sentence summaryChange of PETC to P171L via site directed mutagenesis alters the pH dependency of the photosynthetic control mechanism

plant biology↗