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Ca2+ signaling is central to plant development and acclimation. While Ca2+-responsive proteins have been investigated intensely in plants, only a few Ca2+-permeable channels have been identified, and our understanding of how intracellular Ca2+ fluxes is facilitated remains limited. Arabidopsis thaliana homologs of the mammalian channel-forming mitochondrial calcium uniporter (MCU) protein showed Ca2+ transport activity in vitro. Yet, the evolutionary complexity of MCU proteins, as well as reports about alternative systems and unperturbed mitochondrial Ca2+ uptake in knockout lines of MCU genes, leave critical questions about the in vivo functions of the MCU protein family in plants unanswered. Here, we demonstrate that MCU proteins mediate mitochondrial Ca2+ transport in planta and that this mechanism is the major route for fast Ca2+ uptake. Guided by the subcellular localization, expression, and conservation of MCU proteins, we generated an mcu triple knockout line. Using Ca2+ imaging in living root tips and the stimulation of Ca2+ transients of different amplitudes, we demonstrated that mitochondrial Ca2+ uptake became limiting in the triple mutant. The drastic cell physiological phenotype of impaired subcellular Ca2+ transport coincided with deregulated jasmonic acid-related signaling and thigmomorphogenesis. Our findings establish MCUs as a major mitochondrial Ca2+ entry route in planta and link mitochondrial Ca2+ transport with phytohormone signaling. Monitoring of subcellular Ca2+ dynamics in living Arabidopsis roots reveals that MCU proteins provide the dominant mitochondrial Ca2+ uptake mechanism in vivo.

Recent insights about the transport mechanisms involved in the in and out of calcium ions in plant organelles, and their role in the regulation of cytosolic calcium homeostasis in different signaling pathways.

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.

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.

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.

Redox processes are at the heart of universal life processes, such as metabolism, signaling, or folding of secreted proteins. Redox landscapes differ between cell compartments and are strictly controlled to tolerate changing conditions and to avoid cell dysfunction. While a sophisticated antioxidant network counteracts oxidative stress, our understanding of reductive stress responses remains fragmentary. Here, we observed root growth impairment in Arabidopsis thaliana mutants of mitochondrial alternative oxidase 1a (aox1a) in response to the model thiol reductant dithiothreitol (DTT). Mutants of mitochondrial uncoupling protein 1 (ucp1) displayed a similar phenotype indicating that impaired respiratory flexibility led to hypersensitivity. Endoplasmic reticulum (ER) stress was enhanced in the mitochondrial mutants and limiting ER oxidoreductin capacity in the aox1a background led to synergistic root growth impairment by DTT, indicating that mitochondrial respiration alleviates reductive ER stress. The observations that DTT triggered nicotinamide adenine dinucleotide (NAD) reduction in vivo and that the presence of thiols led to electron transport chain activity in isolated mitochondria offer a biochemical framework of mitochondrion-mediated alleviation of thiol-mediated reductive stress. Ablation of transcription factor Arabidopsis NAC domain-containing protein17 (ANAC017) impaired the induction of AOX1a expression by DTT and led to DTT hypersensitivity, revealing that reductive stress tolerance is achieved by adjusting mitochondrial respiratory capacity via retrograde signaling. Our data reveal an unexpected role for mitochondrial respiratory flexibility and retrograde signaling in reductive stress tolerance involving inter-organelle redox crosstalk.

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is reactivated to drive germination when the external conditions are favorable. Since the switchover from quiescence to reactivation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. The redox state across all Cys peptides was shifted toward reduction from 27.1% down to 13.0% oxidized thiol. A large number of Cys peptides (412) were redox switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid (TCA) cycle. Active site Cys peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.

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.

Much like a factory, the endoplasmic reticulum assembles simple cellular building blocks into complex molecular machines known as proteins. In order to protect the delicate protein folding process and ensure the proper cellular delivery of protein products under environmental stresses, eukaryotes have evolved a set of signaling mechanisms known as the unfolded protein response (UPR) to increase the folding capacity of the endoplasmic reticulum. This process is particularly important in plants, because their sessile nature commands adaptation for survival rather than escape from stress. As such, plants make special use of the UPR, and evidence indicates that the master regulators and downstream effectors of the UPR have distinct roles in mediating cellular processes that affect organism growth and development as well as stress responses. In this review we outline recent developments in this field that support a strong relevance of the UPR to many areas of plant life.

