Stefano D'alessandro
email: stefano.dalessandro.phd@gmail.com

Inès Beaugelin

Michel Havaux
email: michel.havaux@cea.fr

Stefano D ' Alessandro

Tanned or Sunburned: How Excessive Light Triggers Plant Cell Death

Keywords: Arabidopsis, photosynthesis, excessive light, photooxidative stress, carotenoid, plant cell death, stress response PFD, Photon Flux Density, ROS, Reactive Oxygen Species, RWC, Relative Water Content, OEC, Oxygen Evolving Complex, PCD, Programmed Cell Death, RCS, Reactive Carbonyl Species, HNE, 4-Hydroxy-2-nonenal, NPQ, Non-Photochemical Quenching, JA, Jasmonic acid, JA-Ile, Jasmonoyl-Isoleucine, SA, Salicylic acid, OXI1, OXIDATIVE STRESS INDUCIBLE 1, EX1, EXECUTER 1, ER, Endoplasmic Reticulum, UPR, Unfolded Protein Response, ABA, abscisic acid, 1 O2, singlet oxygen

Capturing light intensities exceeding the capacity of photosynthesis can lead to cell death in plant leaves. In this review, we summarize recent findings on the mechanisms mediating light-induced cell death, highlighting the role of reactive carbonyl species, of the endoplasmic reticulum and of phytohormones in the adoption of phototolerance versus cell death pathways.

Introduction

Plants often encounter light intensities that exceed the needs of photosynthesis [START_REF] Ort | When There Is Too Much Light[END_REF]. Light energy that is not used to assimilate CO2 or dissipated can be considered to be in excess and leads to a depression of photosynthetic efficiency, especially at PSII [START_REF] Kato | The Excess Light Energy that is neither Utilized in Photosynthesis nor Dissipated by Photoprotective Mechanisms Determines the Rate of Photoinactivation in Photosystem II[END_REF][START_REF] Murata | Photoinhibition of photosystem II under environmental stress[END_REF].

Excessive light occurs mainly in response to biotic and abiotic factors, such as drought, heat or pests, and lowers the capacity of light energy sinks, first and foremost CO2 fixation. The excess electrons and excitation energy can be transferred to molecular oxygen, generating biologically damaging molecules such as reactive oxygen species (ROS), peroxides and radicals, hence leading to photooxidative stress that can ultimately lead to cell death [START_REF] Apel | REACTIVE OXYGEN SPECIES: Metabolism, Oxidative Stress, and Signal Transduction[END_REF][START_REF] Li | Sensing and Responding to Excess Light[END_REF].

High light under different conditions induces different responses

The real condition of excessive light, which is dependent on photosynthetic activity and can also occur at relatively low light intensities, is often confused with 'high light' treatment. Drought stress, nutrient starvation or pathogens can lead to the same state of overexcitation of the photosystems, without the imposition of high light [START_REF] Garai | Alleviation of Nitrogen and Sulfur Deficiency and Enhancement of Photosynthesis in Arabidopsis thaliana by Overexpression of Uroporphyrinogen III Methyltransferase (UPM1)[END_REF][START_REF] Wang | Effects of drought stress on photosynthesis and photosynthetic electron transport chain in young apple tree leaves[END_REF][START_REF] Pérez-Bueno | Phenotyping Plant Responses to Biotic Stress by Chlorophyll Fluorescence Imaging[END_REF]. Likewise, by playing with the CO2 and O2 composition of the gas phase, [START_REF] Montané | Differential control of xanthophylls and light-induced stress proteins, as opposed to light-harvesting chlorophyll a/b proteins, during photosynthetic acclimation of barley leaves to light irradiance[END_REF] generated the same excitation pressure on the photosystems at different photon flux densities (PFDs) [START_REF] Montané | Differential control of xanthophylls and light-induced stress proteins, as opposed to light-harvesting chlorophyll a/b proteins, during photosynthetic acclimation of barley leaves to light irradiance[END_REF]. In addition, the intensity of sunlight that leaves receive under natural conditions often fluctuates, because of clouds and intermittent shading by other leaves or plants. This "fluctuating light" can be very damaging to the photosynthetic machinery, particularly PSI, even at relatively low PFDs [START_REF] Kono | Roles of the Cyclic Electron Flow Around PSI (CEF-PSI) and O2-Dependent Alternative Pathways in Regulation of the Photosynthetic Electron Flow in Short-Term Fluctuating Light in Arabidopsis thaliana[END_REF][START_REF] Yamori | A physiological role of cyclic electron transport around photosystem I in sustaining photosynthesis under fluctuating light in rice[END_REF].

PFDs used in the literature to induce excessive light range from 500 to 2000 mol photons m -2 s -1 .

These treatments can also increase leaf temperature and, unless air temperature is controlled, will impose a double condition of excessive light and heat stress. Lack of leaf temperature control in experimental protocols increases interlaboratory data variability and makes comparison between different studies more difficult.

Even when leaf temperature is controlled by lowering air temperature, different setups have been adopted, such as 600 mol photons m -2 s -1 PFD at 23°C air temperature [START_REF] Balfagón | Jasmonic Acid Is Required for Plant Acclimation to a Combination of High Light and Heat Stress[END_REF], 1500 mol photons m -2 s -1 at 7°C [START_REF] Ramel | Carotenoid oxidation products are stress signals that mediate gene responses to singlet oxygen in plants[END_REF][START_REF] D'alessandro | Decoding β-Cyclocitral-Mediated Retrograde Signaling Reveals the Role of a Detoxification Response in Plant Tolerance to Photooxidative Stress[END_REF], 1200 mol photons m -2 s -1 at 18°C [START_REF] Lv | β-cyclocitral upregulates salicylic acid signalling to enhance excess light acclimation in Arabidopsis[END_REF], 1200 mol photons m -2 s -1 at 11.5°C [START_REF] Huang | The Arabidopsis Transcriptome Responds Specifically and Dynamically to High Light Stress[END_REF] or 2000 mol photons m -2 s -1 at 4°C [START_REF] Mattila | Effects of low temperature and cold-acclimation on photoinhibition and singlet oxygen production in four natural accessions of Arabidopsis[END_REF]. These studies show that leaf temperature remains at standard levels (20-22°C) when exposed to 1500 mol photons m -2 s -1 at 7°C or 1200 mol photons m -2 s -1 at 11.5°C. However, leaf temperature increases by 4°C (from 23°C to 27°C) when exposed to 600 mol photons m -2 s -1 or 13°C (from 22°C to 35°C) at 1200 mol photons m -2 s -1 if air temperature is not lowered.

Several studies have indicated that there can be significant overlap between high light and heat stress responses when air temperature is maintained at normal growth temperature. Balfagón et al. ( 2019) compared a 7-h high light treatment (HL) with a heat shock treatment (HS, 42°C) and the combination of both [START_REF] Balfagón | Jasmonic Acid Is Required for Plant Acclimation to a Combination of High Light and Heat Stress[END_REF]. The HL treatment caused a 4°C increase in leaf temperature, from 23°C to 27°C, the HS treatment increased leaf temperature by around 10°C, from 23°C to 33°C, and the combined stresses increased leaf temperature by around 10-12°C. Analysis of transcriptomes showed an astonishing resemblance between HL (at 23°C air temperature) and HS, with a large fraction of the modified genes being common between the two stresses, corresponding to 58% (4439) of HL alone. This result suggests that performing HL experiments at 23°C air temperature causes a response partially overlapping with HS, likely due to the increase in leaf temperature. While the combination of HS and HL was equally similar to HS and to HL (64% overlap) on a transcriptional level, other experiments indicated a dominance of HS during HL stress.

In fact, while HL performed at 23°C caused stomatal closure, HS and HL+HS induced stomatal opening. Relative water content (RWC) was unperturbed by HL but reduced from 75% to 60% by HS and HS+HL. The number of starch granules diminished in HL but increased under HS and HS+HL. H2O2 production increased in HS and HS+HL-treated plants but did not in HL-treated plants, in agreement with previous analyses of pure excess light [START_REF] D'alessandro | Decoding β-Cyclocitral-Mediated Retrograde Signaling Reveals the Role of a Detoxification Response in Plant Tolerance to Photooxidative Stress[END_REF]. Similar results were observed in another study when analyzing the systemic response to HL, HS and the combination of both stresses [START_REF] Zandalinas | Systemic signaling during abiotic stress combination in plants[END_REF].

