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Summary: Hydrogen peroxide (H2O2) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H2O2 notoriously hard to detect dynamically, specifically and at high resolution. Genetically encoded sensors overcome persistent shortcomings, but pH sensitivity, silencing of expression and a limited concept of sensor behaviour in vivo have hampered any meaningful H2O2 sensing in living plants.We established H2O2 monitoring in the cytosol and the mitochondria of Arabidopsis with the fusion protein roGFP2‐Orp1 using confocal microscopy and multiwell fluorimetry.We confirmed sensor oxidation by H2O2, show insensitivity to physiological pH changes, and demonstrated that glutathione dominates sensor reduction in vivo. We showed the responsiveness of the sensor to exogenous H2O2, pharmacologically‐induced H2O2 release, and genetic interference with the antioxidant machinery in living Arabidopsis tissues. Monitoring intracellular H2O2 dynamics in response to elicitor exposure reveals the late and prolonged impact of the oxidative burst in the cytosol that is modified in redox mutants.We provided a well defined toolkit for H2O2 monitoring in planta and showed that intracellular H2O2 measurements only carry meaning in the context of the endogenous thiol redox systems. This opens new possibilities to dissect plant H2O2 dynamics and redox regulation, including intracellular NADPH oxidase‐mediated ROS signalling. See also the Commentary on this article by McLachlan, 221: 1180–1181. [ABSTRACT FROM AUTHOR]

The unfolded protein response (UPR) is an ancient signaling pathway designed to protect cells from the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER). Because misregulation of the UPR is potentially lethal, a stringent surveillance signaling system must be in place to modulate the UPR. The major signaling arms of the plant UPR have been discovered and rely on the transcriptional activity of the transcription factors bZIP60 and bZIP28 and on the kinase and ribonuclease activity of IRE1, which splices mRNA to activate bZIP60. Both bZIP28 and bZIP60 modulate UPR gene expression to overcome ER stress. In this study, we demonstrate at a genetic level that the transcriptional role of bZIP28 and bZIP60 in ER-stress responses is antagonized by nonexpressor of PR1 genes 1 (NPR1), a critical redox-regulated master regulator of salicylic acid (SA)-dependent responses to pathogens, independently of its role in SA defense. We also establish that the function of NPR1 in the UPR is concomitant with ER stress-induced reduction of the cytosol and translocation of NPR1 to the nucleus where it interacts with bZIP28 and bZIP60. Our results support a cellular role for NPR1 as well as a model for plant UPR regulation whereby SA-independent ER stress-induced redox activation of NPR1 suppresses the transcriptional role of bZIP28 and bZIP60 in the UPR. [ABSTRACT FROM AUTHOR]

The unfolded protein response ( UPR) is an ancient signaling pathway that commits to life-or-death outcomes in response to proteotoxic stress in the endoplasmic reticulum ( ER). In plants, the membrane-tethered transcription factor bZIP28 and the ribonuclease-kinase IRE1 along with its splicing target, bZIP60, govern the two cytoprotective UPR signaling pathways known to date. The conserved ER membrane-associated BAX inhibitor 1 ( BI1) modulates ER stress-induced programmed cell death through yet-unknown mechanisms. Despite the significance of the UPR for cell homeostasis, in plants the regulatory circuitry underlying ER stress resolution is still largely unmapped. To gain insights into the coordination of plant UPR strategies, we analyzed the functional relationship of the UPR modulators through the analysis of single and higher order mutants of IRE1, bZIP60, bZIP28 and BI1 in experimental conditions causing either temporary or chronic ER stress. We established a functional duality of bZIP28 and bZIP60, as they exert partially independent tissue-specific roles in recovery from ER stress, but redundantly actuate survival strategies in chronic ER stress. We also discovered that BI1 attenuates the pro-survival function of bZIP28 in ER stress resolution and, differently to animal cells, it does not temper the ribonuclease activity of inositol-requiring enzyme 1 ( IRE1) under temporary ER stress. Together these findings reveal a functional independence of bZIP28 and bZIP60 in plant UPR, and identify an antagonizing role of BI1 in the pro-adaptive signaling mediated by bZIP28, bringing to light the distinctive complexity of the unfolded protein response ( UPR) in plants. [ABSTRACT FROM AUTHOR]

Completion of a plant's life cycle depends on successful prioritization of signaling favoring either growth or defense. Although hormones are pivotal regulators of growth-defense tradeoffs, the underlying signaling mechanisms remain obscure. The unfolded protein response (UPR) is essential for physiological growth as well as management of endoplasmic reticulum (ER) stress in unfavorable growth conditions. The plant UPR transducers are the kinase and ribonuclease IRE1 and the transcription factors bZIP28 and bZIP60. We analyzed management of the tradeoff between growth and ER stress defense by the stress response hormone salicylic acid (SA) and the UPR, which is modulated by SA via unknown mechanisms. We show that the plant growth and stress regulator CPR5, which represses accumulation of SA, favors growth in physiological conditions through inhibition of the SA-dependent IRE1-bZIP60 arm that antagonizes organ growth; CPR5 also favors growth in stress conditions through repression of ER stress-induced bZIP28/IRE1-bZIP60 arms. By demonstrating a physical interaction of CPR5 with bZIP60 and bZIP28, we provide mechanistic insights into CPR5-mediated modulation of UPR signaling. These findings define a critical surveillance strategy for plant growth-ER stress defense tradeoffs based on CPR5 and SA-modulated UPR signaling, whereby CPR5 acts as a positive modulator of growth in physiological conditions and in stress by antagonizing SA-dependent growth inhibition through UPR modulation. [ABSTRACT FROM AUTHOR]