The unfolded protein response (UPR), a highly conserved set of eukaryotic intracellular signaling cascades, controls the homeostasis of the endoplasmic reticulum (ER) in normal physiological growth and situations causing accumulation of potentially toxic levels of misfolded proteins in the ER, a condition known as ER stress. During evolution, eukaryotic lineages have acquired multiple UPR effectors, which have increased the pliability of cytoprotective responses to physiological and environmental stresses. The ER‐associated protein kinase and ribonuclease IRE1 is a UPR effector that is conserved from yeast to metazoans and plants. IRE1 assumes dispensable roles in growth in yeast but it is essential in mammals and plants. The Arabidopsis genome encodes two isoforms of IRE1, IRE1A and IRE1B, whose protein functional domains are conserved across eukaryotes. Here, we describe the identification of a third Arabidopsis IRE1 isoform, IRE1C. This protein lacks the ER lumenal domain that has been implicated in sensing ER stress in the IRE1 isoforms known to date. Through functional analyses, we demonstrate that IRE1C is not essential in growth and stress responses when deleted from the genome singularly or in combination with an IRE1A knockout allele. However, we found that IRE1C exerts an essential role in gametogenesis when IRE1B is also depleted. Our results identify a novel, plant‐specific IRE1 isoform and highlight that at least the control of gametogenesis in Arabidopsis requires an unexpected functional coordination of IRE1C and IRE1B. More broadly, our findings support the existence of a functional form of IRE1 that is required for development despite the remarkable absence of a protein domain that is critical for the function of other known IRE1 isoforms.

The new phytologist 224(4), 1668-1684 (2019). doi:10.1111/nph.16093, Published by Wiley-Blackwell, Oxford [u.a.]

Hydrogen peroxide (H2 O2 ) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H2 O2 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 H2 O2 sensing in living plants. We established H2 O2 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 H2 O2 , show insensitivity to physiological pH changes, and demonstrated that glutathione dominates sensor reduction in vivo. We showed the responsiveness of the sensor to exogenous H2 O2 , pharmacologically-induced H2 O2 release, and genetic interference with the antioxidant machinery in living Arabidopsis tissues. Monitoring intracellular H2 O2 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 H2 O2 monitoring in planta and showed that intracellular H2 O2 measurements only carry meaning in the context of the endogenous thiol redox systems. This opens new possibilities to dissect plant H2 O2 dynamics and redox regulation, including intracellular NADPH oxidase-mediated ROS signalling.

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.

To overcome endoplasmic reticulum (ER) stress, ER-localized stress sensors actuate distinct downstream organelle-nucleus signaling pathways to invoke a cytoprotective response, known as the unfolded protein response (UPR). Compared to yeast and metazoans, plant UPR studies are more recent but nevertheless fascinating. Here we discuss recent discoveries in plant UPR, highlight conserved and unique features of the plant UPR as well as critical yet-open questions whose answers will likely make significant contributions to the understanding plant ER stress management.

The freshwater lichens Verrucaria elaeomelaena , V. alpicola, and V. funckii (Verrucariaceae/Ascomycota) have long been confused with V. margacea and V. placida and conclusions on the substratum preference and distribution have been obscured due to misidentifications. Independent phylogenetic analyses of a multigene dataset ( RPB 1, mtSSU, nuLSU) and an ITS-dataset combined with morphological and ecological characters confirm that the Verrucaria elaeomelaena agg. consists of several cryptic taxa. It includes V. elaeomelaena s.str. with mostly grey to mid-brown thalli and transparent exciple base which cannot be distinguished morphologically from several other unnamed clades from low elevations, the semi-cryptic V. humida spec. nov. , which is characterised by smaller perithecia, shorter and more elongated spores compared to other species in this group and V. alpicola with specimens mostly from high elevations, dark brown to black thalli, a brown exciple base and large ascospores. While Verrucaria funckii is confirmed to be restricted to siliceous substrata, limestone and siliceous substrata are both colonised by V. elaeomelaena s.str. as well as by representatives of various other clades. The substratum type cannot be seen as a diagnostic character for taxa within this species group. The taxa included in Verrucaria elaeomelaena agg. are closely related to V. submersella and together with V. funckii they form a sister clade to the “ Endocarpon -group” in the Verrucariaceae. Verrucaria margacea is superficially similar to some specimens of V. elaeomelaena agg. It is nested within an unresolved larger clade with Dermatocarpon , the subclades of the Endocarpon - and the V. elaeomelaena -group. Verrucaria margacea can be distinguished from most of the specimens in the V. elaeomelaena group by its generally non subgelatinous thallus, often with distinct cracks, narrowly ellipsoidal ascospores and the general absence of a black basal layer or dark pigments in the exciple base. The form and size of the ascospores distinguish V. placida from most specimens in the V. elaeomelaena complex, but there is overlap with some forms of V. alpicola . Epitypes are proposed for V. elaeomelaena, V. alpicola and V. margacea and a European key to the species of subgelatinous freshwater Verrucaria with spreading involucrellum and large ascospores is provided.

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.

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 (LjMAMI) 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 LjMAMI 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-LjMAMI chimera was localized to the nuclei of plant protoplasts, whereas in transgenic Lotus roots expressing the eGFP-LjMAMI 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 LjMAMI and LjPT4, a marker gene for mycorrhizal function. To elucidate the role of the LjMAMI 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 LjMAMI in non-symbiotic functions: i.e. root growth.

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.

Mitochondria are key organelles involved in many aspects of plant physiology and, their ability to generate specific Ca²⁺ signatures in response to abiotic and biotic stimuli has been reported as one of their roles. The recent identification of the mammalian mitochondrial Ca²⁺ uniporter opens a new research area in plant biology. To study the mitochondrial Ca²⁺ handling, it is essential to have a reliable probe. Here we have reported the generation of an Arabidopsis transgenic line expressing the genetically encoded probe Cameleon D3cpv targeted to mitochondria, and compared its properties with the already known Cameleon YC3.6.