The lipid problem in excessive light

ROS production under stress conditions initially elicits a signaling cascade and, if sufficient quantities are produced to overwhelm defense mechanisms, also leads to cellular damage. Singlet oxygen ( 1 O2) is a very harmful and likely the major ROS produced under excessive light [START_REF] Triantaphylidès | Singlet Oxygen Is the Major Reactive Oxygen Species Involved in Photooxidative Damage to Plants[END_REF][START_REF] Ramel | Carotenoid oxidation products are stress signals that mediate gene responses to singlet oxygen in plants[END_REF][START_REF] D'alessandro | Sensing β-carotene oxidation in photosystem II to master plant stress tolerance[END_REF][START_REF] Khorobrykh | Oxygen and ROS in Photosynthesis[END_REF]. In fact, a 1 O2 mediated transcriptional response can be detected after a 24-h high light treatment at controlled air temperature, while marker genes for H2O2 are not induced [START_REF] Shumbe | METHYLENE BLUE SENSITIVITY 1 (MBS1) is required for acclimation of Arabidopsis to singlet oxygen and acts downstream of β-cyclocitral[END_REF]. This result is in line with the increased biosynthesis of H2O2 and the induction of APX2 only when high light conditions are combined with heat stress [START_REF] Smirnoff | Hydrogen peroxide metabolism and functions in plants[END_REF][START_REF] Balfagón | Jasmonic Acid Is Required for Plant Acclimation to a Combination of High Light and Heat Stress[END_REF][START_REF] Huang | The Arabidopsis Transcriptome Responds Specifically and Dynamically to High Light Stress[END_REF]. Notably, the O2-evolving-complex (OEC) proteins are particularly susceptible to heat stress [START_REF] Berry | Photosynthetic Response and Adaptation to Temperature in Higher Plants[END_REF][START_REF] Weis | Plants and high temperature stress[END_REF][START_REF] Havaux | Rapid photosynthetic adaptation to heat stress triggered in potato leaves by moderately elevated temperatures[END_REF], and OEC-depleted PSII predominantly generates H2O2 [START_REF] Khorobrykh | Oxygen and ROS in Photosynthesis[END_REF]. Only a prolonged high light stress is therefore likely to cause a combined accumulation of 1 O2 and H2O2 [START_REF] Laloi | Key players of singlet oxygen-induced cell death in plants[END_REF]. ROS, and especially 1 O2, are highly reactive towards carbon-carbon double bonds. ROS are primarily produced at the level of the photosystems in a lipid rich environment, and so oxidized lipids are one of the major products of ROS generation in response to excessive light. Fatty acids are highly unsaturated in plant leaves [START_REF] Douce | 12 -Plant Galactolipids[END_REF] and are therefore very sensitive to oxidation [START_REF] Bour | Lipid Unsaturation Properties Govern the Sensitivity of Membranes to Photoinduced Oxidative Stress[END_REF]. The major fatty acids in leaves are the polyunsaturated fatty acids linolenic acid (C18:3) and linoleic acid (C18:2) containing 3 and 2 double bonds, respectively. At the level of thylakoids, a primary site of ROS production, the ratio between unsaturated to saturated lipids is 9.4 to 1, and linolenic acid corresponds to 70% of total fatty acids [START_REF] Chapman | Analysis of plastoquinone-9 levels in appressed and non-appressed thylakoid membrane regions[END_REF][START_REF] Yalcinkaya | Lipid peroxidation-derived reactive carbonyl species (RCS): Their interaction with ROS and cellular redox during environmental stresses[END_REF]. Considering Arabidopsis whole leaves, linolenic acid can represent up to 50 % of the total fatty acid content [START_REF] Li-Beisson | Acyl-lipid metabolism[END_REF]. Consequently, lipid peroxidation is usually a primary event associated with ROS production and oxidative stress in plants [START_REF] Birtic | Using spontaneous photon emission to image lipid oxidation patterns in plant tissues[END_REF].

The attack of lipids by ROS generates specific peroxides, notably lipid endoperoxides specific to 1 O2, while lipid radicals and lipid peroxyl radicals are specific to reduced oxygen forms [START_REF] Mano | Reactive Carbonyl Species: A Missing Link in ROS Signaling[END_REF][START_REF] Khorobrykh | Oxygen and ROS in Photosynthesis[END_REF]. In a second stage, all these molecules are transformed into lipid hydroperoxides.

High light treatment with controlled air temperature was found to be systematically associated with a lipid peroxidation signature typical of 1 O2 [START_REF] Triantaphylidès | Singlet Oxygen Is the Major Reactive Oxygen Species Involved in Photooxidative Damage to Plants[END_REF], indicating a major role for this ROS in photooxidative degradation of plant lipids. The abundance of polyunsaturated fatty acids has been proposed to act as a sink for ROS, especially 1 O2, in chloroplasts, which could constitute a protection mechanism against propagation of oxidative stress [START_REF] Mène-Saffrané | Nonenzymatic oxidation of trienoic fatty acids contributes to reactive oxygen species management in Arabidopsis[END_REF]. However, lipid peroxides are not only the signature of photooxidation but also directly contribute to signaling and toxicity.

Phytoprostanes and phytofurans are compounds derived from non-enzymatic oxidation of linolenic acid in plants [START_REF] Mueller | Archetype signals in plants: the phytoprostanes[END_REF][START_REF] Durand | Isoprostanes and phytoprostanes: Bioactive lipids[END_REF][START_REF] Collado-González | Effect of the season on the free phytoprostane content in Cornicabra extra virgin olive oil from deficit-irrigated olive trees[END_REF][START_REF] Galano | Isoprostanes, neuroprostanes and phytoprostanes: An overview of 25years of research in chemistry and biology[END_REF] (Fig. 1). Because of their structural analogies with isoprostanes and prostanoids, these lipid derivatives may constitute a new class of bioactive plant compounds in response to oxidative stress [START_REF] Mueller | General Detoxification and Stress Responses Are Mediated by Oxidized Lipids through TGA Transcription Factors in Arabidopsis[END_REF]. Some phytoprostanes contain an electrophilic site, which can react and make adducts with proteins [START_REF] Liebler | Protein damage by reactive electrophiles: targets and consequences[END_REF]. Typical reactions are Schiff base formation and Michael addition. Accumulation of phytoprostanes/phytofurans has been reported in various photosynthetic organisms under oxidative stress [START_REF] Loeffler | B1-Phytoprostanes Trigger Plant Defense and Detoxification Responses[END_REF][START_REF] Yonny | Thermal Stress in Melon Plants: Phytoprostanes and Phytofurans as Oxidative Stress Biomarkers and the Effect of Antioxidant Supplementation[END_REF][START_REF] Lupette | Non-Enzymatic Synthesis of Bioactive Isoprostanoids in the Diatom Phaeodactylum following Oxidative Stress[END_REF], potentially playing a role in plant responses to photooxidative stress.

Lipid hydroperoxides spontaneously fragment into a variety of secondary compounds that can exhibit high reactivity towards biomolecules. Reactive carbonyl species (RCS) are the unsaturated aldehydes and ketones derived from lipid peroxides, which can further hinder the physiological functioning of macromolecules and induce cell death [START_REF] Farmer | ROS-mediated lipid peroxidation and RES-activated signaling[END_REF][START_REF] Mano | Reactive Carbonyl Species: A Missing Link in ROS Signaling[END_REF]. Some of these molecules, such as (E)-2-hexenal, have been studied as Green Leaf Volatiles and previously reviewed [START_REF] Ameye | Green leaf volatile production by plants: a meta-analysis[END_REF]. RCS, such as hydroxynonenal (HNE), acrolein or malondialdehyde (Fig. 1), are electrophiles and hence can react with nucleophilic groups in biomolecules to make covalent bonds [START_REF] Mano | Reactive carbonyl species: Their production from lipid peroxides, action in environmental stress, and the detoxification mechanism[END_REF][START_REF] Mano | Reactive Carbonyl Species: A Missing Link in ROS Signaling[END_REF]. In this way RCS may change protein conformation or alter protein function under stress conditions, consequently affecting metabolic or signaling processes [START_REF] Biswas | Reactive Carbonyl Species Activate Caspase-3-Like Protease to Initiate Programmed Cell Death in Plants[END_REF][START_REF] Yalcinkaya | Lipid peroxidation-derived reactive carbonyl species (RCS): Their interaction with ROS and cellular redox during environmental stresses[END_REF].

Promiscuous, broad-spectrum detoxification of lipid peroxidation by-products

Reactive molecules, both of endogenous or exogenous origin, may constitute a hazard for the cell and lead to cell death. Plants have evolved a detoxification mechanism that can neutralize these dangers. As this mechanism was discovered when studying the response to herbicides, it was called the 'xenobiotic response' [START_REF] Sandermann | Plant metabolism of xenobiotics[END_REF][START_REF] Kreuz | Old Enzymes for a New Job (Herbicide Detoxification in Plants)[END_REF][START_REF] Riechers | Detoxification without Intoxication: Herbicide Safeners Activate Plant Defense Gene Expression[END_REF]. This process is organized in three phases, modification, conjugation and compartmentalization, and enzyme families participating in each phase of the xenobiotic response are reported in Fig. 2.

Enzymes of the modification phase are active towards RCS and catalyze their conversion into less dangerous molecules [START_REF] Mano | Reactive Carbonyl Species: A Missing Link in ROS Signaling[END_REF]. RCS detoxification can occur through different enzymatic reactions: (i) reduction of the α,β-unsaturated bond in an RCS molecule using NAD(P)H as the electron donor, by alkenal reductase (AER) and alkenal/one oxidoreductase (AOR); (ii) reduction of a carbonyl moiety to an alcohol using NAD(P)H, catalyzed with aldo-keto reductase (AKR); (iii) oxidation of an aldehyde to a carboxylic acid using NAD + as the electron acceptor, catalyzed by aldehyde dehydrogenase (ALDH); (iv) oxidation of an aldehyde using O2 as the electron acceptor catalyzed by aldehyde oxidase (AO) [START_REF] Mano | The NADPH:Quinone Oxidoreductase P1-ζ-crystallin in Arabidopsis Catalyzes the α,β-Hydrogenation of 2-Alkenals: Detoxication of the Lipid Peroxide-Derived Reactive Aldehydes[END_REF][START_REF] Stiti | Aldehyde Dehydrogenases in Arabidopsis thaliana: Biochemical Requirements, Metabolic Pathways, and Functional Analysis[END_REF][START_REF] Yamauchi | NADPH-dependent Reductases Involved in the Detoxification of Reactive Carbonyls in Plants[END_REF][START_REF] Yamauchi | Chloroplastic NADPH-dependent alkenal/one oxidoreductase contributes to the detoxification of reactive carbonyls produced under oxidative stress[END_REF][START_REF] Saito | Functional Analysis of the AKR4C Subfamily of Arabidopsis thaliana: Model Structures, Substrate Specificity, Acrolein Toxicity, and Responses to Light and [CO2][END_REF][START_REF] Srivastava | Aldehyde Oxidase 4 Plays a Critical Role in Delaying Silique Senescence by Catalyzing Aldehyde Detoxification[END_REF]. In addition, formation of a glutathione or glucoside adduct of RCS can be catalyzed by enzymes of the conjugation phase (i.e. glutathione transferase (GST)) [START_REF] Mano | Acrolein-detoxifying isozymes of glutathione transferase in plants[END_REF]Mano et al., 2019a).