The interaction between legumes and arbuscular mycorrhizal ( AM) fungi is vital to the development of sustainable plant production systems. Here, we focus on a putative MYB-like ( Lj MAMI) transcription factor ( TF) previously reported to be highly upregulated in Lotus japonicus mycorrhizal roots. Phylogenetic analyses revealed that the protein is related to a group of TFs involved in phosphate ( Pi) starvation responses, the expression of which is independent of the Pi level, such as PHR1. GUS transformed plants and quantitative reverse transcription PCR revealed strong gene induction in arbusculated cells, as well as the presence of Lj MAMI transcripts in lateral root primordia and root meristems, even in the absence of the fungus, and independently of Pi concentration. In agreement with its putative identification as a TF, an eGFP- Lj MAMI chimera was localized to the nuclei of plant protoplasts, whereas in transgenic Lotus roots expressing the eGFP- Lj MAMI fusion protein under the control of the native promoter, the protein was located in the nuclei of the arbusculated cells. Further expression analyses revealed a correlation between Lj MAMI and Lj PT4, a marker gene for mycorrhizal function. To elucidate the role of the Lj MAMI gene in the mycorrhizal process, RNAi and overexpressing root lines were generated. All the lines retained their symbiotic capacity; however, RNAi root lines and composite plants showed an important reduction in root elongation and branching in the absence of the symbiont. The results support the involvement of the AM-responsive Lj MAMI in non-symbiotic functions: i.e. root growth. [ABSTRACT FROM AUTHOR]

The molecular mechanisms responsible for the heightened reactivity of quiescent T cells in human early life remain largely elusive. Our previous research identified that quiescent adult naïve CD4+ T cells express LINE1 (long interspersed nuclear elements 1) spliced in previously unknown isoforms, and their down-regulation marks the transition to activation. Here, we unveil that neonatal naïve T cell quiescence is characterized by enhanced energy production and protein synthesis. This phenotype is associated with the absence of LINE1 expression attributed to tonic T cell receptor/mTOR complex 1 (mTORC1) signaling and (polypyrimidine tract-binding protein 1 (PTBP1)-mediated LINE1 splicing suppression. The absence of LINE1 expression primes these cells for rapid execution of the activation program by directly regulating protein synthesis. LINE1 expression progressively increases in childhood and adults, peaking in elderly individuals, and, by decreasing protein synthesis, contributes to immune senescence in aging. Our study proposes LINE1 as a critical player of human T cell function across the human life span. [ABSTRACT FROM AUTHOR]

The unfolded protein response (UPR) is a highly regulated signaling pathway that is largely conserved across eukaryotes. It is essential for cell homeostasis under environmental and physiological conditions that perturb the protein folding in the endoplasmic reticulum (ER). Arabidopsis is one of the outstanding multicellular model systems in which to investigate the UPR. Here, we described a protocol to induce the UPR in plants, specifically Arabidopsis, and to estimate their ability to cope with ER stress through the quantification of physiological parameters., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Transient transformation assays for the analysis of protein localization are routinely used as rapid and convenient alternatives to stable transformation. In this chapter, we describe two transient gene expression assays (e.g., isolation and transformation of protoplasts, and agroinfiltration of leaves) optimized for Arabidopsis thaliana, and we combine them with fluorescence microscopy, with the final aim to investigate in vivo the subcellular localization of a mitochondrial protein of interest fused to a fluorescent reporter., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Mitochondria host vital cellular functions, including oxidative phosphorylation and co-factor biosynthesis, which are reflected in their proteome. At the cellular level plant mitochondria are organized into hundreds of discrete functional entities, which undergo dynamic fission and fusion. It is the individual organelle that operates in the living cell, yet biochemical and physiological assessments have exclusively focused on the characteristics of large populations of mitochondria. Here, we explore the protein composition of an individual average plant mitochondrion to deduce principles of functional and structural organisation. We perform proteomics on purified mitochondria from cultured heterotrophic Arabidopsis cells with intensity-based absolute quantification and scale the dataset to the single organelle based on criteria that are justified by experimental evidence and theoretical considerations. We estimate that a total of 1.4 million protein molecules make up a single Arabidopsis mitochondrion on average. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40 000 for Voltage-Dependent Anion Channel 1 to sub-stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat proteins that modify mitochondrial transcripts. For our analysis, we consider the physical and chemical constraints of the single organelle and discuss prominent features of mitochondrial architecture, protein biogenesis, oxidative phosphorylation, metabolism, antioxidant defence, genome maintenance, gene expression, and dynamics. While assessing the limitations of our considerations, we exemplify how our understanding of biochemical function and structural organization of plant mitochondria can be connected in order to obtain global and specific insights into how organelles work., (© 2019 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)