An efficient agroinfiltration-based gene silencing assay was established to evaluate candidate genes likely to be involved in resistance to powdery mildew (Uncinula necator) in grapevine (Vitis vinifera L.). Functional assays were performed using two grapevine genotypes, cv. Superior, mildew-susceptible, and cv. Run1 Mtp3294, mildew-resistant. Constructs encoding a self-complementary “hairpin” RNA for phytoene desaturase (PDS) along with the green fluorescent protein gene were introduced into Agrobacterium tumefaciens. These constructs were used for agroinfiltration of leaf tissues, yielding both efficient transformation and gene silencing events. The agroinfiltration procedure did not interfere with the powdery mildew infection steps. Confocal laser-scanning microscopy analysis revealed co-localization of the GFP signal with cells lacking chlorophyll autofluorescence, further supporting PDS gene silencing. The extent of PDS mRNA degradation was evaluated by qRT-PCR in the agroinfiltrated areas and results supported a significant reduction of the gene transcripts in the silenced regions. Findings of the present work suggest that the developed silencing procedures represent a useful tool for functional characterization of grapevine genes involved in powdery mildew resistance.

Agrobacterium-mediated transient assays for the analysis of gene function are used as alternatives to genetic complementation and stable plant transformation. Although such assays are routinely performed in several plant species, they have not yet been successfully applied to grapevines. We explored genetic background diversity of grapevine cultivars and performed agroinfiltration into in vitro cultured plants. By combining different genotypes and physiological conditions, we developed a protocol for efficient transient transformations of selected grapevine cultivars. Among the four cultivars analyzed, Sugraone and Aleatico exhibited high levels of transient transformation. Transient expression occurred in the majority of cells within the infiltrated tissue several days after agroinfiltration and, in a few cases, it later spread to a larger portion of the leaf. Three laboratory strains of Agrobacterium tumefaciens with different virulence levels were used for agroinfiltration assays on grapevine plants. This method promises to be a powerful tool to perform subcellular localization analyses. Grapevine leaf tissues were transformed with fluorescent markers targeted to cytoplasm (free GFP and mRFP1), endoplasmatic reticulum (GFP::HDEL), chloroplast (GAPA1::YFP) and mitochondria (²::GFP). Confocal microscope analyses demonstrated that these subcellular compartments could be easily visualized in grapevine leaf cells. In addition, from leaves of the Sugraone cultivar agroinfiltrated with endoplasmic reticulum-targeted GFP-construct, stable transformed cells were obtained that show the opportunity to convert a transiently transformed leaf tissue into a stably transformed cell line. Electronic supplementary material The online version of this article (doi:10.1007/s00299-008-0510-4) contains supplementary material, which is available to authorized users.

Background: The ripening of fleshy fruits is a complex developmental program characterized by extensive transcriptomic and metabolic remodeling in the pericarp tissues (pulp and skin) making unripe green fruits soft, tasteful and colored. The onset of ripening is regulated by a plethora of endogenous signals tuned to external stimuli. In grapevine and tomato, which are classified as non-climacteric and climacteric species respectively, the accumulation of hydrogen peroxide (H2O2) and extensive modulation of reactive oxygen species (ROS) scavenging enzymes at the onset of ripening has been reported, suggesting that ROS could participate to the regulatory network of fruit development. In order to investigate this hypothesis, a comprehensive biochemical study of the oxidative events occurring at the beginning of ripening in Vitis vinifera cv. Pinot Noir has been undertaken. Results: ROS-specific staining allowed to visualize not only H2O2 but also singlet oxygen (O-1(2)) in berry skin cells just before color change in distinct subcellular locations, i.e. cytosol and plastids. H2O2 peak in sample skins at veraison was confirmed by in vitro quantification and was supported by the concomitant increase of catalase activity. Membrane peroxidation was also observed by HPLC-MS on galactolipid species at veraison. Mono-and digalactosyl diacylglycerols were found peroxidized on one or both a-linolenic fatty acid chains, with a 13(S) absolute configuration implying the action of a specific enzyme. A lipoxygenase (PnLOXA), expressed at veraison and localizing inside the chloroplasts, was indeed able to catalyze membrane galactolipid peroxidation when overexpressed in tobacco leaves. Conclusions: The present work demonstrates the controlled, harmless accumulation of specific ROS in distinct cellular compartments, i.e. cytosol and chloroplasts, at a definite developmental stage, the onset of grape berry ripening. These features strongly candidate ROS as cellular signals in fruit ripening and encourage further studies to identify downstream elements of this cascade. This paper also reports the transient galactolipid peroxidation carried out by a veraison-specific chloroplastic lipoxygenase. The function of peroxidized membranes, likely distinct from that of free fatty acids due to their structural role and tight interaction with photosynthesis protein complexes, has to be ascertained.