Several of these enzymes are induced under stress conditions, in accord with the overproduction of RCS, and plants overexpressing alkenal reductases, aldo-keto reductases and aldehyde dehydrogenases are usually more tolerant to stress conditions, as reviewed in [START_REF] Mano | Detoxification of Reactive Carbonyl Species by Glutathione Transferase Tau Isozymes[END_REF].

In addition, we have recently reported a pathway controlling acclimation to excessive light mediated by the induction of RCS detoxification by the apocarotenoid -cyclocitral (D' Alessandro et al., 2018;[START_REF] D'alessandro | Sensing β-carotene oxidation in photosystem II to master plant stress tolerance[END_REF]. Phytoprostanes and phytofurans may also require the induction of a detoxification response to limit their effects in vivo and the rapid disappearance of phytoprostanoids in tobacco cells is consistent with this idea [START_REF] Loeffler | B1-Phytoprostanes Trigger Plant Defense and Detoxification Responses[END_REF].

Enzymes of the modification phase are usually associated with biosynthetic pathways for secondary metabolites (Cytochrome P450 oxidases, Short Chain Dehydrogenases/Reductases, …). At the same time, part of these large families of enzymes was identified in the response to xenobiotic compounds [START_REF] Fode | The Arabidopsis GRAS Protein SCL14 Interacts with Class II TGA Transcription Factors and Is Essential for the Activation of Stress-Inducible Promoters[END_REF]. It is likely that plants can adopt the less specific steps of the reactions that they perform in secondary metabolism to cope with hazardous molecules, in what could be considered a broad-spectrum (promiscuous) detoxification pathway. Ligand promiscuity and promiscuous detoxification are well-known processes in animal systems where the two promiscuous receptors CAR and PXR can bind to different molecules and controls the expression of several CYP450 genes [START_REF] Tolson | Regulation of drug-metabolizing enzymes by xenobiotic receptors: PXR and CAR[END_REF]. These processes, catalyzed by the same families of enzymes as the xenobiotic response of plants, are active against endogenous toxicants and constitute the base of drug metabolism and pharmacokinetics. We therefore propose to adopt the animal systems nomenclature of promiscuous detoxification for the xenobiotic response in plants.

As promiscuous detoxification is active towards hazardous metabolites generated during photooxidation, such as RCS, this pathway represents a new level of photoprotection. Light-induced ROS generation is inevitably coupled with photosynthesis. ROS quenching and scavenging are continuous, but under stress conditions they could become insufficient compared to ROS production, leading to ROS accumulation. At this point, a first level of photoprotection, including nonphotochemical energy quenching (NPQ), cyclic electron transport, plastid terminal oxidase (PTOX) and state transition, reduces ROS production [START_REF] Alric | Alternative electron transport pathways in photosynthesis: a confluence of regulation[END_REF][START_REF] Roach | Photosynthetic Regulatory Mechanisms for Efficiency and Prevention of Photo-Oxidative Stress[END_REF]. A second level of photoprotection, including ROS scavenging (by SOD and APX, for the superoxide anion radical and H2O2, respectively) and ROS quenching (by carotenoids, tocopherols and plastoquinones for 1 O2), avoids oxidation [START_REF] Apel | REACTIVE OXYGEN SPECIES: Metabolism, Oxidative Stress, and Signal Transduction[END_REF][START_REF] Krieger-Liszkay | Singlet oxygen production in photosystem II and related protection mechanism[END_REF][START_REF] Noctor | ROS-related redox regulation and signaling in plants[END_REF][START_REF] Filiz | Abiotic stress-induced regulation of antioxidant genes in different Arabidopsis ecotypes: microarray data evaluation[END_REF]. The D1 protein of PSII, which is very susceptible to oxidation, can also be considered a component of photoprotection together with the well described D1 repair mechanism [START_REF] Nath | Towards a critical understanding of the photosystem II repair mechanism and its regulation during stress conditions[END_REF][START_REF] Järvi | Photosystem II repair in plant chloroplasts--Regulation, assisting proteins and shared components with photosystem II biogenesis[END_REF][START_REF] Yoshioka-Nishimura | Close Relationships Between the PSII Repair Cycle and Thylakoid Membrane Dynamics[END_REF][START_REF] Kato | The Photosystem II Repair Cycle Requires FtsH Turnover through the EngA GTPase[END_REF]. When these photoprotective processes are insufficient, or inhibited by stress, oxidation of lipids generates RCS. Promiscuous detoxification can metabolize the hazardous reactive molecules to protect the cell (Fig. 3) which, under certain conditions, can trigger programmed cell death (PCD) [START_REF] Biswas | Reactive Carbonyl Species Activate Caspase-3-Like Protease to Initiate Programmed Cell Death in Plants[END_REF].

From RCS to PCD

1 O2-derived peroxidation of lipids, proteins and nucleic acids has been observed in treatments with photosensitizers (methylene blue or rose Bengal) and in Arabidopsis mutant lines overproducing 1 O2 such as flu and ch1 [START_REF] Triantaphylidès | Singlet Oxygen Is the Major Reactive Oxygen Species Involved in Photooxidative Damage to Plants[END_REF][START_REF] Shao | A Mediator of Singlet Oxygen Responses in Chlamydomonas reinhardtii and Arabidopsis Identified by a Luciferase-Based Genetic Screen in Algal Cells[END_REF][START_REF] Laloi | Key players of singlet oxygen-induced cell death in plants[END_REF]. ch1 showed increased lipid peroxidation and photooxidative damage under excess light conditions, whereas 1 O2 is generated under non-photoinhibitory conditions in flu [START_REF] Wagner | The Genetic Basis of Singlet Oxygen Induced Stress Responses of Arabidopsis thaliana[END_REF]Ramel et al., 2013a). In both ch1 and flu, 1 O2-induced cell death was dependent on signaling proteins, OXIDATIVE STRESS INDUCIBLE 1 (OXI1) (in ch1 and in the wild type), EXECUTER1 (EX1) or SAFE1 (in flu), indicating the involvement of a signaling process leading to cell death [START_REF] Shumbe | Singlet Oxygen-Induced Cell Death in Arabidopsis under High-Light Stress Is Controlled by OXI1 Kinase[END_REF][START_REF] Dogra | Oxidative post-translational modification of EXECUTER1 is required for singlet oxygen sensing in plastids[END_REF][START_REF] Beaugelin | OXI1 and DAD Regulate Light-Induced Cell Death Antagonistically through Jasmonate and Salicylate Levels[END_REF][START_REF] Wang | The Arabidopsis SAFEGUARD1 suppresses singlet oxygen-induced stress responses by protecting grana margins[END_REF]. The EX1 protein is located in the grana margins of the thylakoids [START_REF] Wang | Singlet oxygen-and EXECUTER1-mediated signaling is initiated in grana margins and depends on the protease FtsH2[END_REF][START_REF] Dogra | Singlet oxygen-triggered chloroplast-to-nucleus retrograde signalling pathways: An emerging perspective[END_REF][START_REF] Dogra | Singlet Oxygen Metabolism: From Genesis to Signaling[END_REF], relatively distant from the main 1 O2 source under excessive light, the PSII reaction centers in the grana core (D'Alessandro and Havaux, 2019). EX1 can sense 1 O2 produced from free chlorophylls associated with the PSII repair process or released during non-photoinhibitory stress, through oxidative posttranslational modification and proteolysis by the protease FtsH2 [START_REF] Wang | Singlet oxygen-and EXECUTER1-mediated signaling is initiated in grana margins and depends on the protease FtsH2[END_REF][START_REF] Dogra | Oxidative post-translational modification of EXECUTER1 is required for singlet oxygen sensing in plastids[END_REF]. Following those modifications, EX1 can induce PCD. However, PCD in WT and ch1 leaves under high PFDs at controlled temperature is independent of EX1 (Ramel et al., 2013a;[START_REF] Shumbe | Singlet Oxygen-Induced Cell Death in Arabidopsis under High-Light Stress Is Controlled by OXI1 Kinase[END_REF], indicating the existence of at least two 1 O2 signaling pathways leading to cell death.

Prolonged exposure to extreme light conditions would result in an increased accumulation of ROS, principally a combination of 1 O2 and reduced/radical O2 species [START_REF] Laloi | Key players of singlet oxygen-induced cell death in plants[END_REF][START_REF] Khorobrykh | Oxygen and ROS in Photosynthesis[END_REF]. While 1 O2 generates lipid endoperoxides, O2 radicals oxidize lipids to lipid radicals and peroxyl radicals. Peroxyl radicals can react with other lipids generating a new lipid radical, and again a peroxyl radical in a chain reaction [START_REF] Mano | Reactive Carbonyl Species: A Missing Link in ROS Signaling[END_REF][START_REF] Khorobrykh | Oxygen and ROS in Photosynthesis[END_REF].

