Cislaghi, A. P. | 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↗

bioRxiv2025

The Arabidopsis TNL immune receptor BNT1 localizes to the plastid envelope and mediates flg22-induced resistance against Pseudomonas

Precise localization and trafficking of plant immune receptors are critical for their function. We identify the TNL-class nucleotide-binding leucine-rich repeat receptor (NLR) BURNOUT1 (BNT1) from Arabidopsis thaliana as localized to plastids, key organelles for plant immunity. Alternative transcription start site usage generates two isoforms of BNT1: BNT1.2, which is targeted to the plastid envelope via an N-terminal signal-anchored mechanism, and BNT1.1, which resides in the cytoplasm. Moreover, BNT1.2 is predominantly expressed in epidermal cells, where it localizes to the so-called sensory plastids. Functional analysis revealed that bnt1 mutants exhibit compromised PAMP-triggered immunity (PTI) responses, including impaired callose deposition and reduced flg22-induced resistance to Pseudomonas syringae pv. tomato, while flg22-induced apoplastic reactive oxygen species production remains unaffected. Notably, only the plastid-localized BNT1.2 isoform is required for these PTI responses. Our findings reveal a role for NLRs in regulating PTI responses from plastids and highlight these organelles as key hubs for signal(s) integration during plant-pathogen interactions. Significance statementThis study identifies BNT1 as a TNL-class immune receptor localized to the plastid envelope. Two distinct isoforms of BNT1 were characterized: one with a plastid-targeting signal anchor that ensures plastid localization and another confined to the cytoplasm. Notably, only the plastid-localized isoform mediates PTI responses and confers resistance to Pseudomonas, highlighting the critical role of precise NLR localization and the central role of plastids in plant immunity.

plant biology↗