Reactive oxygen species (ROS)-dependent signaling pathways from chloroplasts and mitochondria merge at the nuclear protein RADICAL-INDUCED CELL DEATH1 (RCD1). RCD1 interacts in vivo and suppresses the activity of the transcription factors ANAC013 and ANAC017, which mediate a ROS-related retrograde signal originating from mitochondrial complex III. Inactivation of RCD1 leads to increased expression of mitochondrial dysfunction stimulon (MDS) genes regulated by ANAC013 and ANAC017. Accumulating MDS gene products, including alternative oxidases (AOXs), affect redox status of the chloroplasts, leading to changes in chloroplast ROS processing and increased protection of photosynthetic apparatus. ROS alter the abundance, thiol redox state and oligomerization of the RCD1 protein in vivo, providing feedback control on its function. RCD1-dependent regulation is linked to chloroplast signaling by 3'-phosphoadenosine 5'-phosphate (PAP). Thus, RCD1 integrates organellar signaling from chloroplasts and mitochondria to establish transcriptional control over the metabolic processes in both organelles.

Reactive oxygen species (ROS) are generated in electron transport processes of living organisms in oxygenic environments. Chloroplasts are plant bioenergetics hubs where imbalances between photosynthetic inputs and outputs drive ROS generation upon changing environmental conditions. Plants have harnessed various site-specific thylakoid membrane ROS products into environmental sensory signals. Our current understanding of ROS production in thylakoids suggests that oxygen (O₂) reduction takes place at numerous components of the photosynthetic electron transfer chain (PETC). To refine models of site-specific O₂ reduction capacity of various PETC components in isolated thylakoids of Arabidopsis thaliana, we quantified the stoichiometry of oxygen production and consumption reactions associated with hydrogen peroxide (H₂O₂) accumulation using membrane inlet mass spectrometry and specific inhibitors. Combined with P700 spectroscopy and electron paramagnetic resonance spin trapping, we demonstrate that electron flow to photosystem I (PSI) is essential for H₂O₂ accumulation during the photosynthetic linear electron transport process. Further leaf disc measurements provided clues that H₂O₂ from PETC has a potential of increasing mitochondrial respiration and CO₂ release. Based on gas exchange analyses in control, site-specific inhibitor-, methyl viologen-, and catalase-treated thylakoids, we provide compelling evidence of no contribution of plastoquinone pool or cytochrome b6f to chloroplastic H₂O₂ accumulation. The putative production of H₂O₂ in any PETC location other than PSI is rapidly quenched and therefore cannot function in H₂O₂ translocation to another cellular location or in signaling.

In plant science, 2,4-dinitrophenylether of iodonitrothymol (DNP-INT) is frequently used as an alternative to 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB) to examine the capacity of plastoquinol and semiquinone to reduce O₂. DNP-INT is considered to be an effective inhibitor of the photosynthetic electron transfer chain (PETC) through its binding at the Q₀ site of Cyt-b6f. The binding and action of DNP-INT has been previously characterized spectroscopically in purified Cyt-b6f complex reconstituted with Plastocyanin, PSII membranes and plastoquinone, as well as in isolated thylakoids based on its property to block MV-mediated O₂ consumption. Contrary to the conclusions obtained from these experiments, we observed clear reduction of P700⁺ in samples incubated with DNP-INT during our recent investigation into the sites of oxygen consumption in isolated thylakoids. Therefore, we carried out an extensive investigation of DNP-INT’s chemical efficacy in isolated thylakoids and intact leaves. This included examination of its capacity to block the PETC before PSI, and therefore its inhibition of CO₂ fixation. P700 redox kinetics were measured using Dual-PAM whilst Membrane Inlet Mass Spectrometry (MIMS) was used for simultaneous determination of the rates of O₂ evolution and O₂ consumption in isolated thylakoids and CO₂ fixation in intact leaves, using two stable isotopes of oxygen (¹⁶O₂, ¹⁸O₂) and CO₂ (¹²C, ¹³C), respectively. Based on these investigations we confirmed that DNP-INT is unable to completely block the PETC and CO₂ fixation, therefore its use may produce artifacts if applied to isolated thylakoids or intact cells, especially when determining the locations of reactive oxygen species formation in the photosynthetic apparatus.

Cyanobacteria perform oxygenic photosynthesis using an integrated network of photosynthetic, respiratory, and auxiliary electron transport pathways embedded within the thylakoid membrane. Understanding how electrons are dynamically distributed among these interacting processes and how these flows are regulated under fluctuating environmental conditions requires approaches that can probe electron transport in vivo. In this review, we summarize the current understanding of linear, cyclic, auxiliary, respiratory, and extracellular electron transport in model cyanobacteria and highlight recent insights into the mechanisms that maintain redox balance and protect the photosynthetic apparatus. We critically assess state-of-the-art techniques used to quantify electron transport in vivo, including chlorophyll fluorescence, microscopy, membrane inlet mass spectrometry, differential absorbance spectroscopy, electrochromic shift measurements, photoelectrochemistry, and electron paramagnetic resonance spectroscopy. Finally, to address the major outstanding questions in regulation of photosynthesis, we recommend integration of techniques for simultaneous measurement of multiple processes and identify a need for non-invasive probes and modeling to achieve a systems-level understanding of cyanobacterial bioenergetics. Further study of nonmodel species is also needed to understand the diversity of cyanobacterial photosynthesis.

Mitochondrial retrograde signals control expression of nuclear mitochondrial dysfunction stimulon (MDS) genes. Although MDS gene products mostly affect mitochondrial functions, they also influence production of reactive oxygen species (ROS) and redox status of chloroplasts. To study this inter-organellar interaction, we analysed the response of the Arabidopsis MDS-overexpressor mutant rcd1 to methyl viologen (MV), which catalyses electron transfer from Photosystem I (PSI) to molecular oxygen, generating ROS in Mehler’s reaction. The response of plants to MV was investigated by imaging chlorophyll fluorescence in aerobic and hypoxic environments, and by membrane inlet mass spectrometry. Hypoxic treatment abolished the effect of MV on photosynthetic electron transfer in rcd1 , but not in wild type. A similar reaction to hypoxia was observed in other MDS-activating lines and treatments. This suggests that MDS gene products contribute to oxygen depletion at the PSI electron-acceptor side. In unstressed growth conditions this MDS-related effect is likely masked by endogenous oxygen evolution and gas exchange with the atmosphere. In rcd1 , altered Mehler’s reaction coincided with more reduced state of the chloroplast NADPH-thioredoxin oxidoreductase C (NTRC) and its targets, suggesting that NTRC performs feedback control of photosynthesis. This regulation may represent a novel mechanism whereby mitochondrial retrograde signalling affects chloroplast functions.