Another vicious circle has been highlighted between RCS and H2O2 [START_REF] Biswas | Inactivation of Carbonyl-Detoxifying Enzymes by H2O2 Is a Trigger to Increase Carbonyl Load for Initiating Programmed Cell Death in Plants[END_REF]. RCS can alter protein function by covalent modification [START_REF] Colzani | Mass spectrometric approaches for the identification and quantification of reactive carbonyl species protein adducts[END_REF], and H2O2 scavenging enzymes (catalase and ascorbate peroxidase) were observed in a study as targets of RCS [START_REF] Mano | Identification of oxidatively modified proteins in salt-stressed Arabidopsis: a carbonyl-targeted proteomics approach[END_REF].

In addition, an animal catalase has been shown to form adducts with HNE, leading to inactivation of the enzyme and a compromised H2O2 scavenging [START_REF] Bauer | Revealing mechanisms of selective, concentration-dependent potentials of 4-hydroxy-2-nonenal to induce apoptosis in cancer cells through inactivation of membraneassociated catalase[END_REF][START_REF] Biswas | Inactivation of Carbonyl-Detoxifying Enzymes by H2O2 Is a Trigger to Increase Carbonyl Load for Initiating Programmed Cell Death in Plants[END_REF].

H2O2 itself can also modify the activity of enzymes of the detoxification pathway. Indeed, it was recently demonstrated that, at relatively low concentrations (0.2 mM), H2O2 hinders almost 50% of the HNE-reducing activity present in BY-2 cell cultures, and this can occur within minutes [START_REF] Biswas | Inactivation of Carbonyl-Detoxifying Enzymes by H2O2 Is a Trigger to Increase Carbonyl Load for Initiating Programmed Cell Death in Plants[END_REF]. Thus, RCS production can result in the accumulation of H2O2 that could inhibit RCS detoxification, hence generating a feed-forward amplification mechanism.

H2O2 is a long-lived ROS that can travel to the nucleus and act as a signaling agent [START_REF] Exposito-Rodriguez | Photosynthesis-dependent H 2 O 2 transfer from chloroplasts to nuclei provides a high-light signalling mechanism[END_REF]. Nonetheless, it has been recently shown that RCS mediates H2O2-induced PCD by inducing caspase-3-like activity in BY-2 cell cultures [START_REF] Biswas | Reactive Carbonyl Species Activate Caspase-3-Like Protease to Initiate Programmed Cell Death in Plants[END_REF][START_REF] Biswas | Inactivation of Carbonyl-Detoxifying Enzymes by H2O2 Is a Trigger to Increase Carbonyl Load for Initiating Programmed Cell Death in Plants[END_REF]. Therefore, the overaccumulation of RCS could be a major mechanism for the induction of cell death, and a mechanism for linking photosynthetic inhibition to PCD. Consistent with this, we have observed that a 4-h pretreatment of Arabidopsis plants with HNE increased lipid peroxidation under excessive light [START_REF] D'alessandro | Decoding β-Cyclocitral-Mediated Retrograde Signaling Reveals the Role of a Detoxification Response in Plant Tolerance to Photooxidative Stress[END_REF].

Hormonal control of excessive light-induced cell death

A key regulator of PCD under excessive light is the AGC kinase OXI1. OXI1 mediates cell death in response to both biotic and abiotic stimuli, and it resides at the intersection of the H2O2 and 1 O2 signaling pathways [START_REF] Shumbe | Singlet Oxygen-Induced Cell Death in Arabidopsis under High-Light Stress Is Controlled by OXI1 Kinase[END_REF]. OXI1 is also linked to the signaling pathways of several phytohormones [START_REF] Howden | The phosphoproteome of Arabidopsis plants lacking the oxidative signal-inducible1 (OXI1) protein kinase[END_REF]. OXI1 is required for the full activation of MAPK3 and MAPK6 to induce PCD under H2O2 stress [START_REF] Rentel | OXI1 kinase is necessary for oxidative burst-mediated signalling in Arabidopsis[END_REF] and induces jasmonic acid (JA) and salicylic acid (SA) biosynthesis in high light-exposed leaves [START_REF] Shumbe | Singlet Oxygen-Induced Cell Death in Arabidopsis under High-Light Stress Is Controlled by OXI1 Kinase[END_REF][START_REF] Beaugelin | OXI1 and DAD Regulate Light-Induced Cell Death Antagonistically through Jasmonate and Salicylate Levels[END_REF].

There are many examples in the literature indicating a link between plant/leaf photodamage and induction of the JA pathway. For instance, the transfer of Arabidopsis plants from 80 to 800 mol m -2 s -1 PFD increased the concentration of JA and JA-Ile [START_REF] Alsharafa | Kinetics of retrograde signalling initiation in the high light response of Arabidopsis thaliana[END_REF]. In apple, sun damage was also accompanied by increased concentrations of those compounds [START_REF] Torres | Phytohormone Interaction Modulating Fruit Responses to Photooxidative and Heat Stress on Apple (Malus domestica Borkh.)[END_REF].

Moreover, treatments of leaves with methyl-JA under excessive light enhances PSII photoinhibition and photodamage [START_REF] Wierstra | Differential effects of methyl jasmonate on the expression of the early light-inducible proteins and other light-regulated genes in barley[END_REF]Ramel et al., 2013a;[START_REF] Savchenko | The hydroperoxide lyase branch of the oxylipin pathway protects against photoinhibition of photosynthesis[END_REF].

Photooxidative damage in the 1 O2-overproducing Arabidopsis mutant ch1 is also associated with a marked upregulation of JA biosynthesis genes and by a strong accumulation of OPDA (12-oxophytodienoic acid) and JAs (Ramel et al., 2013a). Very recently, an excessive light transcriptome of Arabidopsis plants further confirmed that plants respond to excessive light by modifying the metabolism of JA, and additionally revealed a potential role for SA and abscisic acid (ABA) [START_REF] Huang | The Arabidopsis Transcriptome Responds Specifically and Dynamically to High Light Stress[END_REF]. Together these results support the idea that JA plays an important role in response to exposure to excess light.

The role of JAs in PCD associated with plant defense against feeding insects, nematodes, fungal and bacterial pathogens is already well documented [START_REF] Rentel | OXI1 kinase is necessary for oxidative burst-mediated signalling in Arabidopsis[END_REF][START_REF] Reinbothe | Plant oxylipins: role of jasmonic acid during programmed cell death, defence and leaf senescence[END_REF] and suggests that it may also mediate PCD under abiotic stress conditions. Indeed, JA accumulation under excess light is associated with the induction of senescence-associated factors, such as SAG12, SAG13, SEN1 and SEN4, as well as PCD inducers including MC8 and gVPE [START_REF] Beaugelin | OXI1 and DAD Regulate Light-Induced Cell Death Antagonistically through Jasmonate and Salicylate Levels[END_REF]. Furthermore, the allene oxide synthase mutant of Arabidopsis thaliana (dde2), deficient in JA biosynthesis, is much more tolerant to high light-induced cell death (at controlled temperature) than the wild-type (Ramel et al., 2013b). Similarly, the acclimation of the Arabidopsis ch1 mutant to excessive light and 1 O2 stress requires the down-regulation of the JA pathway and JA accumulation (Ramel et al., 2013a).

The role of JA in response to excess light appears to be dependent on temperature. When high light is combined with high temperature the dde2 JA mutant is less tolerant than the wild type [START_REF] Balfagón | Jasmonic Acid Is Required for Plant Acclimation to a Combination of High Light and Heat Stress[END_REF]. In line with these results, JA is known to act together with SA to confer basal thermotolerance [START_REF] Clarke | Jasmonates act with salicylic acid to confer basal thermotolerance in Arabidopsis thaliana[END_REF]. This emphasizes further the fact that different light stress conditions induce different, sometimes opposite, physiological responses.

Similarly to JA, both SA and ABA also accumulate in plants exposed to excess light energy [START_REF] Galvez-Valdivieso | The High Light Response in Arabidopsis Involves ABA Signaling between Vascular and Bundle Sheath Cells[END_REF][START_REF] Lv | β-cyclocitral upregulates salicylic acid signalling to enhance excess light acclimation in Arabidopsis[END_REF][START_REF] Beaugelin | OXI1 and DAD Regulate Light-Induced Cell Death Antagonistically through Jasmonate and Salicylate Levels[END_REF]. ABA deficiency in Arabidopsis was reported to enhance plant sensitivity to high light [START_REF] Huang | The Arabidopsis Transcriptome Responds Specifically and Dynamically to High Light Stress[END_REF], suggesting a positive role for ABA in stress tolerance. Conversely, the SA-deficient sid2 mutant of Arabidopsis shows enhanced tolerance to high light stress as shown by a marked reduction of lipid peroxidation compared with the wild type [START_REF] Beaugelin | OXI1 and DAD Regulate Light-Induced Cell Death Antagonistically through Jasmonate and Salicylate Levels[END_REF]. These results support the finding that SA overaccumulation leads to a 1 O2 burst in the chloroplast [START_REF] Lv | Uncoupled Expression of Nuclear and Plastid Photosynthesis-Associated Genes Contributes to Cell Death in a Lesion Mimic Mutant[END_REF], resulting in a possible self-amplifying loop. Furthermore, SA biosynthesis is downstream of JA accumulation [START_REF] Beaugelin | OXI1 and DAD Regulate Light-Induced Cell Death Antagonistically through Jasmonate and Salicylate Levels[END_REF], suggesting that the cell death mechanism under abiotic stress is different from the well-known SA-JA antagonism in biotic stress. Clearly, the role of these two stress hormones in plant tolerance/sensitivity to excess light energy still needs more investigation [START_REF] Liu | Salicylic acid receptors activate jasmonic acid signalling through a non-canonical pathway to promote effector-triggered immunity[END_REF].

Emerging evidence has also implicated cytokinin signaling in abiotic stresses related to photosynthetic dysfunction and ROS production [START_REF] Zwack | Interactions between cytokinin signalling and abiotic stress responses[END_REF][START_REF] Cortleven | Cytokinin action in response to abiotic and biotic stresses in plants[END_REF].

Loss of the cytokinin receptors AHK2 and AHK3 results in greater photooxidative stress tolerance (induced by a combination of water deficit and moderately elevated PFD) as indicated by decreased lipid peroxidation and by chlorophyll loss, and this was interpreted as an indication that cytokinins negatively regulate photooxidative stress tolerance [START_REF] Danilova | Membrane receptors of cytokinin and their regulatory role in Arabidopsis thaliana plant response to photooxidative stress under conditions of water deficit[END_REF]. However, in another study [START_REF] Cortleven | A novel protective function for cytokinin in the light stress response is mediated by the Arabidopsis histidine kinase2 and Arabidopsis histidine kinase3 receptors[END_REF], ahk2,3 and arr1,12 double mutants were hypersensitive to high light stress, indicating that cytokinin signaling may play a positive role in this response. Those contradictory results illustrate even better the need for standardized conditions in imposing high light stress (i.e. temperature) .

The endoplasmic reticulum in the response to excess light energy

Although excessive light is a stress that primarily affects the chloroplast, it induces an extended response that can lead to cell death and senescence of a full organ. Counterintuitively, it was recently shown in Nicotiana tabacum seedlings that chloroplastic gene expression and translation showed only limited changes after an increase in PFD from 350 mol photons m -2 s -1 to 1000 mol m -2 s -1 at 24°C [START_REF] Schuster | Limited Responsiveness of Chloroplast Gene Expression during Acclimation to High Light in Tobacco[END_REF]. These results are in agreement with the genetic response to excessive light of the Arabidopsis 1 O2-overproducer mutant ch1, which showed that only 9% of the total genetic response involved chloroplastic protein-encoding genes [START_REF] Beaugelin | Endoplasmic reticulummediated unfolded protein response is an integral part of singlet oxygen signalling in plants[END_REF]. The vast majority of the response was nuclear, followed by genes encoding mitochondrial proteins and endoplasmic reticulum (ER)-located proteins. A possible explanation for the ER response lies in the extensive modification of proteins caused by RCS.

ER stress is a well-described phenomenon occurring in eukaryotic cells when unfolded or misfolded proteins accumulate in the ER lumen and overwhelm the folding capacity of the ER. Plant signaling in the ER was previously reviewed by [START_REF] Park | Endoplasmic Reticulum Plays a Critical Role in Integrating Signals Generated by Both Biotic and Abiotic Stress in Plants[END_REF]: ER stress triggers a response called the Unfold Protein Response (UPR) by activating two signaling branches via bZIP28 and IRE1/bZIP60, which can sense and transduce the information [START_REF] Park | Endoplasmic Reticulum Plays a Critical Role in Integrating Signals Generated by Both Biotic and Abiotic Stress in Plants[END_REF]. Both branches are activated when BIP chaperones (binding immunoglobulin proteins) are released from bZIP28 and IRE1 inside the ER and bind to unfolded proteins [START_REF] Howell | Endoplasmic reticulum stress responses in plants[END_REF][START_REF] Nawkar | Activation of the Transducers of Unfolded Protein Response in Plants[END_REF].

Both in plant and animal systems, the ER integrates cellular signals and resides at the intersection between tolerance and cell death responses (Su et al., 2013;[START_REF] Li | Endoplasmic reticulum stress is the crossroads of autophagy, inflammation, and apoptosis signaling pathways and participates in liver fibrosis[END_REF][START_REF] Kørner | Endoplasmic Reticulum Stress Signaling in Plant Immunity-At the Crossroad of Life and Death[END_REF].

Evidence that ROS production in chloroplasts and mitochondria triggers the UPR in plants are accumulating, and Ozgur et al. (2015) depicted transcriptional modifications of UPR gene expression upon application of H2O2, rotenone, methyl viologen or 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), mimicking organellar ROS production [START_REF] Ozgur | The effects of induced production of reactive oxygen species in organelles on endoplasmic reticulum stress and on the unfolded protein response in arabidopsis[END_REF]. In agreement with these results, the UPR is triggered in the 1 O2-overproducing ch1 mutant both under physiological growth conditions (chronic induction) and during excessive light stress [START_REF] Beaugelin | Endoplasmic reticulummediated unfolded protein response is an integral part of singlet oxygen signalling in plants[END_REF]. However, 1 O2 lifetime has been estimated in the sub-s range in the thylakoid membranes allowing an estimated diffusion distance of around only 5.5 nm [START_REF] Khorobrykh | Oxygen and ROS in Photosynthesis[END_REF]. Therefore, 1 O2 must react with molecules in its proximity that then constitute the actual signal mediators (e.g. carotene oxidation products, lipid peroxides, RCS). Walley and co-workers demonstrated that MEcPP, an intermediate of isoprenoid biosynthesis in the chloroplast, was able to activate UPR genes in vivo [START_REF] Walley | Plastid-produced interorgannellar stress signal MEcPP potentiates induction of the unfolded protein response in endoplasmic reticulum[END_REF]. In fact, the accumulation of MEcPP in the Arabidopsis ceh1 mutant causes the constitutive accumulation of SA, which triggers the UPR response in the ER and regulates JA-responsive genes [START_REF] Xiao | Retrograde signaling by the plastidial metabolite MEcPP regulates expression of nuclear stress-response genes[END_REF][START_REF] Lemos | The plastidial retrograde signal methyl erythritol cyclopyrophosphate is a regulator of salicylic acid and jasmonic acid crosstalk[END_REF][START_REF] Bjornson | Integrated omics analyses of retrograde signaling mutant delineate interrelated stress-response strata[END_REF] .

Under high light conditions, mutants of the UPR signaling pathway have helped to reveal the key role of the ER in triggering cell death mechanisms [START_REF] Beaugelin | Endoplasmic reticulummediated unfolded protein response is an integral part of singlet oxygen signalling in plants[END_REF]. The double mutant bzip60 bzip28 showed an increased sensitivity towards excessive light stress, while pretreatment with tauroursodeoxycholic acid (TUDCA, a chemical chaperone alleviating UPR) leads to increased tolerance [START_REF] Beaugelin | Endoplasmic reticulummediated unfolded protein response is an integral part of singlet oxygen signalling in plants[END_REF]. Moreover, pre-treatment of plants with the ER stress inducer, tunicamycin, was shown to enhance photosensitivity, with the induction of PCD marker genes and the development of leaf cell death and lipid peroxidation under excessive light [START_REF] Beaugelin | Endoplasmic reticulummediated unfolded protein response is an integral part of singlet oxygen signalling in plants[END_REF]. The strict interconnection between chloroplasts and ER stress under excessive light is further demonstrated by AtDAD1 and AtDAD2, two ER-located proteins, which play a key role as inhibitors of excessive light-induced PCD [START_REF] Beaugelin | OXI1 and DAD Regulate Light-Induced Cell Death Antagonistically through Jasmonate and Salicylate Levels[END_REF]. DAD proteins appear to act as molecular brakes on the 1 O2-induced, OXI1/JA/SA-mediated cell death by inhibiting OXI1.

To date ER-mediated PCD is known to involve several mechanisms such as the regulation of pro-or anti-death members of the Bcl-2 family, the induction of plant specific N-rich proteins (NRPs) cell death signaling and the induction of proteins exhibiting caspase-1-like or caspase-3-like activities [START_REF] Williams | AtBAG7, an Arabidopsis Bcl-2-associated athanogene, resides in the endoplasmic reticulum and is involved in the unfolded protein response[END_REF][START_REF] Reis | N-rich protein (NRP)-mediated cell death signaling: a new branch of the ER stress response with implications for plant biotechnology[END_REF]. AtBAG7 is a member of Bcl-2-associated athanogene (BAG) located in the ER. AtBAG7 directly binds to AtBIP2 during UPR and facilitates heat and cold stress tolerance. [START_REF] Carvalho | The Endoplasmic Reticulum Binding Protein BiP Displays Dual Function in Modulating Cell Death Events[END_REF] showed that overexpression of BIP in soybean induces delayed senescence under developmental and abiotic stress conditions, and leads to the attenuation of NRP-mediated cell death [START_REF] Carvalho | The Endoplasmic Reticulum Binding Protein BiP Displays Dual Function in Modulating Cell Death Events[END_REF]. Accordingly, a knockout mutant of BiP3 exhibited an increased sensitivity to high light stress in Arabidopsis [START_REF] Beaugelin | Endoplasmic reticulummediated unfolded protein response is an integral part of singlet oxygen signalling in plants[END_REF]. In NRP-overexpressing soybean lines, the transcription factor GmNAC81 (homologue of ANAC089 in Arabidopsis thaliana) is strongly induced and promote activation of caspase 1-and caspase 3-like activity [START_REF] Reis | N-rich protein (NRP)-mediated cell death signaling: a new branch of the ER stress response with implications for plant biotechnology[END_REF]). GmNAC81 interacts with GmNAC30 which together bind to the promoter of a vacuolar processing enzyme (VPE) involved in PCD [START_REF] Mendes | GmNAC30 and GmNAC81 integrate the endoplasmic reticulum stress-and osmotic stress-induced cell death responses through a vacuolar processing enzyme[END_REF]. Also in Arabidopsis, ANAC089 controls a number of PCD regulators, including MC5, BAG6 and NAC094, under stress conditions [START_REF] Yang | The Membrane-Associated Transcription Factor NAC089 Controls ER-Stress-Induced Programmed Cell Death in Plants[END_REF]. In agreement, the 1 O2-overproducing ch1 mutant exhibits an increased expression of ANAC089 under excessive light stress [START_REF] Beaugelin | Endoplasmic reticulummediated unfolded protein response is an integral part of singlet oxygen signalling in plants[END_REF]. Finally, caspase-like activity seems to be a pan-maker of plant PCD mediating excessive light-induced PCD, downstream of RCS and prolonged ER stress signaling .

Why do plants kill leaf cells under excessive light?

The biological role of PCD is easy to understand in a phytopathological context-it limits further pathogen spreading and triggers defense mechanisms in non-infected neighbor tissues [START_REF] Morel | The hypersensitive response and the induction of cell death in plants[END_REF][START_REF] Heath | Hypersensitive response-related death[END_REF]. The physiological relevance of excessive light-induced cell death is less clear. A relevant aspect in this context is the fact that the photoprotective capacity strongly depends on leaf age [START_REF] Bielczynski | Leaf and Plant Age Affects Photosynthetic Performance and Photoprotective Capacity[END_REF][START_REF] D'alessandro | Decoding β-Cyclocitral-Mediated Retrograde Signaling Reveals the Role of a Detoxification Response in Plant Tolerance to Photooxidative Stress[END_REF]. In fact, when plants are exposed to excess light, lipid peroxidation is usually much more pronounced in the mature leaves (D'Alessandro et al., 2018) (see Fig. 4). Non-functional photosynthetic tissues become an unnecessary carbon sink, and damaged tissues are more exposed to pathogen attacks [START_REF] Savatin | Wounding in the plant tissue: the defense of a dangerous passage[END_REF]. Therefore, plants appear to choose to sacrifice old/mature leaves when they are photodamaged and recover important nutrients rather than invest in repair. Also, cell membranes damaged under photooxidative conditions could lead to water loss [START_REF] Carvalho | Dehydration rate determines the degree of membrane damage and desiccation tolerance in bryophytes[END_REF][START_REF] Li | The karrikin receptor KAI2 promotes drought resistance in Arabidopsis thaliana[END_REF]. Stomatal and leaf hydraulic systems are known to be very responsive to stress conditions, and they can decline even before cell integrity is lost [START_REF] Trueba | Thresholds for leaf damage due to dehydration: declines of hydraulic function, stomatal conductance and cellular integrity precede those for photochemistry[END_REF]. Therefore, PCD may be a means to avoid dramatic water losses from photodamaged leaves, to preserve water relations and promote plant survival. Therefore, preservation of healthy organs by light induced PCD of photooxidized organs is a defense mechanism that can come into play under many environmental constraints.

Main conclusions and some perspectives

In this review, we provide a broad summary of the literature linking pure excessive light exposure to programmed cell death. We highlight the importance of controlling the conditions of excessive light treatments, particularly leaf temperature, to limit variability between laboratories. In fact, by selecting results based on pure excessive light exposure, and not from the superposition of heat and high light stress, we are able to propose a model explaining how this stress can trigger PCD (Fig. 5).

After an initial phase of stress response, in which the role of 1 O2 is predominant and phototolerance mechanism are activated, damage to PSII and the decay of lipid peroxides to RCS leads to the accumulation of reduced forms of oxygen. H2O2 further enhances the increase in RCS by limiting cellular detoxification, leading to ER stress. If the stress does not cease, ER stress may then lead to the activation of PCD induced by caspase-like activity. At the same time, overproduction of 1 O2 may activate EX1 at the grana margins, together with the OXI1/JA/SA controlled PCD.

Although we were able to identify several steps in the response to excessive light, many questions remain to be elucidated. First of all, we know that our knowledge of phototolerance mechanisms is not exhaustive, and that many molecular players in apocarotenoid sensing remain elusive. It is not clear if the signaling of those metabolites interacts with other known retrograde signaling pathways.

Finally, PCD may be mediated by at least three pathways in our model, and the connections and interrelations between caspase-like inducers of PCD and EX1-and OXI1-induced PCD remain unknown. Therefore, it is pivotal in our opinion to find similar working conditions between laboratories to maximize the compatibility between the results. At the same time, the substantial difference between excessive light alone or in combination with heat shock highlights the importance of studying stress combinations.

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. The xenobiotic detoxification response is composed of three phases involving families of detoxifying enzymes.

Figure 3 .

 3 Figure 3. Different levels of photoprotective mechanisms are present in plants to sequentially (1) limit ROS photoproduction, (2) ROS-induced oxidation and (3) toxicity of photooxidation byproducts.

Figure 4 .

 4 Figure 4. The extent of high light-induced lipid peroxidation is age dependent. A) Autoluminescence image of lipid peroxidation in an Arabidopsis plant exposed to high light (1500 µmol photons m -2 s - 1 ) and low air temperature (7°C), showing that lipid peroxides accumulate in old/mature leaves. B) Autoluminescence images of detached Arabidopsis leaves exposed to a PFD of 1100 µmol photons m -2 s -1 for 16 h in a cold chamber (6 °C), confirming that young leaves are more tolerant to

Figure 5 .

 5 Figure 5. Scheme of the pathways of excessive light induced PCD in plants. Excessive absorption of light energy in the chloroplasts brings about ROS production that triggers several signaling pathways leading to PCD. These pathways involve reactive products derived from lipid peroxidation,

Acknowledgments

We would like to thank Dr. Benjamin Field (CNRS) for improving the use of English in the text. We would like to acknowledge funding from the French National Research Agency (ANR project SLOSAM, 14-CE02-0010-02). No conflict of interest declared.

Author contributions

S.D. and M.H. planned, structured, and wrote the article; I.B. contributed to the article.


Evangelos Balanikas

Lara Martinez-Fernandez

Roberto Improta

Peter Podbevšek

Gérard Baldacchino

Dimitra Markovitsi

Structural Duality of Nucleobases in Guanine Quadruplexes Controls Their Low-energy Photoionization

ABSTRACT. Guanine quadruplexes are four-stranded DNA/RNA structures composed of a guanine core (vertically stacked guanine tetrads) and peripheral groups (dangling ends and/or loops). Such a dual structural arrangement of the nucleobases favors their photoionization at energies significantly lower than the guanine ionization potential. This effect is important both in respect to the oxidative DNA damage and for applications in the field of optoelectronics.

Photoionization quantum yields, determined at 266 nm by nanosecond transient absorption spectroscopy, strongly depend on both the type and position of the peripheral nucleobases; the highest value (1.5×10 -2 ) is found for the tetramolecular structure (AG4A)4 in which, adenines are intermittently stacked on the adjacent guanine tetrads, as attested by NMR spectroscopy. Quantum chemistry calculations show that peripheral nucleobases interfere in a key step preceding electron ejection: charge separation, initiated by the population of charge transfer states during the relaxation of electronic excited states.

TOC GRAPHICS

KEYWORDS. Guanine quadruplexes, DNA, photoionization, photoconductivity, oxidative damage, transient absorption spectroscopy, NMR spectroscopy, quantum chemistry.

Guanine quadruplexes (G4) are four-stranded DNA/RNA structures extensively studied because of their involvement in multiple biological functions [START_REF] Rhodes | G-quadruplexes and their regulatory roles in biology[END_REF][START_REF] Kharel | Properties and biological impact of RNA G-quadruplexes: from order to turmoil and back[END_REF] and in view of applications in the field of nanotechnology. [START_REF] Mergny | DNA Quadruple Helices in Nanotechnology[END_REF] In both respects, their photoionization is important. Generation of electron holes triggers chemical reactions ultimately leading to oxidative damage. [START_REF] Cadet | Oxidatively generated damage to the guanine moiety of DNA: Mechanistic aspects and formation in cells[END_REF][START_REF] Delaney | Charge transport in DNA duplex/quadruplex conjugates[END_REF] Moreover, electron holes intervene in charge transport. [START_REF] Livshits | Long-range charge transport in single G-quadruplex DNA molecules[END_REF][START_REF] Sha | Charge splitters and charge transport junctions based on guanine quadruplexes[END_REF] It was reported recently [START_REF] Banyasz | Absorption of Low-Energy UV Radiation by Human Telomere G-Quadruplexes Generates Long-Lived Guanine Radical Cations[END_REF][START_REF] Balanikas | Guanine Radicals Induced in DNA by Low-Energy Photoionization[END_REF] that G4 undergo photoionization at energies significantly lower compared to the guanine vertical ionization potential. [START_REF] Schroeder | Oxidation Half-Reaction of Aqueous Nucleosides and Nucleotides via Photoelectron Spectroscopy Augmented by ab Initio Calculations[END_REF] In contrast to high-energy photoionization, [START_REF] Candeias | Ionization of polynucleotides and DNA in aqueous solution by 193 nm pulsed laser light: identification of base derived radicals[END_REF][START_REF] Melvin | 193 nm Light Induces Single-strand Breakage of DNA Predominantly at Guanine[END_REF] this low-energy process, also observed for other DNA systems with significantly lower efficiencies, strongly depends on the secondary structure; it could not be detected neither for mononucleotides nor for poorly stacked single strands (<3×10 -4 ). [START_REF] Balanikas | Guanine Radicals Induced in DNA by Low-Energy Photoionization[END_REF][START_REF] Balanikas | DNA photoionization: from high to low energies[END_REF] Here, combining spectroscopic and computational techniques, we show that the arrangement of G4 nucleobases in two distinct structural parts, vertically stacked guanine (G) tetrads, on one hand, and dangling ends and/or loops joining the tetrads, on the other, is essential in this process.

Depending on their nature and position, peripheral nucleobases may be electronically coupled to guanines of the core, leading to the population of excited charge transfer (CT) states. Thus, as positive and negative charges are located on different structural elements, charge separation, which is a key step in low-energy photoionization, is enhanced. The findings of this study should be impactful for the design of photoactive devices.

Experimentally, we studied tetramolecular G4 formed by association of four DNA strands in phosphate buffer containing K + cations. The latter, whose concentration within cells exceeds by far that of Na + ions, were shown to enhance G4 photoionization. [START_REF] Cadet | Oxidatively generated damage to the guanine moiety of DNA: Mechanistic aspects and formation in cells[END_REF][START_REF] Behmand | Potassium Ions Enhance Guanine Radical Generation upon Absorption of Low-Energy Photons by G-quadruplexes and Modify Their Reactivity[END_REF] Symmetric sequences, with thymines (T), adenines (A) or AT groups at both ends, as well as asymmetric ones, with the peripheral nucleobases only at the 3' or the 5' ends, were examined (Table 1 and Figure 1). Their circular dichroism spectra, presented in the Supporting Information (SI; Figure S1), are all typical of parallel G4 as attested by a positive peak at around 264 nm and a negative one at 243 nm. [START_REF] Karsisiotis | Topological Characterization of Nucleic Acid G-Quadruplexes by UV Absorption and Circular Dichroism[END_REF] Table 1. One-photon ionization quantum yields (i) determined for tetramolecular G4 at 266 nm.

Sequence/G4

 i x10 [START_REF] Mergny | DNA Quadruple Helices in Nanotechnology[END_REF] 5'-AG4A-3' 14.8 ± 0.5 5'-AG5A-3' 14.4 ± 0.1 5'-G4A-3' 12.6 ± 0.1 5'-AG4-3' 9.9 ± 0.4 5'-G4T-3' 8.8 ± 0.2 5'-TG4T-3' 8.1 ± 0.5 [START_REF] Behmand | Potassium Ions Enhance Guanine Radical Generation upon Absorption of Low-Energy Photons by G-quadruplexes and Modify Their Reactivity[END_REF] 5'-TAG4AT-3' 7.0 ± 0.51 5'-TG4-3' 4.7 ± 0.7

The propensity of the examined G4 to undergo low-energy photoionization was evaluated by their one-photon ionization quantum yields  i , determined using nanosecond transient absorption spectroscopy with excitation at 266 nm. Briefly, the decays of the hydrated ejected electrons (Figure S2) were recorded for various excitation intensities. [START_REF] Balanikas | Guanine Radicals Induced in DNA by Low-Energy Photoionization[END_REF] Subsequently, ionization curves were constructed and  i values derived from the intercept on the vertical axes (Figures 2 andS3).

The i values found for G4 composed of four G-tetrads exhibit important variations, up to a factor 3 (Table 1). Replacing A by T induces a decrease in  i , whereas their positioning at the 3' ends leads to significantly higher values compared to 5' ends analogs. In contrast, adding an extra Gtetrad has no effect, the  i values of 5'-AG4A-3' and 5'-AG5A-3' being practically the same.

Consequently, it appears that the synergy between central core and peripheral groups plays an important role in the propensity of G4 to undergo low-energy photoionization. 5'-AG4A-3' exhibits the highest i (1.5×10 -2 ) determined so far for any DNA system. It is an order of magnitude larger than that of genomic DNA, whose ionization curve is shown in Figure 2 for comparison. Moreover, it is twice as high compared to 5'-TG4T-3'. The difference between the i of these two G4 scaffolds could possibly arise from formation of A-tetrads. As a matter of fact, NMR and crystallographic studies reported that in 5'-TG4T-3' the position of thymines is either poorly defined or they are rotated away from the G4 core. [START_REF] Aboul-Ela | Solution structure of a parallel-stranded tetraplex formed by d(TG4T) in the presence of sodium ions by nuclear magnetic resonance spectroscopy[END_REF][START_REF] Phillips | The crystal structure of a parallel-stranded guanine tetraplex at 0.95 angstrom resolution[END_REF] But adenines in some G4 were found to self-associate via Hoogsteen bonding. [START_REF] Patel | NMR studies on truncated sequences of human telomeric DNA: observation of a novel A-tetrad[END_REF][START_REF] Searle | Structure and K+ ion-dependent stability of a parallel-stranded DNA quadruplex containing a core A-tetrad[END_REF][START_REF] Pan | Crystal structure of an RNA purine-rich tetraplex containing adenine tetrads: Implications for specific binding in RNA tetraplexes[END_REF] In order to investigate the above question, we utilized solution NMR. Although 1D NMR of 5'-AG4A-3' have been reported, [START_REF] Virgilio | Improved performances of catalytic G-quadruplexes (G4-DNAzymes) vi a the chemical modifications of the DNA backbone to provide G-quadruplexes with double 3 '-external G-quartets[END_REF] no detailed structural study was carried out. The adjacent adenines or guanines, could be observed at temperatures as low as 0 °C. Nevertheless, several NOE cross-peaks were observed between G of the outer G-tetrads and flanking A (Figure 1C). Most notably, amino protons of A1 and A6 exhibit NOE cross-peaks with imino protons of G2 and G5, respectively, which suggest that in a time-averaged manner A1 and A6 nucleobases are spatially close to G of adjacent G-tetrads.

In conclusion, NMR measurements, have not detected hydrogen bonding between adenines,

showing that no stable A-tetrads are formed. Nevertheless, intermittent stacking of individual adenines is likely. Furthermore, adenines could form heterogenous capping structures, protecting their amino proton from exchange with solvent.

Having ruled out the specific role of A-tetrads, we examined whether CT states may be formed between a peripheral nucleobase and a G of the neighboring tetrad. To this end, we first considered a G-tetrad with an adenine at the 3' position (5'-G-tetradA-3'; Figure S6A). The relative position between G and A is comparable to previously reported structures (Figure S6B). [START_REF] Yang | G-Quadruplex DNA and RNA[END_REF] Results obtained for this system are quite similar with those computed for the 5'-GA-3' stacked dinucleotide, except for the presence of additional * G states, resulting from the larger the number of G (Tables S1 to S4). Consequently, we compared the sequence effect on the electronic excited states of stacked dinucleotides: 5'-AG-3', 5'-GA-3', 5'-TG-3' and 5'-GT-3'.

We considered at quantum mechanical (QM) level the entire system (two nucleobases, the connecting backbone and the counter ion), embedded in water through a polarizable continuum model. We used the density functional theory, in particular the M052X functional, and its timedependent version (TD-DFT) combined with the 6-31G(d) basis set. This method was selected due to its good performance in comparable studies. [START_REF] Martinez-Fernandez | Studying the excited electronic states of guanine rich DNA quadruplexes by quantum mechanical methods: main achievements and perspectives[END_REF] We characterized the Franck-Condon (FC) excited states and, then, studied their relaxation pathways.

Important differences among the four dinucleotides appear already at the FC region (Table S1 and Figure S7). We found that in 5'-GA-3' the coupling between the bright and CT states is larger.

Indeed, its spectroscopic state S2 has some CT character (0.3 a.u.). In contrast, this state is completely devoid of CT character in 5'-AG-3'. Instead, 5'-AG-3' presents a low-energy state with an oscillator strength of 2×10 -1 and significant G +  A -CT character (0.6 a.u.) but whose energy difference with respect to the spectroscopic state is ~0.3 eV. In addition, the energy of higher CT states (S9) is ~0.1 eV lower in 5'-GA-3' compared to 5'-AG-3'. For the T analogs, 5'-GT-3' has a clear low-lying CT state (0.8 a.u.), but with very small oscillator strength (10 -2 ), whereas in 5'-TG-3' the first state with significant CT character is higher in energy (+0.5 eV). Our finding that CT states involving transfer of an electron towards the 3' end are more stable than in the opposite direction, agrees with previous reports for other types of dinucleotides. [START_REF] Aquino | The Charge-Transfer States in a Stacked Nucleobase Dimer Complex: A Benchmark Study[END_REF][START_REF] Santoro | The excited electronic states of adenineguanine stacked dimers in aqueous solution: a PCM/TD-DFT study[END_REF][START_REF] Martinez-Fernandez | Novel adenine/thymine photodimerization channels mapped by PCM/TD-DFT calculations on dApT and TpdA dinucleotides[END_REF][START_REF] Martinez-Fernandez | Sequence dependence on DNA photochemistry: a computational study of photodimerization pathways in TpdC and dCpT dinucleotides[END_REF] Figure 3. Excited state CT minima and the associated electronic density difference computed for 5'-GA-3' (left) and 5'-GT-3' (right). Color code: guanine (pink), adenine (green), thymine (cyan), backbone (silver), hole (blue) and electron (red).

Coming to the excited state relaxation, we examined the population of CT states. To this end, we optimized the lowest five excited states as well as the first state with clear G + T -/A -CT character, locating different types of minima. Their stabilities and geometrical features are presented in Table S3 and Figure S8. 5'-GA-3' presents a minimum with important CT character that can be reached from the bright S2 or the S1 state and whose stability is comparable to that of the * (La G)-minimum (Figure 3, Table S3). 5'-AG-3' and 5'-GT-3' exhibit also such stable CT minima resulting from higher and/or dark excited states (S9 and S4). In contrast, in the case of 5'-TG-3', none of the optimizations lead to a CT minimum.

Our computational results show that the probability of populating a CT state during the excited state relaxation is higher if the "peripheral" nucleobase is located in the 3', which matches the ordering of  i (Table 1). But CT population in G4 does not depend only on electronic factors but also on the stacking probability. However, as NMR spectra did not reveal asymmetric stacking of the peripheral nucleobases, electronic factors should prevail. In this respect, we remark that the steady-state absorption spectra of 5'-G4T-3' and 5'-TG4-3' are practically identical (Figure S5).

Yet their i differ by a factor 2, pinpointing the role of the excited state relaxation. Within this scheme, the lower  i value of 5'-TAG4AT-3' compared to 5'-AG4A-3' is explained by both a smaller stacking probability of adenines when attached to thymines and interaction between them during the relaxation process. [START_REF] Martinez-Fernandez | Novel adenine/thymine photodimerization channels mapped by PCM/TD-DFT calculations on dApT and TpdA dinucleotides[END_REF] Other factors, such as competition between A +  T -and G +  A - CT states cannot be excluded. [START_REF] Martinez-Fernandez | Photoinduced long-lived charge transfer excited states in AT-DNA strands[END_REF] Our QM calculations provided a qualitative interpretation for the role of peripheral groups. A quantitative description, encompassing electronic relaxation and conformational motions in the entire G4, could be achieved by integrating molecular dynamics simulations. Such an approach, already applied for the determination of FC states in small G4, [START_REF] Avagliano | Enhanced Rigidity Changes Ultraviolet Absorption: Effect of a Merocyanine Binder on G-Quadruplex Photophysics[END_REF] should become affordable in the near future for the study of dynamical processes in larger systems.

Femtosecond studies detected the population of CT states in telomeric G4. [START_REF] Mcgovern | Picosecond infrared probing of the vibrational spectra of transients formed upon UV excitation of stacked G-tetrad structures[END_REF][START_REF] Martinez-Fernandez | A Comprehensive Study of Guanine Excited State Relaxation and Photoreactivity in G-Quadruplexes[END_REF][START_REF] Ma | Real-time Monitoring Excitation Dynamics of Human Telomeric Guanine Quadruplexes: Effect of Folding Topology, Metal Cation, and Confinement by Nanocavity Water Pool[END_REF] It was also shown that the excited state relaxation pattern remains the same in reversed micelles, mimicking the crowding conditions in cells. [START_REF] Ma | Real-time Monitoring Excitation Dynamics of Human Telomeric Guanine Quadruplexes: Effect of Folding Topology, Metal Cation, and Confinement by Nanocavity Water Pool[END_REF] QM calculations performed for such G4 showed that CT states may be formed between a T in the loop and a G in the core, but experiments have not disentangled this type of mixed CT states from those involving two guanines. [START_REF] Martinez-Fernandez | A Comprehensive Study of Guanine Excited State Relaxation and Photoreactivity in G-Quadruplexes[END_REF] More importantly, the study on intrinsic G4 fluorescence clearly evidenced the existence of interactions between the G core and nucleobases in loops and dangling groups. [START_REF] Kwok | Effect of Loop Sequence and Loop Length on the Intrinsic Fluorescence of G-Quadruplexes[END_REF][START_REF] Gustavsson | Fundamentals of the Intrinsic DNA Fluorescence[END_REF] Our comparative study on tetramolecular G4 formed in the presence of K + cations and characterized by a parallel arrangement of G, unveiled the importance of the peripheral nucleobases for their low-energy photoionization. Comparing the  i values reported previously for monomolecular telomeric G4 formed in the presence of Na + cations and characterized by antiparallel arrangement of G, we realize retrospectively that a similar effect takes place: when the dangling groups TA at the 5' end and TT at the 3' end are removed from the telomeric sequence i decreases from 5.2×10 -3 to 4.5×10 -3 , 9 despite the fact that the G percentage increases from 48%

to 57%.

Duplexes are devoid of the structural duality characterizing G4. Yet, they do undergo lowenergy photoionization (i≤2×10 -3 ). [START_REF] Balanikas | Guanine Radicals Induced in DNA by Low-Energy Photoionization[END_REF] In this case, inhomogeneity is introduced through conformational disorder and also base sequence. Thus, repetitive sequences (homopolymeric AT, alternating AT or alternating GC), exhibit quite similar  i. But  i increases by ca. 40% in systems containing GGG steps. [START_REF] Balanikas | Guanine Radicals Induced in DNA by Low-Energy Photoionization[END_REF] The latter, due to their low oxidation potential, behave as traps for electron holes. [START_REF] Meggers | Sequence dependent long range hole transport in DNA[END_REF][START_REF] Saito | Mapping of the hot spots for DNA damage by one-electron oxidation: Efficacy of GG doublets and GGG triplets as a trap in long-range hole migration[END_REF][START_REF] Kumar | One-electron oxidation of ds(5 '-GGG-3 ') and ds(5 '-G(8OG)G-3 ') and the nature of hole distribution: a density functional theory (DFT) study[END_REF] Accordingly, the trapping of the positive charge is expected to be particularly efficient in G4, where it may be delocalized. [START_REF] Martinez-Fernandez | Studying the excited electronic states of guanine rich DNA quadruplexes by quantum mechanical methods: main achievements and perspectives[END_REF][START_REF] Kumar | One-electron oxidation of ds(5 '-GGG-3 ') and ds(5 '-G(8OG)G-3 ') and the nature of hole distribution: a density functional theory (DFT) study[END_REF][START_REF] Sun | Effects of G-Quadruplex Topology on Electronic Transfer Integrals[END_REF] Eventually, electron ejection occurs from the peripheral nucleobase bearing the negative charge, whose vertical ionization potential is lower compared to the "neutral" analog. [START_REF] Schiedt | Anion spectroscopy of uracil, thymine and the amino-oxo and amino-hydroxy tautomers of cytosine and their water clusters[END_REF] Beyond DNA, the existence of distinct structural units is a prerequisite for applications relying on photoinduced charge transfer, as artificial photosynthesis. [START_REF] Romero | Quantum design of photosynthesis for bio-inspired solar-energy conversion[END_REF][START_REF] Kranz | Characterizing photocatalysts for water splitting: from atoms to bulk and from slow to ultrafast processes[END_REF] In addition, electron photodetachment is closely related to photoconductivity and photoelectrochemistry, used, among others, for bioanalysis. Sensors involving G4 associated with external photoactive molecules have been reported. [START_REF] Liu | Intercalative methylene blue as an efficient signal molecule of tremella-like Bi 2 WO 6: Toward high performance photoelectrochemical biosensing[END_REF][START_REF] Gu | Homogenous cathodic photoelectrochemistry for DNA binding protein analysis[END_REF] The discovery of the low-energy G4 photoionization and the characterization of structural and electronic factors governing this phenomenon presented here open new perspectives in this field through appropriate design of peripheral groups (for example, by joining G4 at their 5', as proposed for DNAzymes). [START_REF] Virgilio | Improved performances of catalytic G-quadruplexes (G4-DNAzymes) vi a the chemical modifications of the DNA backbone to provide G-quadruplexes with double 3 '-external G-quartets[END_REF] ASSOCIATED CONTENT Supporting Information Available. Experimental Details; Additional Experimental Results; Additional Computational Results.

Figure 1 .

 1 Figure 1. Structural properties of 5'-AG4A-3'. A) Imino, aromatic and amino region of the 1D 1H NMR spectrum. B) Schematic structure of parallel tetramolecular G4. C) Representation of NOE cross-peaks between outer G-tetrads and flanking adenines extracted from a 2D NOESY spectrum (τm = 200 ms). Dashed lines represent weak cross-peaks.

Figure 2 .

 2 Figure 2. Ionization curves determined for 5'-AG4A-3' (green) and duplex calf thymus DNA (blue) with 266 nm excitation; [ehyd -]0 and [h] denote, respectively, the concentration of hydrated ejected electrons and absorbed photons per laser pulse. Experimental points (circles) are fitted with linear functions [ehyd -]0/[h]=i + [h](red).

  1D 1 H NMR spectrum of 5'-AG4A-3' revealed four well-resolved resonances in the δ range 10.8-11.5 ppm (Figure1A); they are indicative of G imino protons involved in Hoogsteen hydrogen bonding, suggesting the formation of symmetric tetrameric G4 (Figure1B). Several sets of low-intensity signals (<5%) were also observed; they possibly correspond to alternative folds. Resonances of the major species could be assigned through sequential connectivities in 2D NOESY NMR spectra (FigureS4). Based on NOE connectivities and intensities of intranucleotide H1'n-H8n cross-peaks all nucleotides were assigned the anti glycosidic conformation. Interestingly, A1 and A6 nucleotides exhibit observable broad resonances at δ 6.75 and 6.77 ppm, respectively, indicating that at 25 °C amino protons are partially protected from exchange with solvent. However, amino proton pairs of A1 and A6 are isochronous, thus precluding formation of long-lived hydrogen bonds. No down-field amino resonances, which would suggest hydrogen bonding of adenines with

AUTHOR INFORMATION

Corresponding Authors

lara.martinez@uam.es, peter.podbevsek@ki.si, dimitra.markovitsi@cea.fr

AUTHOR INFORMATION

Notes

The authors declare no competing financial interests.